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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210001	1/6	Homo sapiens	MKN45	(d)(U)C Total Exosome Isolation UF	Li, Bowen	2021	67%
Study summary Full title miR-151a-3p-rich small extracellular vesicles derived from gastric cancer accelerate liver metastasis via initiating a hepatic stemness-enhancing niche All authors Bowen Li, Yiwen Xia, Jialun Lv, Weizhi Wang, Zhe Xuan, Cen Chen, Tianlu Jiang, Lang Fang, Linjun Wang, Zheng Li, Zhongyuan He, Qingya Li, Li Xie, Shengkui Qiu, Lu Zhang, Diancai Zhang, Hao Xu, Zekuan Xu Journal Oncogene Abstract Liver metastasis (LM) severely affects gastric cancer (GC) patients' prognosis. Small extracellular (show more...)Liver metastasis (LM) severely affects gastric cancer (GC) patients' prognosis. Small extracellular vesicles (sEVs) play key roles in intercellular communication. Specific sEV-miRNAs from several types of cancer were found to induce a premetastatic niche in target organs before tumor cell arrive. However, whether the primary GC affects hepatic microenvironment or the role of sEV-miRNAs in GC-LM is yet unclear. We report that GC-derived sEVs are primarily absorbed by Kupffer cells (KCs). sEV-miR-151a-3p is highly expressed in GC-LM patients' plasma and presents poor prognosis. Treating mice with sEVs-enriched in miR-151a-3p promotes GC-LM, whereas has no influence on the proliferation of GC cells in situ. Mechanistically, sEV-miR-151a-3p inhibits SP3 in KCs. Simultaneously, sEV-miR-151a-3p targets YTHDF3 to decrease the transcriptional inhibitory activity of SP3 by reducing SUMO1 translation in a N6-methyladenosine-dependent manner. These factors contribute to TGF-β1 transactivation in KCs, subsequently activating the SMAD2/3 pathway and enhancing the stem cell-like properties of incoming GC cells. Furthermore, sEV-miR-151a-3p induces miR-151a-3p transcription in KCs to form a positive feedback loop. In summary, our results reveal a previously unidentified regulatory axis initiated by sEV-miR-151a-3p that establishes a hepatic stemness-permissive niche to support GC-LM. sEV-miR-151a-3p could be a promising diagnostic biomarker and preventive treatment candidate for GC-LM. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin transfected with miR-151a-3p reconstitution lentivirus Focus vesicles Other / small extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Commercial method Ultrafiltration Protein markers EV: Calnexin/ TSG101/ TSPAN8/ CD63 non-EV: Albumin Proteomics no Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MKN45 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell viability (%) 98 Cell count 2 500 000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 100 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 8 Wash: time (min) 70 Wash: Rotor Type Type 100 Ti Wash: speed (g) 110000 Ultra filtration Cut-off size (kDa) 100 Membrane type Polyethersulfone (PES) Commercial kit Total Exosome Isolation Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ TSPAN8/ TSG101 Not detected EV-associated proteins Calnexin Not detected contaminants Albumin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNA sequencing Database No Proteinase treatment Yes Moment of Proteinase treatment Before Proteinase type Proteinase K Proteinase concentration 20 RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 124,3 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 1,60E+10 EM EM-type Transmission-EM Image type Close-up

	EV210001	2/6	Homo sapiens	MKN45	(d)(U)C Total Exosome Isolation UF	Li, Bowen	2021	67%
Study summary Full title miR-151a-3p-rich small extracellular vesicles derived from gastric cancer accelerate liver metastasis via initiating a hepatic stemness-enhancing niche All authors Bowen Li, Yiwen Xia, Jialun Lv, Weizhi Wang, Zhe Xuan, Cen Chen, Tianlu Jiang, Lang Fang, Linjun Wang, Zheng Li, Zhongyuan He, Qingya Li, Li Xie, Shengkui Qiu, Lu Zhang, Diancai Zhang, Hao Xu, Zekuan Xu Journal Oncogene Abstract Liver metastasis (LM) severely affects gastric cancer (GC) patients' prognosis. Small extracellular (show more...)Liver metastasis (LM) severely affects gastric cancer (GC) patients' prognosis. Small extracellular vesicles (sEVs) play key roles in intercellular communication. Specific sEV-miRNAs from several types of cancer were found to induce a premetastatic niche in target organs before tumor cell arrive. However, whether the primary GC affects hepatic microenvironment or the role of sEV-miRNAs in GC-LM is yet unclear. We report that GC-derived sEVs are primarily absorbed by Kupffer cells (KCs). sEV-miR-151a-3p is highly expressed in GC-LM patients' plasma and presents poor prognosis. Treating mice with sEVs-enriched in miR-151a-3p promotes GC-LM, whereas has no influence on the proliferation of GC cells in situ. Mechanistically, sEV-miR-151a-3p inhibits SP3 in KCs. Simultaneously, sEV-miR-151a-3p targets YTHDF3 to decrease the transcriptional inhibitory activity of SP3 by reducing SUMO1 translation in a N6-methyladenosine-dependent manner. These factors contribute to TGF-β1 transactivation in KCs, subsequently activating the SMAD2/3 pathway and enhancing the stem cell-like properties of incoming GC cells. Furthermore, sEV-miR-151a-3p induces miR-151a-3p transcription in KCs to form a positive feedback loop. In summary, our results reveal a previously unidentified regulatory axis initiated by sEV-miR-151a-3p that establishes a hepatic stemness-permissive niche to support GC-LM. sEV-miR-151a-3p could be a promising diagnostic biomarker and preventive treatment candidate for GC-LM. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin transfected with miR-151a-3p reconstitution lentivirus Focus vesicles Other / small extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Commercial method Ultrafiltration Protein markers EV: Calnexin/ TSG101/ TSPAN8/ CD63 non-EV: Albumin Proteomics no Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MKN45 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell viability (%) 98 Cell count 2 500 000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 100 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 8 Wash: time (min) 70 Wash: Rotor Type Type 100 Ti Wash: speed (g) 110000 Ultra filtration Cut-off size (kDa) 100 Membrane type Polyethersulfone (PES) Commercial kit Total Exosome Isolation EV-subtype Distinction between multiple subtypes Size Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ TSPAN8/ TSG101 Not detected EV-associated proteins Calnexin Not detected contaminants Albumin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNA sequencing Database No Proteinase treatment Yes Moment of Proteinase treatment Before Proteinase type Proteinase K Proteinase concentration 20 RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 124,3 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 1,60E+10 EM EM-type Transmission-EM Image type Close-up

	EV210001	5/6	Homo sapiens	Blood plasma	(d)(U)C Total Exosome Isolation UF	Li, Bowen	2021	67%
Study summary Full title miR-151a-3p-rich small extracellular vesicles derived from gastric cancer accelerate liver metastasis via initiating a hepatic stemness-enhancing niche All authors Bowen Li, Yiwen Xia, Jialun Lv, Weizhi Wang, Zhe Xuan, Cen Chen, Tianlu Jiang, Lang Fang, Linjun Wang, Zheng Li, Zhongyuan He, Qingya Li, Li Xie, Shengkui Qiu, Lu Zhang, Diancai Zhang, Hao Xu, Zekuan Xu Journal Oncogene Abstract Liver metastasis (LM) severely affects gastric cancer (GC) patients' prognosis. Small extracellular (show more...)Liver metastasis (LM) severely affects gastric cancer (GC) patients' prognosis. Small extracellular vesicles (sEVs) play key roles in intercellular communication. Specific sEV-miRNAs from several types of cancer were found to induce a premetastatic niche in target organs before tumor cell arrive. However, whether the primary GC affects hepatic microenvironment or the role of sEV-miRNAs in GC-LM is yet unclear. We report that GC-derived sEVs are primarily absorbed by Kupffer cells (KCs). sEV-miR-151a-3p is highly expressed in GC-LM patients' plasma and presents poor prognosis. Treating mice with sEVs-enriched in miR-151a-3p promotes GC-LM, whereas has no influence on the proliferation of GC cells in situ. Mechanistically, sEV-miR-151a-3p inhibits SP3 in KCs. Simultaneously, sEV-miR-151a-3p targets YTHDF3 to decrease the transcriptional inhibitory activity of SP3 by reducing SUMO1 translation in a N6-methyladenosine-dependent manner. These factors contribute to TGF-β1 transactivation in KCs, subsequently activating the SMAD2/3 pathway and enhancing the stem cell-like properties of incoming GC cells. Furthermore, sEV-miR-151a-3p induces miR-151a-3p transcription in KCs to form a positive feedback loop. In summary, our results reveal a previously unidentified regulatory axis initiated by sEV-miR-151a-3p that establishes a hepatic stemness-permissive niche to support GC-LM. sEV-miR-151a-3p could be a promising diagnostic biomarker and preventive treatment candidate for GC-LM. (hide) EV-METRIC 67% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin gastric cancer patients with liver metastasis Focus vesicles Other / small extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Commercial method Ultrafiltration Protein markers EV: Calnexin/ TSG101/ TSPAN8/ CD63 non-EV: Albumin Proteomics no Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 100 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 8 Wash: time (min) 70 Wash: Rotor Type Type 100 Ti Wash: speed (g) 110000 Ultra filtration Cut-off size (kDa) 100 Membrane type Polyethersulfone (PES) Commercial kit Total Exosome Isolation Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins TSPAN8/ CD63/ TSG101 Not detected EV-associated proteins Calnexin Not detected contaminants Albumin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNAsequencing Database No Proteinase treatment Yes Moment of Proteinase treatment Before Proteinase type Proteinase K Proteinase concentration 20 RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 125,5 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 2,00E+10 EM EM-type Transmission-EM Image type Close-up

	EV210001	6/6	Homo sapiens	Blood plasma	(d)(U)C Total Exosome Isolation UF	Li, Bowen	2021	67%
Study summary Full title miR-151a-3p-rich small extracellular vesicles derived from gastric cancer accelerate liver metastasis via initiating a hepatic stemness-enhancing niche All authors Bowen Li, Yiwen Xia, Jialun Lv, Weizhi Wang, Zhe Xuan, Cen Chen, Tianlu Jiang, Lang Fang, Linjun Wang, Zheng Li, Zhongyuan He, Qingya Li, Li Xie, Shengkui Qiu, Lu Zhang, Diancai Zhang, Hao Xu, Zekuan Xu Journal Oncogene Abstract Liver metastasis (LM) severely affects gastric cancer (GC) patients' prognosis. Small extracellular (show more...)Liver metastasis (LM) severely affects gastric cancer (GC) patients' prognosis. Small extracellular vesicles (sEVs) play key roles in intercellular communication. Specific sEV-miRNAs from several types of cancer were found to induce a premetastatic niche in target organs before tumor cell arrive. However, whether the primary GC affects hepatic microenvironment or the role of sEV-miRNAs in GC-LM is yet unclear. We report that GC-derived sEVs are primarily absorbed by Kupffer cells (KCs). sEV-miR-151a-3p is highly expressed in GC-LM patients' plasma and presents poor prognosis. Treating mice with sEVs-enriched in miR-151a-3p promotes GC-LM, whereas has no influence on the proliferation of GC cells in situ. Mechanistically, sEV-miR-151a-3p inhibits SP3 in KCs. Simultaneously, sEV-miR-151a-3p targets YTHDF3 to decrease the transcriptional inhibitory activity of SP3 by reducing SUMO1 translation in a N6-methyladenosine-dependent manner. These factors contribute to TGF-β1 transactivation in KCs, subsequently activating the SMAD2/3 pathway and enhancing the stem cell-like properties of incoming GC cells. Furthermore, sEV-miR-151a-3p induces miR-151a-3p transcription in KCs to form a positive feedback loop. In summary, our results reveal a previously unidentified regulatory axis initiated by sEV-miR-151a-3p that establishes a hepatic stemness-permissive niche to support GC-LM. sEV-miR-151a-3p could be a promising diagnostic biomarker and preventive treatment candidate for GC-LM. (hide) EV-METRIC 67% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin gastric cancer patients with liver metastasis Focus vesicles Other / small extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Commercial method Ultrafiltration Protein markers EV: Calnexin/ TSG101/ TSPAN8/ CD63 non-EV: Albumin Proteomics no Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 100 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 8 Wash: time (min) 70 Wash: Rotor Type Type 100 Ti Wash: speed (g) 110000 Ultra filtration Cut-off size (kDa) 100 Membrane type Polyethersulfone (PES) Commercial kit Total Exosome Isolation Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins TSPAN8/ CD63/ TSG101 Not detected EV-associated proteins Calnexin Not detected contaminants Albumin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNAsequencing Database No Proteinase treatment Yes Moment of Proteinase treatment Before Proteinase type Proteinase K Proteinase concentration 20 RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 125,5 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 2,00E+10 EM EM-type Transmission-EM Image type Close-up

	EV200186	1/1	Homo sapiens	primary amnion epithelial cells	(d)(U)C Filtration UF	Radnaa, Enkhtuya	2021	67%
Study summary Full title Extracellular vesicle mediated feto-maternal HMGB1 signaling induces preterm birth All authors Enkhtuya Radnaa, Lauren S Richardson, Samantha Sheller-Miller, Tuvshintugs Baljinnyam, Mariana de Castro Silva, Ananth Kumar Kammala, Rheanna Urrabaz-Garza, Talar Kechichian, Sungjin Kim, Arum Han, Ramkumar Menon Journal Lab Chip Abstract Preterm birth (PTB; <37 weeks of gestation) impacts ∼11% of all pregnancies and contributes to 1 m (show more...)Preterm birth (PTB; <37 weeks of gestation) impacts ∼11% of all pregnancies and contributes to 1 million neonatal deaths worldwide annually. An understanding of the feto-maternal (F-M) signals that initiate birthing (parturition) at term is critical to design strategies to prevent their premature activation, resulting in PTB. Although endocrine and immune cell signaling are well-reported, fetal-derived paracrine signals capable of transitioning quiescent uterus to an active state of labor are poorly studied. Recent reports have suggested that senescence of the fetal amnion membrane coinciding with fetal growth and maturation generates inflammatory signals capable of triggering parturition. This is by increasing the inflammatory load at the feto-maternal interface (FMi) tissues (i.e., amniochorion-decidua). High mobility group box 1 protein (HMGB1), an alarmin, is one of the inflammatory signals released by senescent amnion cells via extracellular vesicles (exosomes; 40-160 nm). Increased levels of HMGB1 in the amniotic fluid, cord and maternal blood are associated with term and PTB. This study tested the hypothesis that senescent amnion cells release HMGB1, which is fetal signaling capable of increasing FMi inflammation, predisposing them to parturition. To test this hypothesis, exosomes from amnion epithelial cells (AECs) grown under normal conditions were engineered to contain HMGB1 by electroporation (eHMGB1). eHMGB1 was characterized (quantity, size, shape, markers and loading efficiency), and its propagation through FMi was tested using a four-chamber microfluidic organ-on-a-chip device (FMi-OOC) that contained four distinct cell types (amnion and chorion mesenchymal, chorion trophoblast and decidual cells) connected through microchannels. eHMGB1 propagated through the fetal cells and matrix to the maternal decidua and increased inflammation (receptor expression [RAGE and TLR4] and cytokines). Furthermore, intra-amniotic injection of eHMGB1 (containing 10 ng) into pregnant CD-1 mice on embryonic day 17 led to PTB. Injecting carboxyfluorescein succinimidyl ester (CFSE)-labeled eHMGB1, we determined in vivo kinetics and report that eHMGB1 trafficking resulting in PTB was associated with increased FMi inflammation. This study determined that fetal exosome mediated paracrine signaling can generate inflammation and induce parturition. Besides, in vivo functional validation of FMi-OOC experiments strengthens the reliability of such devices to test physiologic and pathologic systems. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Ultrafiltration Protein markers EV: CD63/ CD81/ HMGB1/ CD9 non-EV: None Proteomics no Show all info Study aim Function/Biomarker/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary amnion epithelial cells EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 960 Pelleting: rotor type Type 70.1Ti Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 5 Wash: time (min) 60 Wash: Rotor Type Type 70.1Ti Wash: speed (g) 100,000 Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100,000 Membrane type Polypropylene;Other Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81 ELISA Antibody details provided? No Detected EV-associated proteins HMGB1 Detected EV-associated proteins CD9/ CD63/ HMGB1/ CD81 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type mode;Other Reported size (nm) 127 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size 70 EV-concentration No

	EV200181	3/3	Homo sapiens	Bronchoalveolar lavage fluid	(d)(U)C	Dlugolecka, Magdalena	2021	67%
Study summary Full title Characterization of Extracellular Vesicles from Bronchoalveolar Lavage Fluid and Plasma of Patients with Lung Lesions Using Fluorescence Nanoparticle Tracking Analysis All authors Magdalena Dlugolecka, Jacek Szymanski, Lukasz Zareba, Zuzanna Homoncik, Joanna Domagala-Kulawik, Malgorzata Polubiec-Kownacka, Malgorzata Czystowska-Kuzmicz Journal Cells Abstract The current lack of reliable methods for quantifying extracellular vesicles (EVs) isolated from comp (show more...)The current lack of reliable methods for quantifying extracellular vesicles (EVs) isolated from complex biofluids significantly hinders translational applications in EV research. The recently developed fluorescence nanoparticle tracking analysis (FL-NTA) allows for the detection of EV-associated proteins, enabling EV content determination. In this study, we present the first comprehensive phenotyping of bronchopulmonary lavage fluid (BALF)-derived EVs from non-small cell lung cancer (NSCLC) patients using classical EV-characterization methods as well as the FL-NTA method. We found that EV immunolabeling for the specific EV marker combined with the use of the fluorescent mode NTA analysis can provide the concentration, size, distribution, and surface phenotype of EVs in a heterogeneous solution. However, by performing FL-NTA analysis of BALF-derived EVs in comparison to plasma-derived EVs, we reveal the limitations of this method, which is suitable only for relatively pure EV isolates. For more complex fluids such as plasma, this method appears to not be sensitive enough and the measurements can be compromised. Our parallel presentation of NTA-based phenotyping of plasma and BALF EVs emphasizes the great impact of sample composition and purity on FL-NTA analysis that has to be taken into account in the further development of FL-NTA toward the detection of EV-associated cancer biomarkers. (hide) EV-METRIC 67% (78th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Bronchoalveolar lavage fluid Sample origin lung cancer Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: TSG101/ CD63/ CD81/ PD-L1/ Syntenin/ CD9 non-EV: Calnexin Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Bronchoalveolar lavage fluid Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 2h Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 8 Wash: time (min) 60 Wash: Rotor Type Type 70.1Ti Wash: speed (g) 110000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ TSG101/ Syntenin/ PD-L1/ CD81 Not detected contaminants Calnexin Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ CD81 Fluorescent NTA Relevant measurements variables specified? NA Antibody details provided? No Detected EV-associated proteins CD81/ CD9/ CD63 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 172 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 8.85E+08 Particle analysis: flow cytometry Flow cytometer type BD FACSVerse 8 color Flow Cytometer (BD) Hardware adjustment Calibration bead size 4.5 Report type Not Reported EM EM-type Cryo-EM Image type Close-up

	EV200159	1/4	Homo sapiens	Expi293F	DG (d)(U)C	Lázaro-Ibáñez, Elisa	2021	67%
Study summary Full title Selection of Fluorescent, Bioluminescent, and Radioactive Tracers to Accurately Reflect Extracellular Vesicle Biodistribution in Vivo All authors Elisa Lázaro-Ibáñez, Farid N Faruqu, Amer F Saleh, Andreia M Silva, Julie Tzu-Wen Wang, Janusz Rak, Khuloud T Al-Jamal, Niek Dekker Journal ACS Nano Abstract The ability to track extracellular vesicles (EVs) in vivo without influencing their biodistribution (show more...)The ability to track extracellular vesicles (EVs) in vivo without influencing their biodistribution is a key requirement for their successful development as drug delivery vehicles and therapeutic agents. Here, we evaluated the effect of five different optical and nuclear tracers on the in vivo biodistribution of EVs. Expi293F EVs were labeled using either a noncovalent fluorescent dye DiR, or covalent modification with 111indium-DTPA, or bioengineered with fluorescent (mCherry) or bioluminescent (Firefly and NanoLuc luciferase) proteins fused to the EV marker, CD63. To focus specifically on the effect of the tracer, we compared EVs derived from the same cell source and administered systemically by the same route and at equal dose into tumor-bearing BALB/c mice. 111Indium and DiR were the most sensitive tracers for in vivo imaging of EVs, providing the most accurate quantification of vesicle biodistribution by ex vivo imaging of organs and analysis of tissue lysates. Specifically, NanoLuc fused to CD63 altered EV distribution, resulting in high accumulation in the lungs, demonstrating that genetic modification of EVs for tracking purposes may compromise their physiological biodistribution. Blood kinetic analysis revealed that EVs are rapidly cleared from the circulation with a half-life below 10 min. Our study demonstrates that radioactivity is the most accurate EV tracking approach for a complete quantitative biodistribution study including pharmacokinetic profiling. In conclusion, we provide a comprehensive comparison of fluorescent, bioluminescent, and radioactivity approaches, including dual labeling of EVs, to enable accurate spatiotemporal resolution of EV trafficking in mice, an essential step in developing EV therapeutics. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Density gradient (Differential) (ultra)centrifugation Protein markers EV: Alix/ CD63/ Flotillin1/ CD9/ CD81 non-EV: Lamin B1 Proteomics no EV density (g/ml) 1.10 - 1.13 Show all info Study aim Technical analysis comparing/optimizing EV-related methods/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Expi293F EV-harvesting Medium Serum free medium Cell viability (%) 85 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 9 Lowest density fraction 10% Highest density fraction 50% Total gradient volume, incl. sample (mL) 17 Sample volume (mL) 1 Orientation Bottom-up Rotor type SW 32.1 Ti Speed (g) 120000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 94 Pelleting: duration (min) 180 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 120000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins Flotillin1/ CD9/ CD63/ Alix/ CD81 Not detected contaminants Lamin B1 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 126-154 EV concentration Yes Particle yield No NA EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 56

	EV200157	7/10	Homo sapiens	MDA-MB-468	(d)(U)C SEC (non-commercial) Polymer-based precipitation	Martínez-Greene, Juan A	2021	67%
Study summary Full title Quantitative proteomic analysis of extracellular vesicle subgroups isolated by an optimized method combining polymer-based precipitation and size exclusion chromatography All authors Juan A Martínez-Greene, Karina Hernández-Ortega, Ricardo Quiroz-Baez, Osbaldo Resendis-Antonio, Israel Pichardo-Casas, David A Sinclair, Bogdan Budnik, Alfredo Hidalgo-Miranda, Eileen Uribe-Querol, María Del Pilar Ramos-Godínez, Eduardo Martínez-Martínez Journal J Extracell Vesicles Abstract The molecular characterization of extracellular vesicles (EVs) has revealed a great heterogeneity in (show more...)The molecular characterization of extracellular vesicles (EVs) has revealed a great heterogeneity in their composition at a cellular and tissue level. Current isolation methods fail to efficiently separate EV subtypes for proteomic and functional analysis. The aim of this study was to develop a reproducible and scalable isolation workflow to increase the yield and purity of EV preparations. Through a combination of polymer-based precipitation and size exclusion chromatography (Pre-SEC), we analyzed two subsets of EVs based on their CD9, CD63 and CD81 content and elution time. EVs were characterized using transmission electron microscopy, nanoparticle tracking analysis, and Western blot assays. To evaluate differences in protein composition between the early- and late-eluting EV fractions, we performed a quantitative proteomic analysis of MDA-MB-468-derived EVs. We identified 286 exclusive proteins in early-eluting fractions and 148 proteins with a differential concentration between early- and late-eluting fractions. A density gradient analysis further revealed EV heterogeneity within each analyzed subgroup. Through a systems biology approach, we found significant interactions among proteins contained in the EVs which suggest the existence of functional clusters related to specific biological processes. The workflow presented here allows the study of EV subtypes within a single cell type and contributes to standardizing the EV isolation for functional studies. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Size-exclusion chromatography (non-commercial) Polymer-based precipitation Protein markers EV: CD9/ CD63/ CD81/ Alix/ TSG101/ ANXA2/ ANXA5 non-EV: Albumin Proteomics no Show all info Study aim New methodological development/Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDA-MB-468 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 95 Cell count 1.50E+06 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 39 Pelleting: rotor type TLA-100.3 Pelleting: speed (g) 118000 Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 1 Resin type Sepharose CL-2B Other Name other separation method Polymer-based precipitation EV-subtype Distinction between multiple subtypes SEC fraction Used subtypes F5-10 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ CD81/ Alix/ TSG101/ ANXA2/ ANXA5 Detected contaminants Albumin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 138 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 4.91E+11 EM EM-type Transmission-EM Image type Wide-field

	EV200157	8/10	Homo sapiens	MDA-MB-468	(d)(U)C SEC (non-commercial) Polymer-based precipitation DG	Martínez-Greene, Juan A	2021	67%
Study summary Full title Quantitative proteomic analysis of extracellular vesicle subgroups isolated by an optimized method combining polymer-based precipitation and size exclusion chromatography All authors Juan A Martínez-Greene, Karina Hernández-Ortega, Ricardo Quiroz-Baez, Osbaldo Resendis-Antonio, Israel Pichardo-Casas, David A Sinclair, Bogdan Budnik, Alfredo Hidalgo-Miranda, Eileen Uribe-Querol, María Del Pilar Ramos-Godínez, Eduardo Martínez-Martínez Journal J Extracell Vesicles Abstract The molecular characterization of extracellular vesicles (EVs) has revealed a great heterogeneity in (show more...)The molecular characterization of extracellular vesicles (EVs) has revealed a great heterogeneity in their composition at a cellular and tissue level. Current isolation methods fail to efficiently separate EV subtypes for proteomic and functional analysis. The aim of this study was to develop a reproducible and scalable isolation workflow to increase the yield and purity of EV preparations. Through a combination of polymer-based precipitation and size exclusion chromatography (Pre-SEC), we analyzed two subsets of EVs based on their CD9, CD63 and CD81 content and elution time. EVs were characterized using transmission electron microscopy, nanoparticle tracking analysis, and Western blot assays. To evaluate differences in protein composition between the early- and late-eluting EV fractions, we performed a quantitative proteomic analysis of MDA-MB-468-derived EVs. We identified 286 exclusive proteins in early-eluting fractions and 148 proteins with a differential concentration between early- and late-eluting fractions. A density gradient analysis further revealed EV heterogeneity within each analyzed subgroup. Through a systems biology approach, we found significant interactions among proteins contained in the EVs which suggest the existence of functional clusters related to specific biological processes. The workflow presented here allows the study of EV subtypes within a single cell type and contributes to standardizing the EV isolation for functional studies. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Size-exclusion chromatography (non-commercial) Polymer-based precipitation Density gradient Protein markers EV: CD9/ CD63/ CD81/ Alix/ TSG101/ ANXA2/ ANXA5 non-EV: Albumin Proteomics no Show all info Study aim New methodological development/Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDA-MB-468 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 95 Cell count 1.50E+06 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 39 Pelleting: rotor type TLA-100.3 Pelleting: speed (g) 118000 Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 1 Resin type Sepharose CL-2B Other Name other separation method Polymer-based precipitation EV-subtype Distinction between multiple subtypes SEC fraction Used subtypes F11-16 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ CD81 Not detected EV-associated proteins Alix/ TSG101/ ANXA2/ ANXA5 Detected contaminants Albumin Characterization: Particle analysis NTA Report type Mean Reported size (nm) 129 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 5.19E+10 EM EM-type Transmission-EM Image type Wide-field

	EV200157	9/10	Homo sapiens	gingival primary fibroblasts	(d)(U)C SEC (non-commercial) Polymer-based precipitation	Martínez-Greene, Juan A	2021	67%
Study summary Full title Quantitative proteomic analysis of extracellular vesicle subgroups isolated by an optimized method combining polymer-based precipitation and size exclusion chromatography All authors Juan A Martínez-Greene, Karina Hernández-Ortega, Ricardo Quiroz-Baez, Osbaldo Resendis-Antonio, Israel Pichardo-Casas, David A Sinclair, Bogdan Budnik, Alfredo Hidalgo-Miranda, Eileen Uribe-Querol, María Del Pilar Ramos-Godínez, Eduardo Martínez-Martínez Journal J Extracell Vesicles Abstract The molecular characterization of extracellular vesicles (EVs) has revealed a great heterogeneity in (show more...)The molecular characterization of extracellular vesicles (EVs) has revealed a great heterogeneity in their composition at a cellular and tissue level. Current isolation methods fail to efficiently separate EV subtypes for proteomic and functional analysis. The aim of this study was to develop a reproducible and scalable isolation workflow to increase the yield and purity of EV preparations. Through a combination of polymer-based precipitation and size exclusion chromatography (Pre-SEC), we analyzed two subsets of EVs based on their CD9, CD63 and CD81 content and elution time. EVs were characterized using transmission electron microscopy, nanoparticle tracking analysis, and Western blot assays. To evaluate differences in protein composition between the early- and late-eluting EV fractions, we performed a quantitative proteomic analysis of MDA-MB-468-derived EVs. We identified 286 exclusive proteins in early-eluting fractions and 148 proteins with a differential concentration between early- and late-eluting fractions. A density gradient analysis further revealed EV heterogeneity within each analyzed subgroup. Through a systems biology approach, we found significant interactions among proteins contained in the EVs which suggest the existence of functional clusters related to specific biological processes. The workflow presented here allows the study of EV subtypes within a single cell type and contributes to standardizing the EV isolation for functional studies. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Size-exclusion chromatography (non-commercial) Polymer-based precipitation Protein markers EV: CD9/ CD63/ CD81/ Alix/ TSG101/ ANXA2/ ANXA5 non-EV: Albumin Proteomics no Show all info Study aim New methodological development/Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells gingival primary fibroblasts EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 95 Cell count 1.50E+06 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 39 Pelleting: rotor type TLA-100.3 Pelleting: speed (g) 118000 Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 1 Resin type Sepharose CL-2B Other Name other separation method Polymer-based precipitation EV-subtype Distinction between multiple subtypes SEC fraction Used subtypes F5-10 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ CD81/ ANXA2/ ANXA5 Not detected EV-associated proteins Alix/ TSG101 Detected contaminants Albumin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 149 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 2.89E+11 EM EM-type Transmission-EM Image type Wide-field

	EV200157	10/10	Homo sapiens	gingival primary fibroblasts	(d)(U)C SEC (non-commercial) Polymer-based precipitation DG	Martínez-Greene, Juan A	2021	67%
Study summary Full title Quantitative proteomic analysis of extracellular vesicle subgroups isolated by an optimized method combining polymer-based precipitation and size exclusion chromatography All authors Juan A Martínez-Greene, Karina Hernández-Ortega, Ricardo Quiroz-Baez, Osbaldo Resendis-Antonio, Israel Pichardo-Casas, David A Sinclair, Bogdan Budnik, Alfredo Hidalgo-Miranda, Eileen Uribe-Querol, María Del Pilar Ramos-Godínez, Eduardo Martínez-Martínez Journal J Extracell Vesicles Abstract The molecular characterization of extracellular vesicles (EVs) has revealed a great heterogeneity in (show more...)The molecular characterization of extracellular vesicles (EVs) has revealed a great heterogeneity in their composition at a cellular and tissue level. Current isolation methods fail to efficiently separate EV subtypes for proteomic and functional analysis. The aim of this study was to develop a reproducible and scalable isolation workflow to increase the yield and purity of EV preparations. Through a combination of polymer-based precipitation and size exclusion chromatography (Pre-SEC), we analyzed two subsets of EVs based on their CD9, CD63 and CD81 content and elution time. EVs were characterized using transmission electron microscopy, nanoparticle tracking analysis, and Western blot assays. To evaluate differences in protein composition between the early- and late-eluting EV fractions, we performed a quantitative proteomic analysis of MDA-MB-468-derived EVs. We identified 286 exclusive proteins in early-eluting fractions and 148 proteins with a differential concentration between early- and late-eluting fractions. A density gradient analysis further revealed EV heterogeneity within each analyzed subgroup. Through a systems biology approach, we found significant interactions among proteins contained in the EVs which suggest the existence of functional clusters related to specific biological processes. The workflow presented here allows the study of EV subtypes within a single cell type and contributes to standardizing the EV isolation for functional studies. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Size-exclusion chromatography (non-commercial) Polymer-based precipitation Density gradient Protein markers EV: CD9/ CD63/ CD81/ Alix/ TSG101/ ANXA2/ ANXA5 non-EV: Albumin Proteomics no Show all info Study aim New methodological development/Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells gingival primary fibroblasts EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 95 Cell count 1.50E+06 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 39 Pelleting: rotor type TLA-100.3 Pelleting: speed (g) 118000 Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 1 Resin type Sepharose CL-2B Other Name other separation method Polymer-based precipitation EV-subtype Distinction between multiple subtypes SEC fraction Used subtypes F11-16 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ CD81 Not detected EV-associated proteins Alix/ TSG101/ ANXA2/ ANXA5 Detected contaminants Albumin Characterization: Particle analysis NTA Report type Mean Reported size (nm) 158.5 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 4.68E+10 EM EM-type Transmission-EM Image type Wide-field

	EV200121	1/6	Homo sapiens	HT29 colon carcinoma	(d)(U)C DG	Keulers, Tom	2021	67%
Study summary Full title Secretion of pro-angiogenic extracellular vesicles during hypoxia is dependent on the autophagy-related protein GABARAPL1 All authors Tom G Keulers, Sten F Libregts, Joel E J Beaumont, Kim G Savelkouls, Johan Bussink, Hans Duimel, Ludwig Dubois, Marijke I Zonneveld, Carmen López-Iglesias, Karel Bezstarosti, Jeroen A Demmers, Marc Vooijs, Marca Wauben, Kasper M A Rouschop Journal J Extracell Vesicles Abstract Tumour hypoxia is a hallmark of solid tumours and contributes to tumour progression, metastasis deve (show more...)Tumour hypoxia is a hallmark of solid tumours and contributes to tumour progression, metastasis development and therapy resistance. In response to hypoxia, tumour cells secrete pro-angiogenic factors to induce blood vessel formation and restore oxygen supply to hypoxic regions. Extracellular vesicles (EVs) are emerging as mediators of intercellular communication in the tumour microenvironment. Here we demonstrate that increased expression of the LC3/GABARAP protein family member GABARAPL1, is required for endosomal maturation, sorting of cargo to endosomes and the secretion of EVs. Silencing GABARAPL1 results in a block in the early endosomal pathway and impaired secretion of EVs with pro-angiogenic properties. Tumour xenografts of doxycycline inducible GABARAPL1 knockdown cells display impaired vascularisation that results in decreased tumour growth, elevated tumour necrosis and increased therapy efficacy. Moreover, our data show that GABARAPL1 is expressed on the EV surface and targeting GABARAPL1+ EVs with GABARAPL1 targeting antibodies results in blockade of pro-angiogenic effects in vitro. In summary, we reveal that GABARAPL1 is required for EV cargo loading and secretion. GABARAPL1+ EVs are detectable and targetable and are therefore interesting to pursue as a therapeutic target. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Protein markers EV: CD81/ CD63/ CD9 non-EV: None Proteomics no EV density (g/ml) 1.14 Show all info Study aim Function/Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HT29 colon carcinoma EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell count 1.00E+06 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Continuous Lowest density fraction 0.4M Highest density fraction 2.0M Total gradient volume, incl. sample (mL) 12.4 Sample volume (mL) 0.15 Orientation Bottom-up Rotor type SW 41 Ti Speed (g) 200000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 12.4 Pelleting: duration (min) 60 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ CD81 Not detected EV-associated proteins CD81/ CD63/ CD9 Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 100 Particle analysis: flow cytometry Flow cytometer type influx Hardware adjustment customized influx Calibration bead size 100 Report type Not Reported EV concentration Yes

	EV200121	2/6	Homo sapiens	HT29 colon carcinoma	(d)(U)C DG	Keulers, Tom	2021	67%
Study summary Full title Secretion of pro-angiogenic extracellular vesicles during hypoxia is dependent on the autophagy-related protein GABARAPL1 All authors Tom G Keulers, Sten F Libregts, Joel E J Beaumont, Kim G Savelkouls, Johan Bussink, Hans Duimel, Ludwig Dubois, Marijke I Zonneveld, Carmen López-Iglesias, Karel Bezstarosti, Jeroen A Demmers, Marc Vooijs, Marca Wauben, Kasper M A Rouschop Journal J Extracell Vesicles Abstract Tumour hypoxia is a hallmark of solid tumours and contributes to tumour progression, metastasis deve (show more...)Tumour hypoxia is a hallmark of solid tumours and contributes to tumour progression, metastasis development and therapy resistance. In response to hypoxia, tumour cells secrete pro-angiogenic factors to induce blood vessel formation and restore oxygen supply to hypoxic regions. Extracellular vesicles (EVs) are emerging as mediators of intercellular communication in the tumour microenvironment. Here we demonstrate that increased expression of the LC3/GABARAP protein family member GABARAPL1, is required for endosomal maturation, sorting of cargo to endosomes and the secretion of EVs. Silencing GABARAPL1 results in a block in the early endosomal pathway and impaired secretion of EVs with pro-angiogenic properties. Tumour xenografts of doxycycline inducible GABARAPL1 knockdown cells display impaired vascularisation that results in decreased tumour growth, elevated tumour necrosis and increased therapy efficacy. Moreover, our data show that GABARAPL1 is expressed on the EV surface and targeting GABARAPL1+ EVs with GABARAPL1 targeting antibodies results in blockade of pro-angiogenic effects in vitro. In summary, we reveal that GABARAPL1 is required for EV cargo loading and secretion. GABARAPL1+ EVs are detectable and targetable and are therefore interesting to pursue as a therapeutic target. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin gabarapl1 KD hypoxia Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Protein markers EV: CD81/ CD63/ CD9 non-EV: None Proteomics no EV density (g/ml) 1.14 Show all info Study aim Function/Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HT29 colon carcinoma EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell count 1.00E+06 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Continuous Lowest density fraction 0.4M Highest density fraction 2.0M Total gradient volume, incl. sample (mL) 12.4 Sample volume (mL) 0.15 Orientation Bottom-up Rotor type SW 41 Ti Speed (g) 200000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 12.4 Pelleting: duration (min) 60 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ CD81 Not detected EV-associated proteins CD81/ CD63/ CD9 Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 200 Particle analysis: flow cytometry Flow cytometer type influx Hardware adjustment customized influx Calibration bead size 100 Report type Not Reported EV concentration Yes

	EV200121	4/6	Homo sapiens	U87 glioblastoma	(d)(U)C DG	Keulers, Tom	2021	67%
Study summary Full title Secretion of pro-angiogenic extracellular vesicles during hypoxia is dependent on the autophagy-related protein GABARAPL1 All authors Tom G Keulers, Sten F Libregts, Joel E J Beaumont, Kim G Savelkouls, Johan Bussink, Hans Duimel, Ludwig Dubois, Marijke I Zonneveld, Carmen López-Iglesias, Karel Bezstarosti, Jeroen A Demmers, Marc Vooijs, Marca Wauben, Kasper M A Rouschop Journal J Extracell Vesicles Abstract Tumour hypoxia is a hallmark of solid tumours and contributes to tumour progression, metastasis deve (show more...)Tumour hypoxia is a hallmark of solid tumours and contributes to tumour progression, metastasis development and therapy resistance. In response to hypoxia, tumour cells secrete pro-angiogenic factors to induce blood vessel formation and restore oxygen supply to hypoxic regions. Extracellular vesicles (EVs) are emerging as mediators of intercellular communication in the tumour microenvironment. Here we demonstrate that increased expression of the LC3/GABARAP protein family member GABARAPL1, is required for endosomal maturation, sorting of cargo to endosomes and the secretion of EVs. Silencing GABARAPL1 results in a block in the early endosomal pathway and impaired secretion of EVs with pro-angiogenic properties. Tumour xenografts of doxycycline inducible GABARAPL1 knockdown cells display impaired vascularisation that results in decreased tumour growth, elevated tumour necrosis and increased therapy efficacy. Moreover, our data show that GABARAPL1 is expressed on the EV surface and targeting GABARAPL1+ EVs with GABARAPL1 targeting antibodies results in blockade of pro-angiogenic effects in vitro. In summary, we reveal that GABARAPL1 is required for EV cargo loading and secretion. GABARAPL1+ EVs are detectable and targetable and are therefore interesting to pursue as a therapeutic target. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin gabarapl1 KD hypoxia Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Protein markers EV: CD81/ CD63/ CD9 non-EV: None Proteomics no EV density (g/ml) 1.14 Show all info Study aim Function/Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells U87 glioblastoma EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell count 1.00E+07 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Continuous Lowest density fraction 0.4M Highest density fraction 2.0M Total gradient volume, incl. sample (mL) 12.4 Sample volume (mL) 0.15 Orientation Bottom-up Rotor type SW 41 Ti Speed (g) 200000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 12.4 Pelleting: duration (min) 60 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ CD81 Not detected EV-associated proteins CD81/ CD63/ CD9 Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type influx Hardware adjustment customized influx Calibration bead size 100 Report type Not Reported EV concentration Yes

	EV200103	1/2	Sus scrofa	seminal plasma	(d)(U)C Filtration	Ding, Yaqun	2021	67%
Study summary Full title MicroRNA-222 Transferred From Semen Extracellular Vesicles Inhibits Sperm Apoptosis by Targeting BCL2L11 All authors Yaqun Ding, Ning Ding, Yu Zhang, Shenmin Xie, Mengna Huang, Xiangdong Ding, Wuzi Dong, Qin Zhang, Li Jiang Journal Front Cell Dev Biol Abstract Seminal plasma contains a large number of extracellular vesicles (EVs). However, the roles of these (show more...)Seminal plasma contains a large number of extracellular vesicles (EVs). However, the roles of these EVs and their interactions with sperm are not clear. To identify the important molecules affecting sperm motility in EVs, we analyzed RNA from seminal plasma EVs of boars with different sperm motility using whole-transcriptome sequencing and proteomic analysis. In total, 7 miRNAs, 67 lncRNAs, 126 mRNAs and 76 proteins were differentially expressed between the two groups. We observed that EV-miR-222 can obviously improve sperm motility. In addition, the results suggested that miR-222 was transferred into sperm by the EVs and that miR-222 affected sperm apoptosis by inhibiting the expression of EGFR, BCL2L11, BAX, CYCs, CASP9 and CASP3. The results of electron microscopy also showed that overexpression of miR-222 in EVs could reduce sperm apoptosis. The study of the whole transcriptomes and proteomes of EVs in boar semen revealed some miRNAs may play an important role in these EVs interactions with Duroc sperm, and the findings suggest that the release of miR-222 by semen EVs is an important mechanism by which sperm viability is maintained and sperm apoptosis is reduced. Our studies provide a new insight of miR-222 in EVs regulation for sperm motility and sperm apoptosis. (hide) EV-METRIC 67% (50th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type seminal plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Protein markers EV: TSG101/ Alix/ CD63/ CD9 non-EV: Calnexin Proteomics no Show all info Study aim Function/Biogenesis/cargo sorting/Biomarker/Identification of content (omics approaches)/Mechanism of uptake/transfer Sample Species Sus scrofa Sample Type seminal plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 270 Pelleting: rotor type SW 28 Pelleting: speed (g) 120000 Wash: volume per pellet (ml) 35 Wash: time (min) 90 Wash: Rotor Type SW 28 Wash: speed (g) 120000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ TSG101/ Alix Not detected contaminants Calnexin Characterization: RNA analysis RNA analysis Type RNA sequencing Database Yes Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 108.7 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 660000000000 EM EM-type Transmission-EM Image type Wide-field

	EV200103	2/2	Sus scrofa	seminal plasma	(d)(U)C Filtration	Ding, Yaqun	2021	67%
Study summary Full title MicroRNA-222 Transferred From Semen Extracellular Vesicles Inhibits Sperm Apoptosis by Targeting BCL2L11 All authors Yaqun Ding, Ning Ding, Yu Zhang, Shenmin Xie, Mengna Huang, Xiangdong Ding, Wuzi Dong, Qin Zhang, Li Jiang Journal Front Cell Dev Biol Abstract Seminal plasma contains a large number of extracellular vesicles (EVs). However, the roles of these (show more...)Seminal plasma contains a large number of extracellular vesicles (EVs). However, the roles of these EVs and their interactions with sperm are not clear. To identify the important molecules affecting sperm motility in EVs, we analyzed RNA from seminal plasma EVs of boars with different sperm motility using whole-transcriptome sequencing and proteomic analysis. In total, 7 miRNAs, 67 lncRNAs, 126 mRNAs and 76 proteins were differentially expressed between the two groups. We observed that EV-miR-222 can obviously improve sperm motility. In addition, the results suggested that miR-222 was transferred into sperm by the EVs and that miR-222 affected sperm apoptosis by inhibiting the expression of EGFR, BCL2L11, BAX, CYCs, CASP9 and CASP3. The results of electron microscopy also showed that overexpression of miR-222 in EVs could reduce sperm apoptosis. The study of the whole transcriptomes and proteomes of EVs in boar semen revealed some miRNAs may play an important role in these EVs interactions with Duroc sperm, and the findings suggest that the release of miR-222 by semen EVs is an important mechanism by which sperm viability is maintained and sperm apoptosis is reduced. Our studies provide a new insight of miR-222 in EVs regulation for sperm motility and sperm apoptosis. (hide) EV-METRIC 67% (50th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type seminal plasma Sample origin low sperm motility Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Protein markers EV: TSG101/ Alix/ CD63/ CD9 non-EV: Calnexin Proteomics no Show all info Study aim Function/Biogenesis/cargo sorting/Biomarker/Identification of content (omics approaches)/Mechanism of uptake/transfer Sample Species Sus scrofa Sample Type seminal plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 270 Pelleting: rotor type SW 28 Pelleting: speed (g) 120000 Wash: volume per pellet (ml) 35 Wash: time (min) 90 Wash: Rotor Type SW 28 Wash: speed (g) 120000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ TSG101/ Alix Not detected contaminants Calnexin Characterization: RNA analysis RNA analysis Type RNAsequencing Database Yes Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 108.7 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 660000000000 EM EM-type Transmission-EM Image type Wide-field

	EV200076	1/5	microalgae	Dinoflagellate	(d)(U)C	Picciotto, Sabrina	2021	67%
Study summary Full title Isolation of extracellular vesicles from microalgae: towards the production of sustainable and natural nanocarriers of bioactive compounds All authors Sabrina Picciotto, Maria E. Barone, David Fierli, Anita Aranyos, Giorgia Adamo, Darja Božič, Daniele P. Romancino, Christopher Stanly, Rachel Parkes, Svenja Morsbach, Samuele Raccosta, Carolina Paganini, Antonella Cusimano, Vincenzo Martorana, Rosina Noto, Rita Carrotta, Fabio Librizzi, Umberto Capasso Palmiero, Pamela Santonicola, Ales Iglič, Meiyu Gai, Laura Corcuera, Annamaria Kisslinger, Elia Di Schiavi, Katharina Landfester, Giovanna L. Liguori, Veronika Kralj-Iglič, Paolo Arosio, Gabriella Pocsfalvi, Mauro Manno, Nicolas Touzet, Antonella Bongiovanni Journal Biomaterials science Abstract Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeuti (show more...)Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeutics, precision medicine and other biotechnology sectors. Novel bio-based nanotechnologies have recently arisen, which are based on the exploitation of extracellular vesicles (EVs). In this context, it has become essential to identify suitable organisms or cellular types to act as reliable sources of EVs and to develop their pilot- to large-scale production. The discovery of new biosources and the optimisation of related bioprocesses for the isolation and functionalisation of nano-delivery vehicles are fundamental to further develop therapeutic and biotechnological applications. Microalgae constitute sustainable sources of bioactive compounds with a range of sectorial applications including for example the formulation of health supplements, cosmetic products or food ingredients. In this study, we demonstrate that microalgae are promising producers of EVs. By analysing the nanosized extracellular nano-objects produced by eighteen microalgal species, we identified seven promising EV-producing strains belonging to distinct lineages, suggesting that the production of EVs in microalgae is an evolutionary conserved trait. Here we report the selection process and focus on one of this seven species, the glaucophyte Cyanophora paradoxa, which returned a protein yield in the small EV fraction of 1 μg of EV proteins per mg of dry weight of microalgal biomass (corresponding to 109 particles per mg of dried biomass) and EVs with a diameter of 130 nm (mode), as determined by the micro bicinchoninic acid assay, nanoparticle tracking and dynamic light scattering analyses. Moreover, the extracellular nanostructures isolated from the conditioned media of microalgae species returned positive immunoblot signals for some commonly used EV-biomarkers such as Alix, Enolase, HSP70, and β-actin. Overall, this work establishes a platform for the efficient production of EVs from a sustainable bioresource and highlights the potential of microalgal EVs as novel biogenic nanovehicles. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: Alix/ HSP70/ beta-actin/ enolase non-EV: None Proteomics no Show all info Study aim Function/Biomarker/novel EV type Sample Species microalgae Sample Type Cell culture supernatant EV-producing cells Dinoflagellate EV-harvesting Medium Serum free medium Cell viability (%) 90 Cell count 1 mg dry weight biomass/ml Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type SW 28 Pelleting: speed (g) 118000 Wash: volume per pellet (ml) 32 Wash: time (min) 70 Wash: Rotor Type SW 28 Wash: speed (g) 118000 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins beta-actin/ enolase/ HSP70/ Alix Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 92 NTA Report type Size range/distribution Reported size (nm) 125 EV concentration Yes Particle yield number of particles per mg dry weight microalgal mass 6.00E+09 EM EM-type Scanning-EM Image type Close-up, Wide-field

	EV200076	2/5	microalgae	Diatom	(d)(U)C	Picciotto, Sabrina	2021	67%
Study summary Full title Isolation of extracellular vesicles from microalgae: towards the production of sustainable and natural nanocarriers of bioactive compounds All authors Sabrina Picciotto, Maria E. Barone, David Fierli, Anita Aranyos, Giorgia Adamo, Darja Božič, Daniele P. Romancino, Christopher Stanly, Rachel Parkes, Svenja Morsbach, Samuele Raccosta, Carolina Paganini, Antonella Cusimano, Vincenzo Martorana, Rosina Noto, Rita Carrotta, Fabio Librizzi, Umberto Capasso Palmiero, Pamela Santonicola, Ales Iglič, Meiyu Gai, Laura Corcuera, Annamaria Kisslinger, Elia Di Schiavi, Katharina Landfester, Giovanna L. Liguori, Veronika Kralj-Iglič, Paolo Arosio, Gabriella Pocsfalvi, Mauro Manno, Nicolas Touzet, Antonella Bongiovanni Journal Biomaterials science Abstract Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeuti (show more...)Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeutics, precision medicine and other biotechnology sectors. Novel bio-based nanotechnologies have recently arisen, which are based on the exploitation of extracellular vesicles (EVs). In this context, it has become essential to identify suitable organisms or cellular types to act as reliable sources of EVs and to develop their pilot- to large-scale production. The discovery of new biosources and the optimisation of related bioprocesses for the isolation and functionalisation of nano-delivery vehicles are fundamental to further develop therapeutic and biotechnological applications. Microalgae constitute sustainable sources of bioactive compounds with a range of sectorial applications including for example the formulation of health supplements, cosmetic products or food ingredients. In this study, we demonstrate that microalgae are promising producers of EVs. By analysing the nanosized extracellular nano-objects produced by eighteen microalgal species, we identified seven promising EV-producing strains belonging to distinct lineages, suggesting that the production of EVs in microalgae is an evolutionary conserved trait. Here we report the selection process and focus on one of this seven species, the glaucophyte Cyanophora paradoxa, which returned a protein yield in the small EV fraction of 1 μg of EV proteins per mg of dry weight of microalgal biomass (corresponding to 109 particles per mg of dried biomass) and EVs with a diameter of 130 nm (mode), as determined by the micro bicinchoninic acid assay, nanoparticle tracking and dynamic light scattering analyses. Moreover, the extracellular nanostructures isolated from the conditioned media of microalgae species returned positive immunoblot signals for some commonly used EV-biomarkers such as Alix, Enolase, HSP70, and β-actin. Overall, this work establishes a platform for the efficient production of EVs from a sustainable bioresource and highlights the potential of microalgal EVs as novel biogenic nanovehicles. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: Alix/ HSP70/ enolase non-EV: None Proteomics no Show all info Study aim Function/Biomarker/novel EV type Sample Species microalgae Sample Type Cell culture supernatant EV-producing cells Diatom EV-harvesting Medium Serum free medium Cell viability (%) 90 Cell count 1 mg dry weight biomass/ml Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type SW 28 Pelleting: speed (g) 118000 Wash: volume per pellet (ml) 32 Wash: time (min) 70 Wash: Rotor Type SW 28 Wash: speed (g) 118000 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins enolase/ HSP70/ Alix Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 135 NTA Report type Size range/distribution Reported size (nm) 90 EV concentration Yes Particle yield number of particles per mg dry weight microalgal mass 2.40E+08 EM EM-type Scanning-EM Image type Close-up, Wide-field

	EV200076	3/5	microalgae	Glaucophyte	(d)(U)C	Picciotto, Sabrina	2021	67%
Study summary Full title Isolation of extracellular vesicles from microalgae: towards the production of sustainable and natural nanocarriers of bioactive compounds All authors Sabrina Picciotto, Maria E. Barone, David Fierli, Anita Aranyos, Giorgia Adamo, Darja Božič, Daniele P. Romancino, Christopher Stanly, Rachel Parkes, Svenja Morsbach, Samuele Raccosta, Carolina Paganini, Antonella Cusimano, Vincenzo Martorana, Rosina Noto, Rita Carrotta, Fabio Librizzi, Umberto Capasso Palmiero, Pamela Santonicola, Ales Iglič, Meiyu Gai, Laura Corcuera, Annamaria Kisslinger, Elia Di Schiavi, Katharina Landfester, Giovanna L. Liguori, Veronika Kralj-Iglič, Paolo Arosio, Gabriella Pocsfalvi, Mauro Manno, Nicolas Touzet, Antonella Bongiovanni Journal Biomaterials science Abstract Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeuti (show more...)Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeutics, precision medicine and other biotechnology sectors. Novel bio-based nanotechnologies have recently arisen, which are based on the exploitation of extracellular vesicles (EVs). In this context, it has become essential to identify suitable organisms or cellular types to act as reliable sources of EVs and to develop their pilot- to large-scale production. The discovery of new biosources and the optimisation of related bioprocesses for the isolation and functionalisation of nano-delivery vehicles are fundamental to further develop therapeutic and biotechnological applications. Microalgae constitute sustainable sources of bioactive compounds with a range of sectorial applications including for example the formulation of health supplements, cosmetic products or food ingredients. In this study, we demonstrate that microalgae are promising producers of EVs. By analysing the nanosized extracellular nano-objects produced by eighteen microalgal species, we identified seven promising EV-producing strains belonging to distinct lineages, suggesting that the production of EVs in microalgae is an evolutionary conserved trait. Here we report the selection process and focus on one of this seven species, the glaucophyte Cyanophora paradoxa, which returned a protein yield in the small EV fraction of 1 μg of EV proteins per mg of dry weight of microalgal biomass (corresponding to 109 particles per mg of dried biomass) and EVs with a diameter of 130 nm (mode), as determined by the micro bicinchoninic acid assay, nanoparticle tracking and dynamic light scattering analyses. Moreover, the extracellular nanostructures isolated from the conditioned media of microalgae species returned positive immunoblot signals for some commonly used EV-biomarkers such as Alix, Enolase, HSP70, and β-actin. Overall, this work establishes a platform for the efficient production of EVs from a sustainable bioresource and highlights the potential of microalgal EVs as novel biogenic nanovehicles. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: Alix/ HSP70/ beta-actin/ enolase non-EV: None Proteomics no Show all info Study aim Function/Biomarker/novel EV type Sample Species microalgae Sample Type Cell culture supernatant EV-producing cells Glaucophyte EV-harvesting Medium Serum free medium Cell viability (%) 90 Cell count 1 mg dry weight biomass/ml Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type SW 28 Pelleting: speed (g) 118000 Wash: volume per pellet (ml) 32 Wash: time (min) 70 Wash: Rotor Type SW 28 Wash: speed (g) 118000 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ HSP70/ beta-actin/ enolase Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 125 NTA Report type Size range/distribution Reported size (nm) 122 EV concentration Yes Particle yield number of particles per mg dry weight microalgal mass 2.00E+09 EM EM-type Scanning-EM Image type Close-up, Wide-field

	EV200076	4/5	microalgae	Haptophyte	(d)(U)C	Picciotto, Sabrina	2021	67%
Study summary Full title Isolation of extracellular vesicles from microalgae: towards the production of sustainable and natural nanocarriers of bioactive compounds All authors Sabrina Picciotto, Maria E. Barone, David Fierli, Anita Aranyos, Giorgia Adamo, Darja Božič, Daniele P. Romancino, Christopher Stanly, Rachel Parkes, Svenja Morsbach, Samuele Raccosta, Carolina Paganini, Antonella Cusimano, Vincenzo Martorana, Rosina Noto, Rita Carrotta, Fabio Librizzi, Umberto Capasso Palmiero, Pamela Santonicola, Ales Iglič, Meiyu Gai, Laura Corcuera, Annamaria Kisslinger, Elia Di Schiavi, Katharina Landfester, Giovanna L. Liguori, Veronika Kralj-Iglič, Paolo Arosio, Gabriella Pocsfalvi, Mauro Manno, Nicolas Touzet, Antonella Bongiovanni Journal Biomaterials science Abstract Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeuti (show more...)Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeutics, precision medicine and other biotechnology sectors. Novel bio-based nanotechnologies have recently arisen, which are based on the exploitation of extracellular vesicles (EVs). In this context, it has become essential to identify suitable organisms or cellular types to act as reliable sources of EVs and to develop their pilot- to large-scale production. The discovery of new biosources and the optimisation of related bioprocesses for the isolation and functionalisation of nano-delivery vehicles are fundamental to further develop therapeutic and biotechnological applications. Microalgae constitute sustainable sources of bioactive compounds with a range of sectorial applications including for example the formulation of health supplements, cosmetic products or food ingredients. In this study, we demonstrate that microalgae are promising producers of EVs. By analysing the nanosized extracellular nano-objects produced by eighteen microalgal species, we identified seven promising EV-producing strains belonging to distinct lineages, suggesting that the production of EVs in microalgae is an evolutionary conserved trait. Here we report the selection process and focus on one of this seven species, the glaucophyte Cyanophora paradoxa, which returned a protein yield in the small EV fraction of 1 μg of EV proteins per mg of dry weight of microalgal biomass (corresponding to 109 particles per mg of dried biomass) and EVs with a diameter of 130 nm (mode), as determined by the micro bicinchoninic acid assay, nanoparticle tracking and dynamic light scattering analyses. Moreover, the extracellular nanostructures isolated from the conditioned media of microalgae species returned positive immunoblot signals for some commonly used EV-biomarkers such as Alix, Enolase, HSP70, and β-actin. Overall, this work establishes a platform for the efficient production of EVs from a sustainable bioresource and highlights the potential of microalgal EVs as novel biogenic nanovehicles. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: Alix/ HSP70/ enolase non-EV: None Proteomics no Show all info Study aim Function/Biomarker/novel EV type Sample Species microalgae Sample Type Cell culture supernatant EV-producing cells Haptophyte EV-harvesting Medium Serum free medium Cell viability (%) 90 Cell count 1 mg dry weight biomass/ml Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type SW 28 Pelleting: speed (g) 118000 Wash: volume per pellet (ml) 32 Wash: time (min) 70 Wash: Rotor Type SW 28 Wash: speed (g) 118000 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ enolase Not detected EV-associated proteins HSP70 Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 87 NTA Report type Size range/distribution Reported size (nm) 165 EV concentration Yes Particle yield number of particles per mg dry weight microalgal mass 1.30E+08 EM EM-type Scanning-EM Image type Close-up, Wide-field

	EV200076	5/5	microalgae	Chlorophyte	(d)(U)C	Picciotto, Sabrina	2021	67%
Study summary Full title Isolation of extracellular vesicles from microalgae: towards the production of sustainable and natural nanocarriers of bioactive compounds All authors Sabrina Picciotto, Maria E. Barone, David Fierli, Anita Aranyos, Giorgia Adamo, Darja Božič, Daniele P. Romancino, Christopher Stanly, Rachel Parkes, Svenja Morsbach, Samuele Raccosta, Carolina Paganini, Antonella Cusimano, Vincenzo Martorana, Rosina Noto, Rita Carrotta, Fabio Librizzi, Umberto Capasso Palmiero, Pamela Santonicola, Ales Iglič, Meiyu Gai, Laura Corcuera, Annamaria Kisslinger, Elia Di Schiavi, Katharina Landfester, Giovanna L. Liguori, Veronika Kralj-Iglič, Paolo Arosio, Gabriella Pocsfalvi, Mauro Manno, Nicolas Touzet, Antonella Bongiovanni Journal Biomaterials science Abstract Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeuti (show more...)Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeutics, precision medicine and other biotechnology sectors. Novel bio-based nanotechnologies have recently arisen, which are based on the exploitation of extracellular vesicles (EVs). In this context, it has become essential to identify suitable organisms or cellular types to act as reliable sources of EVs and to develop their pilot- to large-scale production. The discovery of new biosources and the optimisation of related bioprocesses for the isolation and functionalisation of nano-delivery vehicles are fundamental to further develop therapeutic and biotechnological applications. Microalgae constitute sustainable sources of bioactive compounds with a range of sectorial applications including for example the formulation of health supplements, cosmetic products or food ingredients. In this study, we demonstrate that microalgae are promising producers of EVs. By analysing the nanosized extracellular nano-objects produced by eighteen microalgal species, we identified seven promising EV-producing strains belonging to distinct lineages, suggesting that the production of EVs in microalgae is an evolutionary conserved trait. Here we report the selection process and focus on one of this seven species, the glaucophyte Cyanophora paradoxa, which returned a protein yield in the small EV fraction of 1 μg of EV proteins per mg of dry weight of microalgal biomass (corresponding to 109 particles per mg of dried biomass) and EVs with a diameter of 130 nm (mode), as determined by the micro bicinchoninic acid assay, nanoparticle tracking and dynamic light scattering analyses. Moreover, the extracellular nanostructures isolated from the conditioned media of microalgae species returned positive immunoblot signals for some commonly used EV-biomarkers such as Alix, Enolase, HSP70, and β-actin. Overall, this work establishes a platform for the efficient production of EVs from a sustainable bioresource and highlights the potential of microalgal EVs as novel biogenic nanovehicles. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: Alix/ beta-actin/ enolase non-EV: None Proteomics no Show all info Study aim Function/Biomarker/novel EV type Sample Species microalgae Sample Type Cell culture supernatant EV-producing cells Chlorophyte EV-harvesting Medium Serum free medium Cell viability (%) 90 Cell count 1 mg dry weight biomass/ml Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type SW 28 Pelleting: speed (g) 118000 Wash: volume per pellet (ml) 32 Wash: time (min) 70 Wash: Rotor Type SW 28 Wash: speed (g) 118000 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins beta-actin/ enolase/ Alix Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 75 NTA Report type Size range/distribution Reported size (nm) 137 EV concentration Yes Particle yield number of particles per mg dry weight microalgal mass 2.60E+08 EM EM-type Scanning-EM Image type Close-up, Wide-field

	EV200075	1/2	Tetraselmis chuii	Tetraselmis chuii	(d)(U)C	Adamo, Giorgia	2021	67%
Study summary Full title Nanoalgosomes: Introducing extracellular vesicles produced by microalgae All authors Giorgia Adamo, David Fierli, Daniele P Romancino, Sabrina Picciotto, Maria E Barone, Anita Aranyos, Darja Božič, Svenja Morsbach, Samuele Raccosta, Christopher Stanly, Carolina Paganini, Meiyu Gai, Antonella Cusimano, Vincenzo Martorana, Rosina Noto, Rita Carrotta, Fabio Librizzi, Loredana Randazzo, Rachel Parkes, Umberto Capasso Palmiero, Estella Rao, Angela Paterna, Pamela Santonicola, Ales Iglič, Laura Corcuera, Annamaria Kisslinger, Elia Di Schiavi, Giovanna L Liguori 10 , Katharina Landfester, Veronika Kralj-Iglič, Paolo Arosio, Gabriella Pocsfalvi, Nicolas Touzet, Mauro Manno, Antonella Bongiovanni Journal J Extracell Vesicles Abstract Cellular, inter-organismal and cross kingdom communication via extracellular vesicles (EVs) is inten (show more...)Cellular, inter-organismal and cross kingdom communication via extracellular vesicles (EVs) is intensively studied in basic science with high expectation for a large variety of bio-technological applications. EVs intrinsically possess many attributes of a drug delivery vehicle. Beyond the implications for basic cell biology, academic and industrial interests in EVs have increased in the last few years. Microalgae constitute sustainable and renewable sources of bioactive compounds with a range of sectoral applications, including the formulation of health supplements, cosmetic products and food ingredients. Here we describe a newly discovered subtype of EVs derived from microalgae, which we named nanoalgosomes. We isolated these extracellular nano-objects from cultures of microalgal strains, including the marine photosynthetic chlorophyte Tetraselmis chuii, using differential ultracentrifugation or tangential flow fractionation and focusing on the nanosized small EVs (sEVs). We explore different biochemical and physical properties and we show that nanoalgosomes are efficiently taken up by mammalian cell lines, confirming the cross kingdom communication potential of EVs. This is the first detailed description of such membranous nanovesicles from microalgae. With respect to EVs isolated from other organisms, nanoalgosomes present several advantages in that microalgae are a renewable and sustainable natural source, which could easily be scalable in terms of nanoalgosome production. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles Other / small Extracellular Vesicles - Algosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: Alix/ H/ ATPase/ enolase/ beta-actin/ HSP70 non-EV: None Proteomics no Show all info Study aim Function/Biomarker/novel EV type Sample Species Tetraselmis chuii Sample Type Cell culture supernatant EV-producing cells Tetraselmis chuii EV-harvesting Medium Serum free medium Cell viability (%) 90 Cell count mg dry weight microalgal biomass Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type SW 28 Pelleting: speed (g) 118000 Wash: volume per pellet (ml) 32 Wash: time (min) 70 Wash: Rotor Type SW 28 Wash: speed (g) 118000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins H/ ATPase/ enolase/ beta-actin/ HSP70/ Alix Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 90 NTA Report type Size range/distribution Reported size (nm) 130+/-20 EV concentration Yes Particle yield number of particles per mg dry weight microalgal mass 2.00E+09 EM EM-type Atomic force-EM/ Transmission-EM/ Scanning-EM/ Cryo-EM Image type Close-up, Wide-field

	EV200039	1/4	Homo sapiens	primary B cells	(d)(U)C Filtration DG	Albanese, Manuel	2021	67%
Study summary Full title MicroRNAs are minor constituents of extracellular vesicles that are rarely delivered to target cells All authors Manuel Albanese, Yen-Fu Adam Chen, Corinna Hu, Kathrin Gärtner, Takanobu Tagawa, Ernesto Mejias-Perez, Oliver T. Keppler, Christine Göbel, Reinhard Zeidler, Mikhail Shein, Anne K. Schütz, Wolfgang Hammerschmidt Journal PLoS Genetics Abstract Mammalian cells release different types of vesicles, collectively termed extracellular vesicles (EVs (show more...)Mammalian cells release different types of vesicles, collectively termed extracellular vesicles (EVs). EVs contain cellular microRNAs (miRNAs) with an apparent potential to deliver their miRNA cargo to recipient cells to affect the stability of individual mRNAs and the cells' transcriptome. The extent to which miRNAs are exported via the EV route and whether they contribute to cell-cell communication are controversial. To address these issues, we defined multiple properties of EVs and analyzed their capacity to deliver packaged miRNAs into target cells to exert biological functions. We applied well-defined approaches to produce and characterize purified EVs with or without specific viral miRNAs. We found that only a small fraction of EVs carried miRNAs. EVs readily bound to different target cell types, but EVs did not fuse detectably with cellular membranes to deliver their cargo. We engineered EVs to be fusogenic and document their capacity to deliver functional messenger RNAs. Engineered fusogenic EVs, however, did not detectably alter the functionality of cells exposed to miRNA-carrying EVs. These results suggest that EV-borne miRNAs do not act as effectors of cell-to-cell communication. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin EBV-infected LCLs Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration DG Protein markers EV: TSG101/ LMP1 non-EV: Calnexin Proteomics no EV density (g/ml) 1.05 Show all info Study aim Identification of content (omics approaches)/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary B cells EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 95 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 0% Highest density fraction 42% Total gradient volume, incl. sample (mL) 4 Sample volume (mL) 0.38 Orientation Bottom-up Rotor type SW 60 Ti Speed (g) 160000 Duration (min) 1080 Fraction volume (mL) 0.4 Fraction processing None Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins LMP1/ TSG101 Not detected contaminants Calnexin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes

	EV200047	2/2	Homo sapiens	MCF7	UF DG	Yildizhan, Yagmur	2021	67%
Study summary Full title FO‐SPR biosensor calibrated with recombinant extracellular vesicles enables specific and sensitive detection directly in complex matrices All authors Yagmur Yildizhan, Venkata Suresh Vajrala, Edward Geeurickx, Charles Declerck, Nevena Duskunovic, Delphine De Sutter, Sam Noppen, Filip Delport, Dominique Schols, Johannes V. Swinnen, Sven Eyckerman, An Hendrix, Jeroen Lammertyn, Dragana Spasic Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) have drawn huge attention for diagnosing myriad of diseases, including (show more...)Extracellular vesicles (EVs) have drawn huge attention for diagnosing myriad of diseases, including cancer. However, the EV detection and analyses procedures often lack much desired sample standardization. To address this, we used well‐characterized recombinant EVs (rEVs) for the first time as a biological reference material in developing a fiber optic surface plasmon resonance (FO‐SPR) bioassay. In this context, EV binding on the FO‐SPR probes was achieved only with EV‐specific antibodies (e.g. anti‐CD9 and anti‐CD63) but not with non‐specific anti‐IgG. To increase detection sensitivity, we tested six different combinations of EV‐specific antibodies in a sandwich bioassay. Calibration curves were generated with two most effective combinations (anti‐CD9/Banti‐CD81 and anti‐CD63/Banti‐CD9), resulting in 103 and 104 times higher sensitivity than the EV concentration in human blood plasma from healthy or cancer patients, respectively. Additionally, by using anti‐CD63/Banti‐CD9, we detected rEVs spiked in cell culture medium and HEK293 endogenous EVs in the same matrix without any prior EV purification or enrichment. Lastly, we selectively captured breast cancer cell EVs spiked in blood plasma using anti‐EpCAM antibody on the FO‐SPR surface. The obtained results combined with FO‐SPR real‐time monitoring, fast response time and ease of operation, demonstrate its outstanding potential for EV quantification and analysis. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Rab27B-GFP Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Ultrafiltration Density gradient Protein markers EV: None non-EV: None Proteomics no EV density (g/ml) 1.086-1.119 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MCF7 EV-harvesting Medium Serum free medium Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 11 Lowest density fraction 6 Highest density fraction 18 Total gradient volume, incl. sample (mL) 15 Sample volume (mL) 1 Orientation Top-down Rotor type SW 32.1 Ti Speed (g) 186700 Duration (min) 116 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: duration (min) 180 Pelleting: rotor type SW 32.1 Ti Pelleting: speed (g) 100000 Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Not determined Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV200004	2/10	Homo sapiens	MEC 1	DC (d)(U)C	Elgamal, Sara	2021	67%
Study summary Full title Optimizing extracellular vesicles’ isolation from chronic lymphocytic leukemia patient plasma and cell line supernatant All authors Sara Elgamal, Emanuele Cocucci, Ellen J Sass, Xiaokui M Mo, Angela R Blissett, Edward P Calomeni, Kerry A Rogers, Jennifer A Woyach, Seema A Bhat, Natarajan Muthusamy, Amy J Johnson, Karilyn T Larkin, John C Byrd Journal JCI insight Abstract In chronic lymphocytic leukemia (CLL) and very likely all cancer types, extracellular vesicles (EVs) (show more...)In chronic lymphocytic leukemia (CLL) and very likely all cancer types, extracellular vesicles (EVs) are a common mechanism by which intercellular messages are communicated between normal, diseased, and transformed cells. Studies of EVs in CLL and other cancers have great variability and often lack reproducibility. For CLL patient plasma and cell lines, we sought to characterize current approaches used in isolating EV products and understand whether cell culture–conditioned media or complex biological fluids confound results. Utilizing nanoparticle tracking analysis, protein quantification, and electron microscopy, we show that ultracentrifugation with an OptiPrep cushion can effectively minimize contaminants from starting materials including plasma and conditioned media of CLL cell lines grown in EV-depleted complete RPMI media but not grown in the serum-free media AIM V commonly used in CLL experimental work. Moreover, we confirm the benefit of including 25 mM trehalose in PBS during EV isolation steps to reduce EV aggregation, to preserve function for downstream applications and characterization. Furthermore, we report the highest particles/μg EVs were obtained from our CLL cell lines utilizing the CELLine bioreactor flask. Finally, we optimized a proliferation assay that offers a functional evaluation of our EVs with minimal sample requirements. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin RPMI Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DC (d)(U)C Protein markers EV: TSG101/ CD81/ CD63/ GAPDH non-EV: Calnexin/ Albumin Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MEC 1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 88 Cell count 2.60E+08 +/- 7.59E+07 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 22 Wash: time (min) 70 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Density cushion Density medium Iodixanol Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ GAPDH/ TSG101/ CD81 Not detected contaminants Calnexin/ Albumin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution EV concentration Yes Particle yield Particles/ug;Yes, other: 1.11E+09 +/- 6.52E+08 EM EM-type Transmission-EM Image type Wide-field

	EV200004	3/10	Homo sapiens	MEC 1	(d)(U)C	Elgamal, Sara	2021	67%
Study summary Full title Optimizing extracellular vesicles’ isolation from chronic lymphocytic leukemia patient plasma and cell line supernatant All authors Sara Elgamal, Emanuele Cocucci, Ellen J Sass, Xiaokui M Mo, Angela R Blissett, Edward P Calomeni, Kerry A Rogers, Jennifer A Woyach, Seema A Bhat, Natarajan Muthusamy, Amy J Johnson, Karilyn T Larkin, John C Byrd Journal JCI insight Abstract In chronic lymphocytic leukemia (CLL) and very likely all cancer types, extracellular vesicles (EVs) (show more...)In chronic lymphocytic leukemia (CLL) and very likely all cancer types, extracellular vesicles (EVs) are a common mechanism by which intercellular messages are communicated between normal, diseased, and transformed cells. Studies of EVs in CLL and other cancers have great variability and often lack reproducibility. For CLL patient plasma and cell lines, we sought to characterize current approaches used in isolating EV products and understand whether cell culture–conditioned media or complex biological fluids confound results. Utilizing nanoparticle tracking analysis, protein quantification, and electron microscopy, we show that ultracentrifugation with an OptiPrep cushion can effectively minimize contaminants from starting materials including plasma and conditioned media of CLL cell lines grown in EV-depleted complete RPMI media but not grown in the serum-free media AIM V commonly used in CLL experimental work. Moreover, we confirm the benefit of including 25 mM trehalose in PBS during EV isolation steps to reduce EV aggregation, to preserve function for downstream applications and characterization. Furthermore, we report the highest particles/μg EVs were obtained from our CLL cell lines utilizing the CELLine bioreactor flask. Finally, we optimized a proliferation assay that offers a functional evaluation of our EVs with minimal sample requirements. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin AIMV Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: TSG101/ CD81/ CD63/ GAPDH non-EV: Calnexin/ Albumin Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MEC 1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 88 Cell count 2.60E+08 +/- 7.59E+07 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 22 Wash: time (min) 70 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ GAPDH/ CD81 Not detected EV-associated proteins TSG101 Detected contaminants Albumin Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution EV concentration Yes Particle yield Particles/ug;Yes, other: 5.97E+08 +/- 4.36E+08 EM EM-type Transmission-EM Image type Wide-field

	EV200004	4/10	Homo sapiens	MEC 1	(d)(U)C	Elgamal, Sara	2021	67%
Study summary Full title Optimizing extracellular vesicles’ isolation from chronic lymphocytic leukemia patient plasma and cell line supernatant All authors Sara Elgamal, Emanuele Cocucci, Ellen J Sass, Xiaokui M Mo, Angela R Blissett, Edward P Calomeni, Kerry A Rogers, Jennifer A Woyach, Seema A Bhat, Natarajan Muthusamy, Amy J Johnson, Karilyn T Larkin, John C Byrd Journal JCI insight Abstract In chronic lymphocytic leukemia (CLL) and very likely all cancer types, extracellular vesicles (EVs) (show more...)In chronic lymphocytic leukemia (CLL) and very likely all cancer types, extracellular vesicles (EVs) are a common mechanism by which intercellular messages are communicated between normal, diseased, and transformed cells. Studies of EVs in CLL and other cancers have great variability and often lack reproducibility. For CLL patient plasma and cell lines, we sought to characterize current approaches used in isolating EV products and understand whether cell culture–conditioned media or complex biological fluids confound results. Utilizing nanoparticle tracking analysis, protein quantification, and electron microscopy, we show that ultracentrifugation with an OptiPrep cushion can effectively minimize contaminants from starting materials including plasma and conditioned media of CLL cell lines grown in EV-depleted complete RPMI media but not grown in the serum-free media AIM V commonly used in CLL experimental work. Moreover, we confirm the benefit of including 25 mM trehalose in PBS during EV isolation steps to reduce EV aggregation, to preserve function for downstream applications and characterization. Furthermore, we report the highest particles/μg EVs were obtained from our CLL cell lines utilizing the CELLine bioreactor flask. Finally, we optimized a proliferation assay that offers a functional evaluation of our EVs with minimal sample requirements. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Bioreactor Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: CD52/ TSG101/ CD63/ CD45/ GAPDH/ CD81/ MHC2/ CD19 non-EV: Calnexin/ Albumin Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MEC 1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 88 Cell count 2.60E+08 +/- 7.59E+07 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 22 Wash: time (min) 70 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins GAPDH/ CD63/ TSG101/ CD81 Not detected contaminants Albumin/ Calnexin Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins MHC2/ CD19/ CD45/ CD52/ CD81 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution EV concentration Yes Particle yield Particles/ug;Yes, other: 2.4E+09 +/- 3.64E+08 EM EM-type Transmission-EM Image type Wide-field

	EV200004	9/10	Homo sapiens	Blood plasma	(d)(U)C	Elgamal, Sara	2021	67%
Study summary Full title Optimizing extracellular vesicles’ isolation from chronic lymphocytic leukemia patient plasma and cell line supernatant All authors Sara Elgamal, Emanuele Cocucci, Ellen J Sass, Xiaokui M Mo, Angela R Blissett, Edward P Calomeni, Kerry A Rogers, Jennifer A Woyach, Seema A Bhat, Natarajan Muthusamy, Amy J Johnson, Karilyn T Larkin, John C Byrd Journal JCI insight Abstract In chronic lymphocytic leukemia (CLL) and very likely all cancer types, extracellular vesicles (EVs) (show more...)In chronic lymphocytic leukemia (CLL) and very likely all cancer types, extracellular vesicles (EVs) are a common mechanism by which intercellular messages are communicated between normal, diseased, and transformed cells. Studies of EVs in CLL and other cancers have great variability and often lack reproducibility. For CLL patient plasma and cell lines, we sought to characterize current approaches used in isolating EV products and understand whether cell culture–conditioned media or complex biological fluids confound results. Utilizing nanoparticle tracking analysis, protein quantification, and electron microscopy, we show that ultracentrifugation with an OptiPrep cushion can effectively minimize contaminants from starting materials including plasma and conditioned media of CLL cell lines grown in EV-depleted complete RPMI media but not grown in the serum-free media AIM V commonly used in CLL experimental work. Moreover, we confirm the benefit of including 25 mM trehalose in PBS during EV isolation steps to reduce EV aggregation, to preserve function for downstream applications and characterization. Furthermore, we report the highest particles/μg EVs were obtained from our CLL cell lines utilizing the CELLine bioreactor flask. Finally, we optimized a proliferation assay that offers a functional evaluation of our EVs with minimal sample requirements. (hide) EV-METRIC 67% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin DUC Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: TSG101/ CD63/ CD45/ GAPDH/ Alix/ CD81/ CD235A/ HSP70/ MHC2/ CD9 non-EV: Calnexin Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 22 Wash: time (min) 70 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ GAPDH/ TSG101/ CD9/ CD63/ HSP70 Not detected contaminants Calnexin Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins CD235A/ CD45/ MHC2/ CD9/ CD81 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution EV concentration Yes Particle yield Particles/ug;Yes, other: 3.40E+08 +/- 2.85E+08 EM EM-type Transmission-EM Image type Wide-field

	EV200004	10/10	Homo sapiens	Blood plasma	(d)(U)C	Elgamal, Sara	2021	67%
Study summary Full title Optimizing extracellular vesicles’ isolation from chronic lymphocytic leukemia patient plasma and cell line supernatant All authors Sara Elgamal, Emanuele Cocucci, Ellen J Sass, Xiaokui M Mo, Angela R Blissett, Edward P Calomeni, Kerry A Rogers, Jennifer A Woyach, Seema A Bhat, Natarajan Muthusamy, Amy J Johnson, Karilyn T Larkin, John C Byrd Journal JCI insight Abstract In chronic lymphocytic leukemia (CLL) and very likely all cancer types, extracellular vesicles (EVs) (show more...)In chronic lymphocytic leukemia (CLL) and very likely all cancer types, extracellular vesicles (EVs) are a common mechanism by which intercellular messages are communicated between normal, diseased, and transformed cells. Studies of EVs in CLL and other cancers have great variability and often lack reproducibility. For CLL patient plasma and cell lines, we sought to characterize current approaches used in isolating EV products and understand whether cell culture–conditioned media or complex biological fluids confound results. Utilizing nanoparticle tracking analysis, protein quantification, and electron microscopy, we show that ultracentrifugation with an OptiPrep cushion can effectively minimize contaminants from starting materials including plasma and conditioned media of CLL cell lines grown in EV-depleted complete RPMI media but not grown in the serum-free media AIM V commonly used in CLL experimental work. Moreover, we confirm the benefit of including 25 mM trehalose in PBS during EV isolation steps to reduce EV aggregation, to preserve function for downstream applications and characterization. Furthermore, we report the highest particles/μg EVs were obtained from our CLL cell lines utilizing the CELLine bioreactor flask. Finally, we optimized a proliferation assay that offers a functional evaluation of our EVs with minimal sample requirements. (hide) EV-METRIC 67% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Opti Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: TSG101/ CD63/ CD45/ GAPDH/ Alix/ CD81/ CD235A/ HSP70/ MHC2/ CD9 non-EV: Calnexin Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 22 Wash: time (min) 70 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ GAPDH/ HSP70/ TSG101/ CD63/ CD9 Not detected contaminants Calnexin Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins MHC2/ CD235A/ CD45/ CD81/ CD9 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution EV concentration Yes Particle yield Particles/ug;Yes, other: 2.46E+08 +/- 7.57E+07 EM EM-type Transmission-EM Image type Wide-field

	EV190107	2/3	Homo sapiens	Jurkat	DG (d)(U)C	Martin-Jaular, Lorena	2021	67%
Study summary Full title Unbiased proteomic profiling of host cell extracellular vesicle composition and dynamics upon HIV-1 infection All authors Lorena Martin-Jaular, Nathalie Nevo, Julia P Schessner, Mercedes Tkach, Mabel Jouve, Florent Dingli, Damarys Loew, Kenneth W Witwer, Matias Ostrowski, Georg H H Borner, Clotilde Théry Journal EMBO J Abstract Cells release diverse types of extracellular vesicles (EVs), which transfer complex signals to surro (show more...)Cells release diverse types of extracellular vesicles (EVs), which transfer complex signals to surrounding cells. Specific markers to distinguish different EVs (e.g. exosomes, ectosomes, enveloped viruses like HIV) are still lacking. We have developed a proteomic profiling approach for characterizing EV subtype composition and applied it to human Jurkat T cells. We generated an interactive database to define groups of proteins with similar profiles, suggesting release in similar EVs. Biochemical validation confirmed the presence of preferred partners of commonly used exosome markers in EVs: CD81/ADAM10/ITGB1, and CD63/syntenin. We then compared EVs from control and HIV-1-infected cells. HIV infection altered EV profiles of several cellular proteins, including MOV10 and SPN, which became incorporated into HIV virions, and SERINC3, which was re-routed to non-viral EVs in a Nef-dependent manner. Furthermore, we found that SERINC3 controls the surface composition of EVs. Our workflow provides an unbiased approach for identifying candidate markers and potential regulators of EV subtypes. It can be widely applied to in vitro experimental systems for investigating physiological or pathological modifications of EV release. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Protein markers EV: "cd45/ AChE/ CD63/ CD9/ syntenin-1" non-EV: Proteomics no EV density (g/ml) 1.001-1.097 Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Jurkat EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) 87 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 106750 Wash: volume per pellet (ml) 37 Wash: time (min) 90 Wash: Rotor Type SW 32 Ti Wash: speed (g) 106750 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 6% Highest density fraction 18% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1 Orientation Top-down Rotor type SW 41 Ti Speed (g) 200000 Duration (min) 60 Fraction volume (mL) 1 or 2 Fraction processing Centrifugation Pelleting: volume per fraction 37 Pelleting: duration (min) 90 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 106750 EV-subtype Distinction between multiple subtypes Used subtypes Characterization: Protein analysis Protein Concentration Method "Other;Gel stain free assay" Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins "CD9/ CD63/ syntenin-1/ cd45" Not detected EV-associated proteins AChE Characterization: Lipid analysis No

	EV190048	1/1	Homo sapiens	NA	DC (d)(U)C Filtration	Shaihov-Teper, Olga	2021	67%
Study summary Full title Extracellular Vesicles from Epicardial Fat Facilitate Atrial Fibrillation All authors Olga Shaihov-Teper, Eilon Ram, Nimer Ballan, Rafael Y Brzezinski, Nili Naftali-Shani, Rula Masoud, Tamar Ziv, Nir Lewis, Yeshai Schary, La-Paz Levin-Kotler, David Volvovitch, Elchanan M Zuroff, Sergei Amunts, Neta Regev-Rudzki, Leonid Sternik, Ehud Raanani, Lior Gepstein, Jonathan Leor Journal Circulation Abstract Background: The role of epicardial fat (eFat)-derived extracellular vesicles (EVs) in the pathogenes (show more...)Background: The role of epicardial fat (eFat)-derived extracellular vesicles (EVs) in the pathogenesis of atrial fibrillation (AF) has never been studied. We tested the hypothesis that eFat-EVs transmit proinflammatory, profibrotic, and proarrhythmic molecules that induce atrial myopathy and fibrillation. Methods: We collected eFat specimens from patients with (n=32) and without AF (n=30) during elective heart surgery. eFat samples were grown as organ cultures, and the culture medium was collected every two days. We then isolated and purified eFat-EVs from the culture medium, and analyzed the EV number, size, morphology, specific markers, encapsulated cytokines, proteome, and miRNAs. Next, we evaluated the biological effects of unpurified and purified EVs on atrial mesenchymal stromal cells (MSCs) and endothelial cells (ECs) in vitro. To establish a causal association between eFat-EVs and vulnerability to AF, we modeled AF in vitro using induced pluripotent stem cell-derived cardiomyocytes (iCMs). Results: Microscopic examination revealed excessive inflammation, fibrosis, and apoptosis in fresh and cultured eFat tissues. Cultured explants from patients with AF secreted more EVs and harbored greater amounts of proinflammatory and profibrotic cytokines, as well as profibrotic miRNA, than those without AF. The proteomic analysis confirmed the distinctive profile of purified eFat-EVs from patients with AF. In vitro, purified and unpurified eFat-EVs from patients with AF had a greater effect on proliferation and migration of human MSCs and ECs, compared to eFat-EVs from patients without AF. Finally, while eFat-EVs from patients with and without AF shortened the action potential duration of iCMs, only eFat-EVs from patients with AF induced sustained reentry (rotor) in iCMs. Conclusions: We show, for the first time, a distinctive proinflammatory, profibrotic, and proarrhythmic signature of eFat-EVs from patients with AF. Our findings uncover another pathway by which eFat promotes the development of atrial myopathy and fibrillation. (hide) EV-METRIC 67% (50th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Epicardial fat tissue culture supernatant Sample origin cardiac disease Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DC (d)(U)C Filtration Protein markers EV: TSG101/ CD63/ CD81/ IL-10/ Alix/ TNF-A/ IL1A/ CD9 non-EV: Calreticulin Proteomics no Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Epicardial fat tissue culture supernatant Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 960 Pelleting: rotor type Type 50.2 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 39 Wash: time (min) 70 Wash: Rotor Type Type 50 Wash: speed (g) 100000 Density cushion Density medium Sucrose Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ TSG101/ Alix/ CD81 Detected contaminants Calreticulin ELISA Antibody details provided? No Detected EV-associated proteins TNF-A/ IL-10/ IL1A Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment Yes Moment of Proteinase treatment After Proteinase type Proteinase K Proteinase concentration 1 tablet per 10 ml extraction solution RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-170 EV concentration Yes EM EM-type Transmission-EM Image type Close-up Report size (nm) 30-170

	EV220194	1/6	Mus musculus	Liver tissue	(d)(U)C Filtration ExoQuick	Matejovič A	2021	63%
Study summary Full title Comparison of separation methods for tissue-derived extracellular vesicles in the liver, heart, and skeletal muscle. All authors Matejovič A, Wakao S, Kitada M, Kushida Y, Dezawa M Journal FEBS Open Bio Abstract Extracellular vesicles (EVs), which are nanosized vesicles released by cells as intracellular messen (show more...)Extracellular vesicles (EVs), which are nanosized vesicles released by cells as intracellular messengers, have high potential as biomarkers. EVs are usually collected from in vitro sources, such as cell culture media or biofluids, and not from tissues. Techniques enabling direct collection of EVs from tissues will extend the applications of EVs. We compared methods for separating EVs from solid liver, heart, and skeletal muscle. Compared with a precipitation method, an ultracentrifugation-based method for collection of EVs from solid tissues yielded a higher proportion of EVs positive for EV-related markers, with minimum levels of intracellular organelle-related markers. Some tissue-specific modifications, such as a sucrose cushion step, may improve the yield and purity of the collected EVs. (hide) EV-METRIC 63% (50th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Liver tissue Sample origin Intact liver - CCL4-induced hepatotoxicity Focus vesicles Extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration ExoQuick Protein markers EV: Alix/ CD63/ HSP70/ TSG101 non-EV: Calnexin/ RPL5 Proteomics no Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Liver tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Filtration steps 0.2 or 0.22 µm Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD63/ HSP70/ TSG101 Detected contaminants Calnexin Not detected contaminants Calnexin/ RPL5 Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Mean Reported size (nm) 346 ± 152.9 nm EV concentration Yes EM EM-type Transmission-EM Image type Wide-field Extra information Tissue processing: ex vivo culture of intact tissue (48 h)

	EV220162	1/1	Homo sapiens	Serum	Hydrophobic interaction chromatography UF	Ji X	2021	63%
Study summary Full title A novel method of high-purity extracellular vesicle enrichment from microliter-scale human serum for proteomic analysis. All authors Ji X, Huang S, Zhang J, Bruce TF, Tan Z, Wang D, Zhu J, Marcus RK, Lubman DM Journal Electrophoresis Abstract We have developed a rapid, low-cost, and simple separation strategy to separate extracellular vesicl (show more...)We have developed a rapid, low-cost, and simple separation strategy to separate extracellular vesicles (EVs) from a small amount of serum (i.e.,<100 μL) with minimal contamination by serum proteins and lipoprotein particles to meet the high purity requirement for EV proteome analysis. EVs were separated by a novel polyester capillary channel polymer (PET C-CP) fiber phase/hydrophobic interaction chromatography (HIC) method which is rapid and can process small size samples. The collected EV fractions were subjected to a post-column cleanup protocol using a centrifugal filter to perform buffer exchange and eliminate potential coeluting non-EV proteins while minimizing EV sample loss. Downstream characterization demonstrated that our current strategy can separate EVs with the anticipated exosome-like particle size distribution and high yield (∼1 × 10 EV particles per mL of serum) in approximately 15 min. Proteome profiling of the EVs reveals that a group of genuine EV components were identified that have significantly less high-abundance blood proteins and lipoprotein particle contamination in comparison to traditional separation methods. The use of this methodology appears to address the major challenges facing EV separation for proteomics analysis. In addition, the EV post-column cleanup protocol proposed in the current work has the potential to be combined with other separation methods, such as ultracentrifugation (UC), to further purify the separated EV samples. (hide) EV-METRIC 63% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Hydrophobic interaction chromatography Ultrafiltration Protein markers EV: CD9/ CD63 non-EV: None Proteomics yes Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Serum Separation Method Ultra filtration Cut-off size (kDa) 10/ 100 Membrane type Regenerated cellulose Other Name other separation method Hydrophobic interaction chromatography Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD9/ CD63 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mode Reported size (nm) 112.3 EV concentration Yes Particle yield as number of particles per milliliter of starting sample: 1.00e+11 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 30-150

	EV210462	1/3	Homo sapiens	PLX stromal cells	(d)(U)C Tangential flow filtration	Wolf, Martin	2021	63%
Study summary Full title A functional corona around extracellular vesicles enhances angiogenesis during skin regeneration and signals in immune cells All authors Martin Wolf, Rodolphe W Poupardin, Patricia Ebner-Peking, André Cronemberger Andrade, Constantin Blöchl, Astrid Obermayer, Fausto Gueths Gomes, Balazs Vari, Essi Eminger, Heide-Marie Binder, Anna M Raninger, Sarah Hochmann, Gabriele Brachtl, Andreas Spittler, Thomas Heuser, Racheli Ofir, Christian G Huber, Zami Aberman, Katharina Schallmoser, Hans-Dieter Volk, Dirk Strunk Journal bioRxiv Abstract Nanoparticles can acquire a protein corona defining their biological identity. Corona functions were (show more...)Nanoparticles can acquire a protein corona defining their biological identity. Corona functions were not yet considered for cell-derived extracellular vesicles (EVs). Here we demonstrate that nanosized EVs from therapy-grade human placental-expanded (PLX) stromal cells are surrounded by an imageable and functional protein corona when enriched with permissive technology. Scalable EV separation from cell-secreted soluble factors via tangential flow-filtration and subtractive tandem mass-tag proteomics revealed significant enrichment of predominantly immunomodulatory and proangiogenic proteins. Western blot, calcein-based flow cytometry, super-resolution and electron microscopy verified EV identity. PLX-EVs protected corona proteins from protease digestion. EVs significantly ameliorated human skin regeneration and angiogenesis in vivo, induced differential signaling in immune cells, and dose-dependently inhibited T cell proliferation in vitro. Corona removal by size-exclusion or ultracentrifugation abrogated angiogenesis. Re-establishing an artificial corona by cloaking EVs with defined proangiogenic proteins served as a proof-of-concept. Understanding EV corona formation will improve rational EV-inspired nanotherapy design (hide) EV-METRIC 63% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Tangential flow filtration Protein markers EV: CD9/ CD63/ CD81/ Flotillin-1/ CD49e/ CD44/ CD29/ uPA/ DPP IV/ IGBP-3/ Serpin E1/ PF4/ TSP-1/ Serpin F1/ TF/ PTX3/ CD105 non-EV: GRP94/ Calnexin/ Albumin/ ApoA1 Proteomics yes Show all info Study aim Function/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-?related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells PLX stromal cells EV-harvesting Medium EV-depleted medium Preparation of EDS tangential flow filtration Cell count 60000000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Pelleting performed No Filtration steps Between 800 g and 10,000 g Other Name other separation method Tangential flow filtration Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) 1000-38000 Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ CD81/ Flotillin-1 Detected contaminants Albumin/ ApoA1 Not detected contaminants GRP94/ Calnexin Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Detected EV-associated proteins CD9/ CD63/ CD81/ CD49e/ CD44/ CD29 Proteomics database Yes Detected EV-associated proteins CD9/ CD63/ CD81 Detected EV-associated proteins uPA/ DPP IV/ IGBP-3/ Serpin E1/ PF4/ TSP-1/ Serpin F1/ TF/ PTX3/ CD105 Characterization: RNA analysis RNA analysis Type Capillary electrophoresis (e.g. Bioanalyzer) Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Modus Reported size (nm) 141 EV concentration Yes EM EM-type Cryo-EM Image type Wide-field

	EV210292	1/1	Homo sapiens	Urine	Filtration qEV	Sedej, Ivana	2021	63%
Study summary Full title Optimization of isolation protocol and characterization of urinary extracellular vesicles as biomarkers of kidney allograft injury All authors Ivana Sedej, Magda Tušek Žnidarič, Vita Dolžan, Metka Lenassi, Miha Arnol Journal Clinical Nephrology Abstract Aims: Long-term kidney allograft survival requires a personalized approach to allograft injury recog (show more...)Aims: Long-term kidney allograft survival requires a personalized approach to allograft injury recognition in a timely and reliable manner. Kidney biopsy is invasive and unsuitable for continuous function assessment. Alternatively, in urine, we find extracellular vesicles (uEVs), stable carriers of kidney pathology signals. Analysis of uEVs and their cargo could allow for more frequent and non-invasive assessment of allograft function. We aimed to optimize the uEVs isolation method applicable for kidney allograft injury biomarker studies. Materials and methods: To this end, we optimized several steps of size-exclusion chromatography (SEC)-based method for uEVs isolation from second morning urine of kidney allograft recipients. uEVs were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), western analysis, and quantitative PCR. Results: According to TEM and NTA, SEC isolated high concentrations (8.64 × 108 EVs/mL of urine) of EVs that showed typical morphology and mean size (171 nm), but addition of EDTA and filtration step were needed to remove impurities. Additionally, typical EV proteins Hsc70, CD63, flotillin, tubulin, GAPDH, and miR hsa-let-7i were detected in isolated uEVs, further confirming their identity. Conclusion: Optimized method based on SEC was effective and adequate in isolating pure EVs from urine of kidney allograft recipients and could be used in future biomarker studies. (hide) EV-METRIC 63% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin Kidney transplantation Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Filtration qEV Protein markers EV: CD63/ Flotillin-1/ Tubulin/ HSC70/ GAPDH/ CD9/ CD81 non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Urine Separation Method Filtration steps 0.2 or 0.22 µm Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) 7.5µg (from 60 mL urine) Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ Flotillin-1/ Tubulin/ HSC70/ GAPDH Not detected EV-associated proteins CD9/ CD81 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 171 nm EV concentration Yes Particle yield particles per milliliter of starting sample: 8.64e+8 EM Image type Close-up, Wide-field

	EV210162	1/2	Homo sapiens	HCT116	Total Exosome Isolation UF Filtration	Clerici, Stefano	2021	63%
Study summary Full title Colorectal Cancer Cell-Derived Small Extracellular Vesicles Educate Human Fibroblasts to Stimulate Migratory Capacity All authors Stefano Piatto Clerici 1 , Maikel Peppelenbosch 2 , Gwenny Fuhler 2 , Sílvio Roberto Consonni 1 , Carmen Veríssima Ferreira-Halder 1 Journal Front Cell Dev Biol Abstract Colorectal cancer (CRC) is in the top 10 cancers most prevalent worldwide, affecting equally men and (show more...)Colorectal cancer (CRC) is in the top 10 cancers most prevalent worldwide, affecting equally men and women. Current research on tumor-derived extracellular vesicles (EVs) suggests that these small extracellular vesicles (sEVs) play an important role in mediating cell-to-cell communication and thus potentially affecting cancer progression via multiple pathways. In the present study, we hypothesized that sEVs derived from different CRC cell lines differ in their ability to reprogram normal human fibroblasts through a process called tumor education. The sEVs derived from CRC cell lines (HT29 and HCT116) were isolated by a combination of ultrafiltration and polymeric precipitation, followed by characterization based on morphology, size, and the presence or absence of EV and non-EV markers. It was observed that the HT29 cells displayed a higher concentration of sEVs compared with HCT116 cells. For the first time, we demonstrated that HT29-derived sEVs were positive for low-molecular-weight protein tyrosine phosphatase (Lmwptp). CRC cell-derived sEVs were uptake by human fibroblasts, stimulating migratory ability via Rho-Fak signaling in co-incubated human fibroblasts. Another important finding showed that HT29 cell-derived sEVs are much more efficient in activating human fibroblasts to cancer-associated fibroblasts (CAFs). Indeed, the sEVs produced by the HT29 cells that are less responsive to a cytotoxic agent display higher efficiency in educating normal human fibroblasts by providing them advantages such as activation and migratory ability. In other words, these sEVs have an influence on the CRC microenvironment, in part, due to fibroblasts reprogramming. (hide) EV-METRIC 63% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles Other / small extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Commercial method UF Filtration Protein markers EV: Alix/ TSG101/ B-actin/ CD63/ CD81 non-EV: GM130 Proteomics no Show all info Study aim Function/Biogenesis/cargo sorting/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HCT116 EV-harvesting Medium Serum free medium Cell viability (%) 80 Cell count 1.50E+10 Separation Method Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Commercial kit Total Exosome Isolation Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins B-actin/ TSG101/ CD81 Not detected EV-associated proteins CD63/ Alix Not detected contaminants GM130 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 135 EV concentration Yes Particle yield Yes, as number of particles per million cells 81 EM EM-type Transmission-EM Image type Close-up Report size (nm) 135

	EV210162	2/2	Homo sapiens	HT29	Total Exosome Isolation UF Filtration	Clerici, Stefano	2021	63%
Study summary Full title Colorectal Cancer Cell-Derived Small Extracellular Vesicles Educate Human Fibroblasts to Stimulate Migratory Capacity All authors Stefano Piatto Clerici 1 , Maikel Peppelenbosch 2 , Gwenny Fuhler 2 , Sílvio Roberto Consonni 1 , Carmen Veríssima Ferreira-Halder 1 Journal Front Cell Dev Biol Abstract Colorectal cancer (CRC) is in the top 10 cancers most prevalent worldwide, affecting equally men and (show more...)Colorectal cancer (CRC) is in the top 10 cancers most prevalent worldwide, affecting equally men and women. Current research on tumor-derived extracellular vesicles (EVs) suggests that these small extracellular vesicles (sEVs) play an important role in mediating cell-to-cell communication and thus potentially affecting cancer progression via multiple pathways. In the present study, we hypothesized that sEVs derived from different CRC cell lines differ in their ability to reprogram normal human fibroblasts through a process called tumor education. The sEVs derived from CRC cell lines (HT29 and HCT116) were isolated by a combination of ultrafiltration and polymeric precipitation, followed by characterization based on morphology, size, and the presence or absence of EV and non-EV markers. It was observed that the HT29 cells displayed a higher concentration of sEVs compared with HCT116 cells. For the first time, we demonstrated that HT29-derived sEVs were positive for low-molecular-weight protein tyrosine phosphatase (Lmwptp). CRC cell-derived sEVs were uptake by human fibroblasts, stimulating migratory ability via Rho-Fak signaling in co-incubated human fibroblasts. Another important finding showed that HT29 cell-derived sEVs are much more efficient in activating human fibroblasts to cancer-associated fibroblasts (CAFs). Indeed, the sEVs produced by the HT29 cells that are less responsive to a cytotoxic agent display higher efficiency in educating normal human fibroblasts by providing them advantages such as activation and migratory ability. In other words, these sEVs have an influence on the CRC microenvironment, in part, due to fibroblasts reprogramming. (hide) EV-METRIC 63% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles Other / small extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Commercial method UF Filtration Protein markers EV: TSG101/ CD63/ CD81/ Lmwptp/ Alix/ B-actin non-EV: GM130 Proteomics no Show all info Study aim Function/Biogenesis/cargo sorting/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HT29 EV-harvesting Medium Serum free medium Cell viability (%) 90 Cell count 1.30E+10 Separation Method Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Commercial kit Total Exosome Isolation Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins Lmwptp/ B-actin/ TSG101/ CD81 Not detected EV-associated proteins CD63/ Alix Not detected contaminants GM130 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 140 EV concentration Yes Particle yield Yes, as number of particles per million cells 214 EM EM-type Transmission-EM Image type Close-up Report size (nm) 140

	EV210144	1/9	Homo sapiens	Blood plasma	(d)(U)C DC	Kumar, Awanit	2021	63%
Study summary Full title The polysaccharide chitosan facilitates the isolation of small extracellular vesicles from multiple biofluids All authors Awanit Kumar, Surendar Reddy Dhadi, Ngoc‐Nu Mai, Catherine Taylor, Jeremy W. Roy, David A. Barnett, Stephen M. Lewis, Anirban Ghosh, and Rodney J. Ouellette Journal J Extracell Vesicles Abstract Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biop (show more...)Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biopsy‐based diagnostic tests and therapeutic applications; however, clinical use of EVs presents a challenge as many currently‐available EV isolation methods have limitations related to efficiency, purity, and complexity of the methods. Moreover, many EV isolation methods do not perform efficiently in all biofluids due to their differential physicochemical properties. Thus, there continues to be a need for novel EV isolation methods that are simple, robust, non‐toxic, and/or clinically‐amenable. Here we demonstrate a rapid and efficient method for small extracellular vesicle (sEV) isolation that uses chitosan, a linear cationic polyelectrolyte polysaccharide that exhibits biocompatibility, non‐immunogenicity, biodegradability, and low toxicity. Chitosan‐precipitated material was characterized using Western blotting, nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and relevant proteomic‐based gene ontology analyses. We find that chitosan facilitates the isolation of sEVs from multiple biofluids, including cell culture‐conditioned media, human urine, plasma and saliva. Overall, our data support the potential for chitosan to isolate a population of sEVs from a variety of biofluids and may have the potential to be a clinically amenable sEV isolation method. (hide) EV-METRIC 63% (91st percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DC Protein markers EV: CD63/ Flotillin1/ CD9/ HSC70 non-EV: FCN3/ SAA/ APOB/ APOA1/ TETN Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Density cushion Density medium Sucrose Sample volume 10 Cushion volume 0.7 Density of the cushion 30% Centrifugation time 120 Centrifugation speed 138,000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins Flotillin1/ CD9/ CD63/ HSC70 Detected contaminants APOB/ FCN3 Not detected contaminants APOA1/ TETN/ SAA Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 222.1 +/- 6.1 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 2.18E+08 EM EM-type Transmission-EM Image type Close-up

	EV210144	2/9	Homo sapiens	Blood plasma	(d)(U)C Chitosan-based	Kumar, Awanit	2021	63%
Study summary Full title The polysaccharide chitosan facilitates the isolation of small extracellular vesicles from multiple biofluids All authors Awanit Kumar, Surendar Reddy Dhadi, Ngoc‐Nu Mai, Catherine Taylor, Jeremy W. Roy, David A. Barnett, Stephen M. Lewis, Anirban Ghosh, and Rodney J. Ouellette Journal J Extracell Vesicles Abstract Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biop (show more...)Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biopsy‐based diagnostic tests and therapeutic applications; however, clinical use of EVs presents a challenge as many currently‐available EV isolation methods have limitations related to efficiency, purity, and complexity of the methods. Moreover, many EV isolation methods do not perform efficiently in all biofluids due to their differential physicochemical properties. Thus, there continues to be a need for novel EV isolation methods that are simple, robust, non‐toxic, and/or clinically‐amenable. Here we demonstrate a rapid and efficient method for small extracellular vesicle (sEV) isolation that uses chitosan, a linear cationic polyelectrolyte polysaccharide that exhibits biocompatibility, non‐immunogenicity, biodegradability, and low toxicity. Chitosan‐precipitated material was characterized using Western blotting, nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and relevant proteomic‐based gene ontology analyses. We find that chitosan facilitates the isolation of sEVs from multiple biofluids, including cell culture‐conditioned media, human urine, plasma and saliva. Overall, our data support the potential for chitosan to isolate a population of sEVs from a variety of biofluids and may have the potential to be a clinically amenable sEV isolation method. (hide) EV-METRIC 63% (91st percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Chitosan-based Protein markers EV: CD63/ Flotillin1/ CD9/ HSC70 non-EV: FCN3/ APOA1/ TETN Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Other Name other separation method Chitosan-based Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins Flotillin1/ CD9/ CD63/ HSC70 Not detected contaminants APOA1/ FCN3/ TETN Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 89.8 +/- 2.5 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 2.94E+08 EM EM-type Transmission-EM Image type Close-up

	EV210144	8/9	Homo sapiens	HEK293	(d)(U)C DG	Kumar, Awanit	2021	63%
Study summary Full title The polysaccharide chitosan facilitates the isolation of small extracellular vesicles from multiple biofluids All authors Awanit Kumar, Surendar Reddy Dhadi, Ngoc‐Nu Mai, Catherine Taylor, Jeremy W. Roy, David A. Barnett, Stephen M. Lewis, Anirban Ghosh, and Rodney J. Ouellette Journal J Extracell Vesicles Abstract Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biop (show more...)Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biopsy‐based diagnostic tests and therapeutic applications; however, clinical use of EVs presents a challenge as many currently‐available EV isolation methods have limitations related to efficiency, purity, and complexity of the methods. Moreover, many EV isolation methods do not perform efficiently in all biofluids due to their differential physicochemical properties. Thus, there continues to be a need for novel EV isolation methods that are simple, robust, non‐toxic, and/or clinically‐amenable. Here we demonstrate a rapid and efficient method for small extracellular vesicle (sEV) isolation that uses chitosan, a linear cationic polyelectrolyte polysaccharide that exhibits biocompatibility, non‐immunogenicity, biodegradability, and low toxicity. Chitosan‐precipitated material was characterized using Western blotting, nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and relevant proteomic‐based gene ontology analyses. We find that chitosan facilitates the isolation of sEVs from multiple biofluids, including cell culture‐conditioned media, human urine, plasma and saliva. Overall, our data support the potential for chitosan to isolate a population of sEVs from a variety of biofluids and may have the potential to be a clinically amenable sEV isolation method. (hide) EV-METRIC 63% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Protein markers EV: CD63/ CD9 non-EV: None Proteomics yes Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK293 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell count 7.00E+07 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 12 Lowest density fraction 10% Highest density fraction 90% Total gradient volume, incl. sample (mL) 12.1 Sample volume (mL) 0.1 Orientation Bottom-up Rotor type SW 40 Ti Speed (g) 200,000 Duration (min) 960 Fraction volume (mL) 2 Fraction processing Centrifugation Pelleting: volume per fraction 10 Pelleting: duration (min) 90 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 138,000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210144	9/9	Homo sapiens	HEK293	(d)(U)C Chitosan-based	Kumar, Awanit	2021	63%
Study summary Full title The polysaccharide chitosan facilitates the isolation of small extracellular vesicles from multiple biofluids All authors Awanit Kumar, Surendar Reddy Dhadi, Ngoc‐Nu Mai, Catherine Taylor, Jeremy W. Roy, David A. Barnett, Stephen M. Lewis, Anirban Ghosh, and Rodney J. Ouellette Journal J Extracell Vesicles Abstract Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biop (show more...)Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biopsy‐based diagnostic tests and therapeutic applications; however, clinical use of EVs presents a challenge as many currently‐available EV isolation methods have limitations related to efficiency, purity, and complexity of the methods. Moreover, many EV isolation methods do not perform efficiently in all biofluids due to their differential physicochemical properties. Thus, there continues to be a need for novel EV isolation methods that are simple, robust, non‐toxic, and/or clinically‐amenable. Here we demonstrate a rapid and efficient method for small extracellular vesicle (sEV) isolation that uses chitosan, a linear cationic polyelectrolyte polysaccharide that exhibits biocompatibility, non‐immunogenicity, biodegradability, and low toxicity. Chitosan‐precipitated material was characterized using Western blotting, nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and relevant proteomic‐based gene ontology analyses. We find that chitosan facilitates the isolation of sEVs from multiple biofluids, including cell culture‐conditioned media, human urine, plasma and saliva. Overall, our data support the potential for chitosan to isolate a population of sEVs from a variety of biofluids and may have the potential to be a clinically amenable sEV isolation method. (hide) EV-METRIC 63% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Chitosan-based Protein markers EV: CD63/ Flotillin1/ CD9/ HSC70 non-EV: CANX Proteomics yes Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK293 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell count 7.00E+07 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Other Name other separation method Chitosan-based Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins Flotillin1/ HSC70/ CD9/ CD63 Not detected contaminants CANX Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 133.0 +/- 5.2 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 2.02E+08 EM EM-type Transmission-EM Image type Close-up

	EV210089	1/2	Homo sapiens	Blood plasma	(d)(U)C qEV	Sabbagh, Quentin	2021	63%
Study summary Full title The von Willebrand factor stamps plasmatic extracellular vesicles from glioblastoma patients All authors Quentin Sabbagh, Gwennan André-Grégoire, Carolina Alves-Nicolau, Aurélien Dupont, Nicolas Bidère, Emmanuel Jouglar, Laëtitia Guével, Jean-Sébastien Frénel, Julie Gavard Journal Sci Rep Abstract Glioblastoma is a devastating tumor of the central nervous system characterized by a poor survival a (show more...)Glioblastoma is a devastating tumor of the central nervous system characterized by a poor survival and an extremely dark prognosis, making its diagnosis, treatment and monitoring highly challenging. Numerous studies have highlighted extracellular vesicles (EVs) as key players of tumor growth, invasiveness and resistance, as they carry and disseminate oncogenic material in the local tumor microenvironment and at distance. However, whether their quality and quantity reflect individual health status and changes in homeostasis is still not fully elucidated. Here, we separated EVs from plasma collected at different time points alongside with the clinical management of GBM patients. Our findings confirm that plasmatic EVs could be separated and characterized with standardized protocols, thereby ensuring the reliability of measuring vesiclemia, i.e. extracellular vesicle concentration in plasma. This unveils that vesiclemia is a dynamic parameter, which could be reflecting tumor burden and/or response to treatments. Further label-free liquid chromatography tandem mass spectrometry unmasks the von Willebrand Factor (VWF) as a selective protein hallmark for GBM-patient EVs. Our data thus support the notion that EVs from GBM patients showed differential protein cargos that can be further surveyed in circulating EVs, together with vesiclemia. (hide) EV-METRIC 63% (91st percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Commercial method Protein markers EV: CD63/ CD9 non-EV: GM130 Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type MLA-130 Pelleting: speed (g) 100000 Commercial kit qEV Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9 Not detected contaminants GM130 ELISA Antibody details provided? No Detected EV-associated proteins CD63 Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Detected EV-associated proteins CD9/ CD63 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Cryo-EM Image type Close-up, Wide-field Report size (nm) 60 Report type Not Reported EV-concentration Yes

	EV200181	2/3	Homo sapiens	Blood plasma	SEC (non-commercial)	Dlugolecka, Magdalena	2021	63%
Study summary Full title Characterization of Extracellular Vesicles from Bronchoalveolar Lavage Fluid and Plasma of Patients with Lung Lesions Using Fluorescence Nanoparticle Tracking Analysis All authors Magdalena Dlugolecka, Jacek Szymanski, Lukasz Zareba, Zuzanna Homoncik, Joanna Domagala-Kulawik, Malgorzata Polubiec-Kownacka, Malgorzata Czystowska-Kuzmicz Journal Cells Abstract The current lack of reliable methods for quantifying extracellular vesicles (EVs) isolated from comp (show more...)The current lack of reliable methods for quantifying extracellular vesicles (EVs) isolated from complex biofluids significantly hinders translational applications in EV research. The recently developed fluorescence nanoparticle tracking analysis (FL-NTA) allows for the detection of EV-associated proteins, enabling EV content determination. In this study, we present the first comprehensive phenotyping of bronchopulmonary lavage fluid (BALF)-derived EVs from non-small cell lung cancer (NSCLC) patients using classical EV-characterization methods as well as the FL-NTA method. We found that EV immunolabeling for the specific EV marker combined with the use of the fluorescent mode NTA analysis can provide the concentration, size, distribution, and surface phenotype of EVs in a heterogeneous solution. However, by performing FL-NTA analysis of BALF-derived EVs in comparison to plasma-derived EVs, we reveal the limitations of this method, which is suitable only for relatively pure EV isolates. For more complex fluids such as plasma, this method appears to not be sensitive enough and the measurements can be compromised. Our parallel presentation of NTA-based phenotyping of plasma and BALF EVs emphasizes the great impact of sample composition and purity on FL-NTA analysis that has to be taken into account in the further development of FL-NTA toward the detection of EV-associated cancer biomarkers. (hide) EV-METRIC 63% (91st percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin lung cancer Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Size-exclusion chromatography (non-commercial) Protein markers EV: TSG101/ CD63/ CD81/ PD-L1/ Syntenin/ CD9 non-EV: Calnexin Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 1 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ Syntenin/ PD-L1/ TSG101/ CD81 Not detected contaminants Calnexin Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Detected EV-associated proteins CD9/ CD63/ CD81 Fluorescent NTA Relevant measurements variables specified? NA Antibody details provided? No Detected EV-associated proteins CD81/ CD9 Not detected EV-associated proteins CD63 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 98.4 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 2.44E+11 Particle analysis: flow cytometry Flow cytometer type BD FACSVerse 8 color Flow Cytometer (BD) Hardware adjustment Calibration bead size 4.5 Report type Not Reported EM EM-type Cryo-EM Image type Close-up

	EV200157	4/10	Homo sapiens	MDA-MB-468	Polymer-based precipitation SEC (non-commercial) UF	Martínez-Greene, Juan A	2021	63%
Study summary Full title Quantitative proteomic analysis of extracellular vesicle subgroups isolated by an optimized method combining polymer-based precipitation and size exclusion chromatography All authors Juan A Martínez-Greene, Karina Hernández-Ortega, Ricardo Quiroz-Baez, Osbaldo Resendis-Antonio, Israel Pichardo-Casas, David A Sinclair, Bogdan Budnik, Alfredo Hidalgo-Miranda, Eileen Uribe-Querol, María Del Pilar Ramos-Godínez, Eduardo Martínez-Martínez Journal J Extracell Vesicles Abstract The molecular characterization of extracellular vesicles (EVs) has revealed a great heterogeneity in (show more...)The molecular characterization of extracellular vesicles (EVs) has revealed a great heterogeneity in their composition at a cellular and tissue level. Current isolation methods fail to efficiently separate EV subtypes for proteomic and functional analysis. The aim of this study was to develop a reproducible and scalable isolation workflow to increase the yield and purity of EV preparations. Through a combination of polymer-based precipitation and size exclusion chromatography (Pre-SEC), we analyzed two subsets of EVs based on their CD9, CD63 and CD81 content and elution time. EVs were characterized using transmission electron microscopy, nanoparticle tracking analysis, and Western blot assays. To evaluate differences in protein composition between the early- and late-eluting EV fractions, we performed a quantitative proteomic analysis of MDA-MB-468-derived EVs. We identified 286 exclusive proteins in early-eluting fractions and 148 proteins with a differential concentration between early- and late-eluting fractions. A density gradient analysis further revealed EV heterogeneity within each analyzed subgroup. Through a systems biology approach, we found significant interactions among proteins contained in the EVs which suggest the existence of functional clusters related to specific biological processes. The workflow presented here allows the study of EV subtypes within a single cell type and contributes to standardizing the EV isolation for functional studies. (hide) EV-METRIC 63% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Polymer-based precipitation Size-exclusion chromatography (non-commercial) Ultrafiltration Protein markers EV: CD9/ CD81/ ANXA2/ TSG101 non-EV: Calnexin/ Albumin Proteomics no Show all info Study aim New methodological development/Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDA-MB-468 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 95 Cell count 1.50E+06 Separation Method Ultra filtration Cut-off size (kDa) 3 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 1 Resin type Sepharose CL-2B Other Name other separation method Polymer-based precipitation Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD81/ ANXA2/ TSG101 Detected contaminants Albumin Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 204.17 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 4.68E+11 EM EM-type Transmission-EM Image type Wide-field

	EV200100	1/1	Homo sapiens	colostrum	(d)(U)C ExoQuick	Civra, Andrea; Francese, Rache	2021	63%
Study summary Full title Human Colostrum and Derived Extracellular Vesicles Prevent Infection by Human Rotavirus and Respiratory Syncytial Virus in Vitro All authors Andrea Civra, Rachele Francese, Manuela Donalisio, Paola Tonetto, Alessandra Coscia, Stefano Sottemano, Raffaella Balestrini, Antonella Faccio, Laura Cavallarin, Guido E Moro, Enrico Bertino, David Lembo Journal J hum Lact Abstract Background: It is known that breastfeeding protects the infant from enteric and respiratory infectio (show more...)Background: It is known that breastfeeding protects the infant from enteric and respiratory infections; however, the antiviral properties of human milk against enteric and respiratory viruses are largely unexplored. Research aims: To explore the antiviral activity of human preterm colostrum against rotavirus and respiratory syncytial virus and to assess whether the derived extracellular vesicle contribute to this activity. Methods: We used a cross-sectional, prospective two-group non-experimental design. Colostra were collected from mothers of preterm newborns (N = 10) and extracellular vesicles were purified and characterized. The antiviral activity of colostra and derived extracellular vesicles were tested in vitro against rotavirus and respiratory syncytial virus and the step of viral replication inhibited by extracellular vesicles was investigated. Results: Each sample of colostrum and colostrum-derived extracellular vesicles had significant antiviral activity with a wide interpersonal variability. Mechanism of action studies demonstrated that extracellular vesicles acted by interfering with the early steps of the viral replicative cycle. Conclusion: We demonstrated the intrinsic antiviral activity of human colostrum against rotavirus and respiratory syncytial virus and we showed that extracellular vesicles substantially contribute to the overall protective effect. Our results contribute to unravelling novel mechanisms underlying the functional role of human milk as a protective and therapeutic agent in preterm infants. (hide) EV-METRIC 63% (50th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type colostrum Sample origin preterm mothers Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Commercial method Protein markers EV: CD81/ CD63/ CD9 non-EV: calnexin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type colostrum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ CD81 Not detected contaminants calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 258 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 7.40E+11 EM EM-type Transmission-EM Image type Wide-field

	EV200093	10/10	Homo sapiens	Blood plasma	(d)(U)C Filtration DG	Dong, Liang	2021	63%
Study summary Full title Comprehensive evaluation of methods for small extracellular vesicles separation from human plasma, urine and cell culture medium All authors Liang Dong, Richard C. Zieren, Kengo Horie, Chi‐Ju Kim, Emily Mallick, Yuezhou Jing, Mingxiao Feng, Morgan D. Kuczler, Jordan Green, Sarah R. Amend, Kenneth W. Witwer, Theo M. de Reijke, Yoon‐Kyoung Cho, Kenneth J. Pienta, Wei Xue Journal J Extracell Vesicles Abstract One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the l (show more...)One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the lack of a consensus method for EV separation. This may also explain the diversity of the experimental results, as co‐separated soluble proteins and lipoproteins may impede the interpretation of experimental findings. In this study, we comprehensively evaluated the EV yields and sample purities of three most popular EV separation methods, ultracentrifugation, precipitation and size exclusion chromatography combined with ultrafiltration, along with a microfluidic tangential flow filtration device, Exodisc, in three commonly used biological samples, cell culture medium, human urine and plasma. Single EV phenotyping and density‐gradient ultracentrifugation were used to understand the proportion of true EVs in particle separations. Our findings suggest Exodisc has the best EV yield though it may co‐separate contaminants when the non‐EV particle levels are high in input materials. We found no 100% pure EV preparations due to the overlap of their size and density with many non‐EV particles in biofluids. Precipitation has the lowest sample purity, regardless of sample type. The purities of the other techniques may vary in different sample types and are largely dependent on their working principles and the intrinsic composition of the input sample. Researchers should choose the proper separation method according to the sample type, downstream analysis and their working scenarios. (hide) EV-METRIC 63% (91st percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration DG Protein markers EV: CD81/ Flotillin1 non-EV: ApoA1 Proteomics no EV density (g/ml) 1.10-1.15 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Density gradient Only used for validation of main results Yes Type Continuous Lowest density fraction 5% Highest density fraction 60% Total gradient volume, incl. sample (mL) 38 Sample volume (mL) 2 Orientation Bottom-up Rotor type SW 32 Ti Speed (g) 100000 Duration (min) 230 Fraction volume (mL) 4.75 Fraction processing Centrifugation Pelleting: volume per fraction 28 Pelleting: duration (min) 60 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 120000 Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Flotillin1/ CD81 Not detected contaminants ApoA1 Flow cytometry Type of Flow cytometry NanoFCM Hardware adaptation to ~100nm EV's Please refer to the publication below: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanopar Calibration bead size 0.2 Antibody details provided? No Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type NanoFCM Hardware adjustment Please refer to the publication below: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanoparticles. ACS nano. 2014 Oct 28;8(10):10998-1006. Calibration bead size 0.2 Report type Not Reported

	EV200075	2/2	Tetraselmis chuii	Tetraselmis chuii	Tangential flow filtration	Adamo, Giorgia	2021	63%
Study summary Full title Nanoalgosomes: Introducing extracellular vesicles produced by microalgae All authors Giorgia Adamo, David Fierli, Daniele P Romancino, Sabrina Picciotto, Maria E Barone, Anita Aranyos, Darja Božič, Svenja Morsbach, Samuele Raccosta, Christopher Stanly, Carolina Paganini, Meiyu Gai, Antonella Cusimano, Vincenzo Martorana, Rosina Noto, Rita Carrotta, Fabio Librizzi, Loredana Randazzo, Rachel Parkes, Umberto Capasso Palmiero, Estella Rao, Angela Paterna, Pamela Santonicola, Ales Iglič, Laura Corcuera, Annamaria Kisslinger, Elia Di Schiavi, Giovanna L Liguori 10 , Katharina Landfester, Veronika Kralj-Iglič, Paolo Arosio, Gabriella Pocsfalvi, Nicolas Touzet, Mauro Manno, Antonella Bongiovanni Journal J Extracell Vesicles Abstract Cellular, inter-organismal and cross kingdom communication via extracellular vesicles (EVs) is inten (show more...)Cellular, inter-organismal and cross kingdom communication via extracellular vesicles (EVs) is intensively studied in basic science with high expectation for a large variety of bio-technological applications. EVs intrinsically possess many attributes of a drug delivery vehicle. Beyond the implications for basic cell biology, academic and industrial interests in EVs have increased in the last few years. Microalgae constitute sustainable and renewable sources of bioactive compounds with a range of sectoral applications, including the formulation of health supplements, cosmetic products and food ingredients. Here we describe a newly discovered subtype of EVs derived from microalgae, which we named nanoalgosomes. We isolated these extracellular nano-objects from cultures of microalgal strains, including the marine photosynthetic chlorophyte Tetraselmis chuii, using differential ultracentrifugation or tangential flow fractionation and focusing on the nanosized small EVs (sEVs). We explore different biochemical and physical properties and we show that nanoalgosomes are efficiently taken up by mammalian cell lines, confirming the cross kingdom communication potential of EVs. This is the first detailed description of such membranous nanovesicles from microalgae. With respect to EVs isolated from other organisms, nanoalgosomes present several advantages in that microalgae are a renewable and sustainable natural source, which could easily be scalable in terms of nanoalgosome production. (hide) EV-METRIC 63% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles Other / small Extracellular Vesicles - Algosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Tangential flow filtration Protein markers EV: Alix/ H/ ATPase/ enolase/ beta-actin/ HSP70 non-EV: None Proteomics no Show all info Study aim Function/Biomarker/novel EV type Sample Species Tetraselmis chuii Sample Type Cell culture supernatant EV-producing cells Tetraselmis chuii EV-harvesting Medium Serum free medium Cell viability (%) 90 Cell count mg dry weight microalgal biomass Separation Method Other Name other separation method Tangential flow filtration Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins H/ ATPase/ enolase/ beta-actin/ HSP70/ Alix Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 75 NTA Report type Size range/distribution Reported size (nm) 115+/-5 EV concentration Yes Particle yield per mg dry weight microalgal mass 1.00E+09 EM EM-type Atomic force-EM/ Transmission-EM/ Scanning-EM/ Cryo-EM Image type Close-up, Wide-field

	EV190087	1/6	Homo sapiens	Saliva	(d)(U)C Sonication	Hiraga, Chiho	2021	63%
Study summary Full title Pentapartite fractionation of particles in oral fluids by differential centrifugation All authors Chiho Hiraga, Satoshi Yamamoto, Sadamitsu Hashimoto, Masataka Kasahara, Tamiko Minamisawa, Sachiko Matsumura, Akira Katakura, Yasutomo Yajima, Takeshi Nomura, Kiyotaka Shiba Journal Sci Rep Abstract Oral fluids (OFs) contain small extracellular vesicles (sEVs or exosomes) that carry disease-associa (show more...)Oral fluids (OFs) contain small extracellular vesicles (sEVs or exosomes) that carry disease-associated diagnostic molecules. However, cells generate extracellular vesicles (EVs) other than sEVs, so the EV population is quite heterogeneous. Furthermore, molecules not packaged in EVs can also serve as diagnostic markers. For these reasons, developing a complete picture of particulate matter in the oral cavity is important before focusing on specific subtypes of EVs. Here, we used differential centrifugation to fractionate human OFs from healthy volunteers and patients with oral squamous cell carcinoma into 5 fractions, and we characterized the particles, nucleic acids, and proteins in each fraction. Canonical exosome markers, including CD63, CD9, CD133, and HSP70, were found in all fractions, whereas CD81 and AQP5 were enriched in the 160K fraction, with non-negligible amounts in the 2K fraction. The 2K fraction also contained its characteristic markers that included short derivatives of EGFR and E-cadherin, as well as an autophagosome marker, LC3, and large multi-layered vesicles were observed by electronic microscopy. Most of the DNA and RNA was recovered from the 0.3K and 2K fractions, with some in the 160K fraction. These results can provide guideline information for development of purpose-designed OF-based diagnostic systems. (hide) EV-METRIC 63% (82nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Saliva Sample origin Control condition Focus vesicles Other / cell debris Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Sonication Protein markers EV: Alix/ HSP70/ CD63/ CD9/ CD81 non-EV: Argonaute2 Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Saliva Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 2000 Other Name other separation method Sonication EV-subtype Distinction between multiple subtypes Used subtypes Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ HSP70/ CD81 Not detected EV-associated proteins Alix Detected contaminants Argonaute2 Characterization: RNA analysis RNA analysis Type Capillary electrophoresis (e.g. Bioanalyzer);Other Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No EM EM-type Atomic force-EM/ Transmission-EM Image type Close-up, Wide-field

	EV190087	2/6	Homo sapiens	Saliva	(d)(U)C Sonication	Hiraga, Chiho	2021	63%
Study summary Full title Pentapartite fractionation of particles in oral fluids by differential centrifugation All authors Chiho Hiraga, Satoshi Yamamoto, Sadamitsu Hashimoto, Masataka Kasahara, Tamiko Minamisawa, Sachiko Matsumura, Akira Katakura, Yasutomo Yajima, Takeshi Nomura, Kiyotaka Shiba Journal Sci Rep Abstract Oral fluids (OFs) contain small extracellular vesicles (sEVs or exosomes) that carry disease-associa (show more...)Oral fluids (OFs) contain small extracellular vesicles (sEVs or exosomes) that carry disease-associated diagnostic molecules. However, cells generate extracellular vesicles (EVs) other than sEVs, so the EV population is quite heterogeneous. Furthermore, molecules not packaged in EVs can also serve as diagnostic markers. For these reasons, developing a complete picture of particulate matter in the oral cavity is important before focusing on specific subtypes of EVs. Here, we used differential centrifugation to fractionate human OFs from healthy volunteers and patients with oral squamous cell carcinoma into 5 fractions, and we characterized the particles, nucleic acids, and proteins in each fraction. Canonical exosome markers, including CD63, CD9, CD133, and HSP70, were found in all fractions, whereas CD81 and AQP5 were enriched in the 160K fraction, with non-negligible amounts in the 2K fraction. The 2K fraction also contained its characteristic markers that included short derivatives of EGFR and E-cadherin, as well as an autophagosome marker, LC3, and large multi-layered vesicles were observed by electronic microscopy. Most of the DNA and RNA was recovered from the 0.3K and 2K fractions, with some in the 160K fraction. These results can provide guideline information for development of purpose-designed OF-based diagnostic systems. (hide) EV-METRIC 63% (82nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Saliva Sample origin Control condition Focus vesicles (shedding) microvesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Sonication Protein markers EV: CD81/ Alix/ CD63/ CD9/ HSP70 non-EV: Argonaute2 Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Saliva Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 10000 Other Name other separation method Sonication EV-subtype Distinction between multiple subtypes Used subtypes Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ HSP70 Not detected EV-associated proteins CD81/ CD9/ Alix Detected contaminants Argonaute2 Characterization: RNA analysis RNA analysis Type Capillary electrophoresis (e.g. Bioanalyzer);Other Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 291.3 ± 22.8 EV concentration Yes EM EM-type Atomic force-EM/ Transmission-EM Image type Wide-field

	EV190087	3/6	Homo sapiens	Saliva	(d)(U)C Sonication	Hiraga, Chiho	2021	63%
Study summary Full title Pentapartite fractionation of particles in oral fluids by differential centrifugation All authors Chiho Hiraga, Satoshi Yamamoto, Sadamitsu Hashimoto, Masataka Kasahara, Tamiko Minamisawa, Sachiko Matsumura, Akira Katakura, Yasutomo Yajima, Takeshi Nomura, Kiyotaka Shiba Journal Sci Rep Abstract Oral fluids (OFs) contain small extracellular vesicles (sEVs or exosomes) that carry disease-associa (show more...)Oral fluids (OFs) contain small extracellular vesicles (sEVs or exosomes) that carry disease-associated diagnostic molecules. However, cells generate extracellular vesicles (EVs) other than sEVs, so the EV population is quite heterogeneous. Furthermore, molecules not packaged in EVs can also serve as diagnostic markers. For these reasons, developing a complete picture of particulate matter in the oral cavity is important before focusing on specific subtypes of EVs. Here, we used differential centrifugation to fractionate human OFs from healthy volunteers and patients with oral squamous cell carcinoma into 5 fractions, and we characterized the particles, nucleic acids, and proteins in each fraction. Canonical exosome markers, including CD63, CD9, CD133, and HSP70, were found in all fractions, whereas CD81 and AQP5 were enriched in the 160K fraction, with non-negligible amounts in the 2K fraction. The 2K fraction also contained its characteristic markers that included short derivatives of EGFR and E-cadherin, as well as an autophagosome marker, LC3, and large multi-layered vesicles were observed by electronic microscopy. Most of the DNA and RNA was recovered from the 0.3K and 2K fractions, with some in the 160K fraction. These results can provide guideline information for development of purpose-designed OF-based diagnostic systems. (hide) EV-METRIC 63% (82nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Saliva Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Sonication Protein markers EV: Alix/ HSP70/ CD63/ CD9/ CD81 non-EV: Argonaute2 Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Saliva Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 160000 Other Name other separation method Sonication EV-subtype Distinction between multiple subtypes Used subtypes Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD9/ CD63/ HSP70/ CD81 Detected contaminants Argonaute2 Characterization: RNA analysis RNA analysis Type Other;Capillary electrophoresis (e.g. Bioanalyzer) Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No EM EM-type Atomic force-EM/ Transmission-EM Image type Wide-field Report size (nm) 52.7 ± 30.7

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