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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV180041	1/1	Mus musculus	4T1	DG (d)(U)C	Keklikoglou I	2018	100%
Study summary Full title Chemotherapy elicits pro-metastatic extracellular vesicles in breast cancer models. All authors Keklikoglou I, Cianciaruso C, Güç E, Squadrito ML, Spring LM, Tazzyman S, Lambein L, Poissonnier A, Ferraro GB, Baer C, Cassará A, Guichard A, Iruela-Arispe ML, Lewis CE, Coussens LM, Bardia A, Jain RK, Pollard JW, De Palma M Journal Nat Cell Biol Abstract Cytotoxic chemotherapy is an effective treatment for invasive breast cancer. However, experimental s (show more...)Cytotoxic chemotherapy is an effective treatment for invasive breast cancer. However, experimental studies in mice also suggest that chemotherapy has pro-metastatic effects. Primary tumours release extracellular vesicles (EVs), including exosomes, that can facilitate the seeding and growth of metastatic cancer cells in distant organs, but the effects of chemotherapy on tumour-derived EVs remain unclear. Here we show that two classes of cytotoxic drugs broadly employed in pre-operative (neoadjuvant) breast cancer therapy, taxanes and anthracyclines, elicit tumour-derived EVs with enhanced pro-metastatic capacity. Chemotherapy-elicited EVs are enriched in annexin A6 (ANXA6), a Ca²⁺-dependent protein that promotes NF-κB-dependent endothelial cell activation, Ccl2 induction and Ly6C⁺CCR2⁺ monocyte expansion in the pulmonary pre-metastatic niche to facilitate the establishment of lung metastasis. Genetic inactivation of Anxa6 in cancer cells or Ccr2 in host cells blunts the pro-metastatic effects of chemotherapy-elicited EVs. ANXA6 is detected, and potentially enriched, in the circulating EVs of breast cancer patients undergoing neoadjuvant chemotherapy. (hide) EV-METRIC 100% (99th 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 Adj. k-factor 191 (pelleting) / 191 (washing) Protein markers EV: CD81/ CD9/ Syntenin-1 non-EV: Gp96 Proteomics yes Show all info Study aim Function, Biomarker, Biogenesis/cargo sorting, Mechanism of uptake/transfer, Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells 4T1 EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) NA 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) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 134000 Pelleting: adjusted k-factor 191.0 Wash: time (min) 70 Wash: Rotor Type SW 32 Ti Wash: speed (g) 134000 Wash: adjusted k-factor 191.0 Density gradient Only used for validation of main results Yes Type Continuous Lowest density fraction 10 Highest density fraction 50 Sample volume (mL) 0.1 Orientation Bottom-up (sample migrates upwards) Rotor type SW 40 Ti Speed (g) 100000 Duration (min) 70 Fraction volume (mL) 2 Fraction processing Centrifugation Pelleting: volume per fraction 10 Pelleting: duration (min) 70 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 110000 Pelleting: adjusted k-factor 251.4 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) 7.3 Western Blot Detected EV-associated proteins CD9, CD81, Syntenin-1 Not detected contaminants Gp96 ELISA Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 140 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV180007	1/1	Bos taurus	Embryo culture	DG UF	Pavani KC	2018	100%
Study summary Full title Isolation and Characterization of Functionally Active Extracellular Vesicles from Culture Medium Conditioned by Bovine Embryos In Vitro. All authors Pavani KC, Hendrix A, Van Den Broeck W, Couck L, Szymanska K, Lin X, De Koster J, Van Soom A, Leemans B Journal Int J Mol Sci Abstract Extracellular vesicles (EVs) play a possible role in cell⁻cell communication and are found in vari (show more...)Extracellular vesicles (EVs) play a possible role in cell⁻cell communication and are found in various body fluids and cell conditioned culture media. The aim of this study was to isolate and characterize EVs in culture medium conditioned by bovine embryos in group and to verify if these EVs are functionally active. Initially, ultracentrifuged bovine serum albumin (BSA) containing medium was selected as suitable EV-free embryo culture medium. Next, EVs were isolated from embryo conditioned culture medium by OptiPrep^TM density gradient ultracentrifugation. Isolated EVs were characterized by nanoparticle tracking analysis, western blotting, transmission, and immunoelectron microscopy. Bovine embryo-derived EVs were sizing between 25⁻230 nm with an average concentration of 236.5 ± 1.27 × 10⁸ particles/mL. Moreover, PKH67 EV pre-labeling showed that embryo-secreted EVs were uptaken by zona-intact bovine embryos. Since BSA did not appear to be a contaminating EV source in culture medium, EV functionality was tested in BSA containing medium. Individual embryo culture in BSA medium enriched with EVs derived from conditioned embryo culture medium showed significantly higher blastocyst rates at day 7 and 8 together with a significantly lower apoptotic cell ratio. In conclusion, our study shows that EVs play an important role in inter embryo communication during bovine embryo culture in group. (hide) EV-METRIC 100% (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 Embryo culture 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 UF Protein markers EV: TSG101/ CD63/ CD9 non-EV: ApoA1/ Argonaute-2 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type Embryo culture Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Sample volume (mL) 1 Orientation Top-down (sample migrates downwards) Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: duration (min) 180 Pelleting: speed (g) 100000 Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9, CD63, TSG101 Not detected contaminants Argonaute-2, ApoA1 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 133.8±6.8 EV concentration Yes EM EM-type Transmission-EM/ Immune-EM EM protein CD63 Image type Close-up, Wide-field

	EV170030	1/1	Mus musculus	primary bone marrow dendritic cells	DG (d)(U)C	Driedonks, Tom A. P.	2018	100%
Study summary Full title Immune stimuli shape the small non-coding transcriptome of extracellular vesicles released by dendritic cells. All authors Driedonks TAP, van der Grein SG, Ariyurek Y, Buermans HPJ, Jekel H, Chow FWN, Wauben MHM, Buck AH, 't Hoen PAC, Nolte-'t Hoen ENM Journal Cell Mol Life Sci Abstract The release and uptake of nano-sized extracellular vesicles (EV) is a highly conserved means of inte (show more...)The release and uptake of nano-sized extracellular vesicles (EV) is a highly conserved means of intercellular communication. The molecular composition of EV, and thereby their signaling function to target cells, is regulated by cellular activation and differentiation stimuli. EV are regarded as snapshots of cells and are, therefore, in the limelight as biomarkers for disease. Although research on EV-associated RNA has predominantly focused on microRNAs, the transcriptome of EV consists of multiple classes of small non-coding RNAs with potential gene-regulatory functions. It is not known whether environmental cues imposed on cells induce specific changes in a broad range of EV-associated RNA classes. Here, we investigated whether immune-activating or -suppressing stimuli imposed on primary dendritic cells affected the release of various small non-coding RNAs via EV. The small RNA transcriptomes of highly pure EV populations free from ribonucleoprotein particles were analyzed by RNA sequencing and RT-qPCR. Immune stimulus-specific changes were found in the miRNA, snoRNA, and Y-RNA content of EV from dendritic cells, whereas tRNA and snRNA levels were much less affected. Only part of the changes in EV-RNA content reflected changes in cellular RNA, which urges caution in interpreting EV as snapshots of cells. By comprehensive analysis of RNA obtained from highly purified EV, we demonstrate that multiple RNA classes contribute to genetic messages conveyed via EV. The identification of multiple RNA classes that display cell stimulation-dependent association with EV is the prelude to unraveling the function and biomarker potential of these EV-RNAs. (hide) EV-METRIC 100% (99th 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 Adj. k-factor 253.9 (pelleting) Protein markers EV: MHC2/ CD63/ CD9/ Galectin-3 non-EV: beta-actin Proteomics no Show all info Study aim Biomarker, Biogenesis/cargo sorting, Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells primary bone marrow dendritic cells EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) NA 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) 65 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 253.9 Density gradient Type Continuous Lowest density fraction 0.4M Highest density fraction 2.5M Sample volume (mL) 0.15 Orientation Bottom-up (sample migrates upwards) Rotor type SW 40 Ti Speed (g) 192000 Duration (min) 900-1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 1 Pelleting: duration (min) 65 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 192000 Pelleting: adjusted k-factor 144.0 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9, CD63, MHC2, Galectin-3 Not detected contaminants beta-actin Characterization: RNA analysis Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100 - 200 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type BD Influx Hardware adjustment see van der Vlist et al. 2012 Nature Protocols;and Nolte-'t Hoen 2012 Nanomedicine Calibration bead size 0.1,0.2 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV170013	1/1	Mus musculus	endothelial progenitor cells	DG (d)(U)C	Degosserie, Jonathan	2018	100%
Study summary Full title Extracellular vesicles from endothelial progenitor cells promote thyroid follicle formation. All authors Degosserie J, Heymans C, Spourquet C, Halbout M, D'Auria L, Van Der Smissen P, Vertommen D, Courtoy PJ, Tyteca D, Pierreux CE. Journal J Extracell Vesicles Abstract Organogenesis is a complex and dynamic process requiring reciprocal communication between different (show more...)Organogenesis is a complex and dynamic process requiring reciprocal communication between different cell types. In the thyroid, thyrocyte progenitors secrete the angiocrine factor, VEGFA, to recruit endothelial cells. In return, endothelial cells promote thyrocyte organisation into spherical follicular structures, which are responsible for thyroid hormone synthesis and storage. Medium conditioned by endothelial progenitor cells (EPCs) can promote follicle formation and lumen expansion (i.e. folliculogenesis) in an ex vivo culture system of thyroid lobes. Here, we postulated that endothelial cells instruct thyrocyte progenitors by producing extracellular vesicles (EVs). We found that medium conditioned by EPCs contain EVs with exosomal characteristics and that these vesicles can be incorporated into thyrocyte progenitors. By mass spectrometry, laminin peptides were abundantly identified in the EV preparations, probably co-sedimenting with EVs. Laminin-α1 silencing in EPC abrogated the folliculogenic effect of EVs. However, density gradient separation of EVs from laminins revealed that both EV-rich and laminin-rich fractions exhibited folliculogenic activity. In conclusion, we suggest that endothelial cells can produce EVs favouring thyrocyte organisation into follicles and lumen expansion, a mechanism promoted by laminin-α1. (hide) EV-METRIC 100% (99th 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 Adj. k-factor 84.53 (pelleting) / 84.53 (washing) Protein markers EV: Flotillin-1/ CD63/ CD9 non-EV: Calnexin Proteomics yes Show all info Study aim Function, Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells endothelial progenitor cells EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps 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) 90 Pelleting: rotor type Type 80 Ti Pelleting: speed (g) 150000 Pelleting: adjusted k-factor 84.53 Wash: time (min) 90 Wash: Rotor Type Type 80 Ti Wash: speed (g) 150000 Wash: adjusted k-factor 84.53 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 10% Highest density fraction 30% Sample volume (mL) 1.3 Orientation Bottom-up (sample migrates upwards) Rotor type SW 55 Ti Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 0.625 Fraction processing Centrifugation Pelleting: volume per fraction 8 Pelleting: duration (min) 90 Pelleting: rotor type Type 80 Ti Pelleting: speed (g) 150000 Pelleting: adjusted k-factor 84.53 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD9, CD63, Flotillin-1 Not detected contaminants Calnexin Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-150 EV concentration Yes Particle yield 3.60E+08 particles/million cells EM EM-type Scanning-EM Image type Close-up, Wide-field

	EV180010	2/2	Homo sapiens	Serum	DG (d)(U)C	Busatto, Sara	2018	88%
Study summary Full title Uptake Profiles of Human Serum Exosomes by Murine and Human Tumor Cells through Combined Use of Colloidal Nanoplasmonics and Flow Cytofluorimetric Analysis All authors Sara Busatto, Arianna Giacomini, Costanza Montis, Roberto Ronca, Paolo Bergese Journal Anal Chem Abstract Understanding extracellular vesicle (EV) internalization mechanisms and pathways in cells is of capi (show more...)Understanding extracellular vesicle (EV) internalization mechanisms and pathways in cells is of capital importance for both EV basic biology and clinical translation, but still presents analytical hurdles, such as undetermined purity grade and/or concentration of the EV samples and lack of standard protocols. We report an accessible, robust, and versatile method for resolving dose-dependent uptake profiles of exosomes-the nanosized (30-150 nm) subtypes of EVs of intracellular origin which are more intensively investigated for diagnostic and therapeutic applications-by cultured cells. The method is based on incubating recipient cells with consistently increasing doses of exosomes which are graded for purity and titrated by a COlorimetric NANoplasmonic (CONAN) assay followed by cell flow cytofluorimetric analysis. The proposed method allowed evaluation and comparison of the uptake of human serum exosomes by cancer cell lines of murine (TRAMP-C2) and human (LNCaP, DU145, MDA-MB-231, and A375) origin, setting a firmer footing for better characterization and understanding of exosome biology in different in vitro and (potentially) in vivo models of cancer growth. (hide) EV-METRIC 88% (99th 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 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 DG (d)(U)C Adj. k-factor 89.2 (pelleting) Protein markers EV: Alix/ CD81/ ANXA11/ CD63 non-EV: GM130/ APOA1 Proteomics no Show all info Study aim Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Serum 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 TLA-55 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 89.20 Density gradient Type Discontinuous Number of initial discontinuous layers 6 Lowest density fraction 0.15 Highest density fraction 0.6 Sample volume (mL) 0.8 Orientation Bottom-up (sample migrates upwards) Rotor type MLS-50 Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 0.4 Fraction processing Centrifugation Pelleting: volume per fraction 0.4 Pelleting: duration (min) 120 Pelleting: rotor type TLA-55 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 89.20 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Alix, CD63, CD81, ANXA11 Not detected contaminants GM130, APOA1 Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 70 EM EM-type Atomic force-EM Image type Wide-field Report size (nm) 30-150 Other particle analysis name(2) Colorimetric nanoplasmonic assay (CONAN) EV-concentration Yes Particle yield 5.30E+12

	EV180059	6/6	Gut microbiota	Stool	DG SEC	Tulkens J	2018	87%
Study summary Full title Increased levels of systemic LPS-positive bacterial extracellular vesicles in patients with intestinal barrier dysfunction. All authors Tulkens J, Vergauwen G, Van Deun J, Geeurickx E, Dhondt B, Lippens L, De Scheerder MA, Miinalainen I, Rappu P, De Geest BG, Vandecasteele K, Laukens D, Vandekerckhove L, Denys H, Vandesompele J, De Wever O, Hendrix A. Journal Gut Abstract (show more...) (hide) EV-METRIC 87% (96th 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 Stool Sample origin Control condition Focus vesicles gut bacteria-derived EV 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 SEC Protein markers EV: Proteomics/ Western blot/ LPS/ OmpA/ Limulus Amebocyte Lysate Assay non-EV: Alix/ flotilin-1/ CD9 Proteomics yes EV density (g/ml) 1.141-1.186 Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Gut microbiota Sample Type Stool Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 10 Highest density fraction 50 Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 0.667 Orientation Bottom-up Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins OmpA Not detected contaminants Alix/ flotilin-1/ CD9 Proteomics database No Detected EV-associated proteins LPS Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-300 EV concentration Yes Particle yield particles/ml start sample EM EM-type Transmission-EM Image type Wide-field

	EV170041	1/1	Homo sapiens	'SW480 Dukes' type B, colorectal adenocarcinoma;SW620, colon; derived from metastatic site: lymph node	DG	Yingkuan Shao	2018	87%
Study summary Full title Colorectal cancer-derived small extracellular vesicles establish an inflammatory premetastatic niche in liver metastasis All authors Yingkuan Shao, Ting Chen, Xi Zheng, Sheng Yang, Kailun Xu, Xuewen Chen, Fei Xu, Lantian Wang, Yanwei Shen, Tingyang Wang, Mengwen Zhang, Wangxiong Hu, Chenyang Ye, XiaoFang Yu, Jimin Shao, Shu Zheng Journal Carcinogenesis Abstract Liver metastases develop in more than half of the patients with colorectal cancer (CRC) and are asso (show more...)Liver metastases develop in more than half of the patients with colorectal cancer (CRC) and are associated with a poor prognosis. The factors influencing liver metastasis of CRC are poorly characterized, but this information is urgently needed. We have now discovered that small extracellular vesicles (sEVs; exosomes) derived from CRC can be specifically targeted to liver tissue and induce liver macrophage polarization toward an interleukin-6 (IL-6)-secreting proinflammatory phenotype. More importantly, we found that microRNA-21-5p (miR-21) was highly enriched in CRC-derived sEVs and was essential for creating a liver proinflammatory phenotype and liver metastasis of CRC. Silencing either miR-21 in CRC-sEVs or Toll-like receptor 7 (TLR7) in macrophages, to which miR-21 binds, abolished CRC-sEVs' induction of proinflammatory macrophages. Furthermore, miR-21 expression in plasma-derived sEVs was positively correlated with liver metastasis in CRC patients. Collectively, our data demonstrate a pivotal role of CRC-sEVs in promoting liver metastasis by inducing an inflammatory premetastatic niche through the miR-21-TLR7-IL-6 axis. Thus, sEVs-miR-21 represents a potential prognostic marker and therapeutic target for CRC patients with liver metastasis. (hide) EV-METRIC 87% (98th 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 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 DG Protein markers EV: Alix/ HSP70/ CD63/ CD9 non-EV: Calreticulin Proteomics no Show all info Study aim Function, Biomarker, Mechanism of uptake/transfer, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells 'SW480 Dukes' type B, colorectal adenocarcinoma / SW620, colon / derived from metastatic site: lymph node EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) NA Separation Method Density gradient Only used for validation of main results Yes Density medium 69.83 Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 0.25M Highest density fraction 1.3M Sample volume (mL) 1 Orientation Bottom-up (sample migrates upwards) Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 150 Fraction volume (mL) 2 Fraction processing Centrifugation Pelleting: volume per fraction 5 Pelleting: duration (min) 150 Pelleting: rotor type MLS-50 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 142.1 Pelleting-wash: volume per pellet (mL) 1 Pelleting-wash: duration (min) 150 Pelleting-wash: rotor type 69.83 Pelleting-wash: speed (g) MLA-130 Pelleting-wash: adjusted k-factor 69.83 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) 2-5 Western Blot Detected EV-associated proteins Alix, CD9, CD63, HSP70 Not detected contaminants Calreticulin Characterization: RNA analysis Database Yes Proteinase treatment Yes Moment of Proteinase treatment After Proteinase type Proteinase K Proteinase concentration 10 RNAse treatment Yes Moment of RNAse treatment After RNAse type RNase A RNAse concentration 10 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-160 EV concentration Yes Particle yield 1.0-2.0E11 EM EM-type Transmission-EM Image type Wide-field Report size (nm) 30-160 EV concentration Yes

	EV180083	1/2	Mus musculus	5TGM1	DG (d)(U)C ExoQuick	Faict S	2018	78%
Study summary Full title Exosomes play a role in multiple myeloma bone disease and tumor development by targeting osteoclasts and osteoblasts. All authors Faict S, Muller J, De Veirman K, De Bruyne E, Maes K, Vrancken L, Heusschen R, De Raeve H, Schots R, Vanderkerken K, Caers J, Menu E. Journal blood cancer j Abstract Progression of multiple myeloma (MM) is largely dependent on the bone marrow (BM) microenvironment w (show more...)Progression of multiple myeloma (MM) is largely dependent on the bone marrow (BM) microenvironment wherein communication through different factors including extracellular vesicles takes place. This cross-talk not only leads to drug resistance but also to the development of osteolysis. Targeting vesicle secretion could therefore simultaneously ameliorate drug response and bone disease. In this paper, we examined the effects of MM exosomes on different aspects of osteolysis using the 5TGM1 murine model. We found that 5TGM1 sEVs, or 'exosomes', not only enhanced osteoclast activity, they also blocked osteoblast differentiation and functionality in vitro. Mechanistically, we could demonstrate that transfer of DKK-1 led to a reduction in Runx2, Osterix, and Collagen 1A1 in osteoblasts. In vivo, we uncovered that 5TGM1 exosomes could induce osteolysis in a similar pattern as the MM cells themselves. Blocking exosome secretion using the sphingomyelinase inhibitor GW4869 not only increased cortical bone volume, but also it sensitized the myeloma cells to bortezomib, leading to a strong anti-tumor response when GW4869 and bortezomib were combined. Altogether, our results indicate an important role for exosomes in the BM microenvironment and suggest a novel therapeutic target for anti-myeloma therapy. (hide) EV-METRIC 78% (97th 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 ExoQuick Protein markers EV: / TSG101/ CD63/ Syntenin/ CD81 non-EV: / Calreticulin Proteomics no EV density (g/ml) 1.08 Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells 5TGM1 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 180 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 5 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 0.5 Orientation Bottom-up Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 10800 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 10 Pelleting: duration (min) 180 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD63/ TSG101/ Syntenin/ CD81 Not detected EV-associated proteins Detected contaminants Not detected contaminants Calreticulin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 114 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 100

	EV190002	1/6	Homo sapiens	MIA PaCa-2	DG (d)(U)C Filtration	Sachiko Matsumura	2018	78%
Study summary Full title Subtypes of tumour cell-derived small extracellular vesicles having differently externalized phosphatidylserine All authors Sachiko Matsumura, Tamiko Minamisawa, Kanako Suga, Hiromi Kishita, Takanori Akagi, Takanori Ichiki, Yuki Ichikawa & Kiyotaka Shiba Journal J Extracell Vesicles Abstract Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially locate (show more...)Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially located in the inner leaflet of normal cells. Tumour cells, however, expose PS at the outer leaflet of cell surfaces, thereby potentially modulating the bio-signalling of cells. Interestingly, exosomes – or, more properly, small extracellular vesicles (sEVs) – which are secreted from tumour cells, are enriched with externalized PS, have been proposed as being involved in the progression of cancers, and could be used as a marker for tumour diagnostics. However, the sEV fractions prepared from various methods are composed of different subtypes of vesicles, and knowledge about the subtypes enriched with exposed PS is still limited. Here, we differentiated sEVs from cancer cell lines by density gradient centrifugation and characterized the separated fractions by using gold-labelling of PS in atomic force microscopy, thrombin generation assay, size and zeta potential measurements, and western blot analysis. These analyses revealed a previously unreported PS+-enriched sEV subtype, which is characterized by a lower density than that of canonical exosomes (1.06 g/ml vs. 1.08 g/ml), larger size (122 nm vs. 105 nm), more negative zeta potential (−28 mV vs. −21 mV), and lower abundance of canonical exosomal markers. The identification of the PS-exposed subtype of sEVs will provide deeper insight into the role of EVs in tumour biology and enhance the development of EV-based tumour diagnosis and therapy. (hide) EV-METRIC 78% (97th 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 DG (d)(U)C Filtration Protein markers EV: TSG101/ CD63/ MFGE8/ CD81/ Alix/ HSP70/ beta-actin non-EV: Histon H2B Proteomics no EV density (g/ml) 1.05-1.1 Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MIA PaCa-2 EV-harvesting Medium Serum free medium 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 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 Density gradient Type Continuous Lowest density fraction 8% Highest density fraction 40% Total gradient volume, incl. sample (mL) 32 Sample volume (mL) 2 Orientation Bottom-up Rotor type SW 32 Ti Speed (g) 100000 Duration (min) 1020 Fraction volume (mL) 3.2 Fraction processing Centrifugation Pelleting: volume per fraction 33.2 Pelleting: duration (min) 120 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 160000 Filtration steps 0.22µm or 0.2µm EV-subtype Distinction between multiple subtypes Density Used subtypes < 1.06 g/ml Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD63/ MFGE8/ beta-actin/ HSP70/ CD81 Not detected EV-associated proteins TSG101/ Alix Not detected contaminants Histon H2B Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 136 EV concentration Yes EM EM-type Atomic force-EM Image type Wide-field Report size (nm) height: 39.1, diameter: 135.9

	EV190002	2/6	Homo sapiens	MIA PaCa-2	DG (d)(U)C Filtration	Sachiko Matsumura	2018	78%
Study summary Full title Subtypes of tumour cell-derived small extracellular vesicles having differently externalized phosphatidylserine All authors Sachiko Matsumura, Tamiko Minamisawa, Kanako Suga, Hiromi Kishita, Takanori Akagi, Takanori Ichiki, Yuki Ichikawa & Kiyotaka Shiba Journal J Extracell Vesicles Abstract Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially locate (show more...)Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially located in the inner leaflet of normal cells. Tumour cells, however, expose PS at the outer leaflet of cell surfaces, thereby potentially modulating the bio-signalling of cells. Interestingly, exosomes – or, more properly, small extracellular vesicles (sEVs) – which are secreted from tumour cells, are enriched with externalized PS, have been proposed as being involved in the progression of cancers, and could be used as a marker for tumour diagnostics. However, the sEV fractions prepared from various methods are composed of different subtypes of vesicles, and knowledge about the subtypes enriched with exposed PS is still limited. Here, we differentiated sEVs from cancer cell lines by density gradient centrifugation and characterized the separated fractions by using gold-labelling of PS in atomic force microscopy, thrombin generation assay, size and zeta potential measurements, and western blot analysis. These analyses revealed a previously unreported PS+-enriched sEV subtype, which is characterized by a lower density than that of canonical exosomes (1.06 g/ml vs. 1.08 g/ml), larger size (122 nm vs. 105 nm), more negative zeta potential (−28 mV vs. −21 mV), and lower abundance of canonical exosomal markers. The identification of the PS-exposed subtype of sEVs will provide deeper insight into the role of EVs in tumour biology and enhance the development of EV-based tumour diagnosis and therapy. (hide) EV-METRIC 78% (97th 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 DG (d)(U)C Filtration Protein markers EV: TSG101/ CD63/ MFGE8/ CD81/ Alix/ HSP70/ beta-actin non-EV: Histon H2B Proteomics no EV density (g/ml) 1.05-1.1 Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MIA PaCa-2 EV-harvesting Medium Serum free medium 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 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 Density gradient Type Continuous Lowest density fraction 8% Highest density fraction 40% Total gradient volume, incl. sample (mL) 32 Sample volume (mL) 2 Orientation Bottom-up Rotor type SW 32 Ti Speed (g) 100000 Duration (min) 1020 Fraction volume (mL) 3.2 Fraction processing Centrifugation Pelleting: volume per fraction 33.2 Pelleting: duration (min) 120 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 160000 Filtration steps 0.22µm or 0.2µm EV-subtype Distinction between multiple subtypes Density Used subtypes ca. 1.1 g/ml Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins Alix/ CD63/ MFGE8/ beta-actin/ TSG101/ HSP70/ CD81 Not detected contaminants Histon H2B Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 124 EV concentration Yes EM EM-type Atomic force-EM Image type Wide-field Report size (nm) height: 30.1, diameter: 96.4

	EV190002	3/6	Homo sapiens	MIA PaCa-2	DG (d)(U)C Filtration	Sachiko Matsumura	2018	78%
Study summary Full title Subtypes of tumour cell-derived small extracellular vesicles having differently externalized phosphatidylserine All authors Sachiko Matsumura, Tamiko Minamisawa, Kanako Suga, Hiromi Kishita, Takanori Akagi, Takanori Ichiki, Yuki Ichikawa & Kiyotaka Shiba Journal J Extracell Vesicles Abstract Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially locate (show more...)Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially located in the inner leaflet of normal cells. Tumour cells, however, expose PS at the outer leaflet of cell surfaces, thereby potentially modulating the bio-signalling of cells. Interestingly, exosomes – or, more properly, small extracellular vesicles (sEVs) – which are secreted from tumour cells, are enriched with externalized PS, have been proposed as being involved in the progression of cancers, and could be used as a marker for tumour diagnostics. However, the sEV fractions prepared from various methods are composed of different subtypes of vesicles, and knowledge about the subtypes enriched with exposed PS is still limited. Here, we differentiated sEVs from cancer cell lines by density gradient centrifugation and characterized the separated fractions by using gold-labelling of PS in atomic force microscopy, thrombin generation assay, size and zeta potential measurements, and western blot analysis. These analyses revealed a previously unreported PS+-enriched sEV subtype, which is characterized by a lower density than that of canonical exosomes (1.06 g/ml vs. 1.08 g/ml), larger size (122 nm vs. 105 nm), more negative zeta potential (−28 mV vs. −21 mV), and lower abundance of canonical exosomal markers. The identification of the PS-exposed subtype of sEVs will provide deeper insight into the role of EVs in tumour biology and enhance the development of EV-based tumour diagnosis and therapy. (hide) EV-METRIC 78% (97th 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 DG (d)(U)C Filtration Protein markers EV: TSG101/ Src/ CD63/ MFGE8/ CD81/ Alix/ HSP70/ beta-actin/ CD9 non-EV: Argonaute2/ Histon H2B Proteomics no EV density (g/ml) 1.05-1.1 Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MIA PaCa-2 EV-harvesting Medium Serum free medium Cell viability (%) 95 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 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 Density gradient Type Continuous Lowest density fraction 8% Highest density fraction 40% Total gradient volume, incl. sample (mL) 32 Sample volume (mL) 2 Orientation Bottom-up Rotor type SW 32 Ti Speed (g) 100000 Duration (min) 1020 Fraction volume (mL) 3.2 Fraction processing Centrifugation Pelleting: volume per fraction 33.2 Pelleting: duration (min) 120 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 160000 Filtration steps 0.22µm or 0.2µm EV-subtype Distinction between multiple subtypes Density Used subtypes < 1.06 g/ml Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD9/ CD63/ HSP70/ MFGE8/ Src/ beta-actin/ CD81 Not detected EV-associated proteins TSG101/ Alix Not detected contaminants Histon H2B/ Argonaute2 Detected EV-associated proteins CD63 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 148 EV concentration Yes EM EM-type Atomic force-EM Image type Wide-field Report size (nm) height: 50.5, length: 190.0

	EV190002	4/6	Homo sapiens	MIA PaCa-2	DG (d)(U)C Filtration	Sachiko Matsumura	2018	78%
Study summary Full title Subtypes of tumour cell-derived small extracellular vesicles having differently externalized phosphatidylserine All authors Sachiko Matsumura, Tamiko Minamisawa, Kanako Suga, Hiromi Kishita, Takanori Akagi, Takanori Ichiki, Yuki Ichikawa & Kiyotaka Shiba Journal J Extracell Vesicles Abstract Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially locate (show more...)Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially located in the inner leaflet of normal cells. Tumour cells, however, expose PS at the outer leaflet of cell surfaces, thereby potentially modulating the bio-signalling of cells. Interestingly, exosomes – or, more properly, small extracellular vesicles (sEVs) – which are secreted from tumour cells, are enriched with externalized PS, have been proposed as being involved in the progression of cancers, and could be used as a marker for tumour diagnostics. However, the sEV fractions prepared from various methods are composed of different subtypes of vesicles, and knowledge about the subtypes enriched with exposed PS is still limited. Here, we differentiated sEVs from cancer cell lines by density gradient centrifugation and characterized the separated fractions by using gold-labelling of PS in atomic force microscopy, thrombin generation assay, size and zeta potential measurements, and western blot analysis. These analyses revealed a previously unreported PS+-enriched sEV subtype, which is characterized by a lower density than that of canonical exosomes (1.06 g/ml vs. 1.08 g/ml), larger size (122 nm vs. 105 nm), more negative zeta potential (−28 mV vs. −21 mV), and lower abundance of canonical exosomal markers. The identification of the PS-exposed subtype of sEVs will provide deeper insight into the role of EVs in tumour biology and enhance the development of EV-based tumour diagnosis and therapy. (hide) EV-METRIC 78% (97th 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 DG (d)(U)C Filtration Protein markers EV: TSG101/ Src/ CD63/ MFGE8/ CD81/ Alix/ HSP70/ beta-actin/ CD9 non-EV: Argonaute2/ Histon H2B Proteomics no EV density (g/ml) 1.05-1.1 Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MIA PaCa-2 EV-harvesting Medium Serum free medium Cell viability (%) 95 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 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 Density gradient Type Continuous Lowest density fraction 8% Highest density fraction 40% Total gradient volume, incl. sample (mL) 32 Sample volume (mL) 2 Orientation Bottom-up Rotor type SW 32 Ti Speed (g) 100000 Duration (min) 1020 Fraction volume (mL) 3.2 Fraction processing Centrifugation Pelleting: volume per fraction 33.2 Pelleting: duration (min) 120 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 160000 Filtration steps 0.22µm or 0.2µm EV-subtype Distinction between multiple subtypes Density Used subtypes ca. 1.1 g/ml Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins Alix/ MFGE8/ Src/ beta-actin/ CD9/ CD63/ TSG101/ HSP70/ CD81 Not detected contaminants Histon H2B/ Argonaute2 Detected EV-associated proteins CD9/ CD63 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 127 EV concentration Yes EM EM-type Atomic force-EM Image type Wide-field Report size (nm) height 32.1, length: 120.0

	EV180026	5/7	Homo sapiens	Serum	(d)(U)C	Wenzhe Li	2018	77%
Study summary Full title Noninvasive Diagnosis and Molecular Phenotyping of Breast Cancer through Microbead‐Assisted Flow Cytometry Detection of Tumor‐Derived Extracellular Vesicles All authors Wenzhe Li, Bin Shao, Changliang Liu, Huayi Wang, Wangshu Zheng, Weiyao Kong, Xiaoran Liu, Guobin Xu, Chen Wang, Huiping Li, Ling Zhu, Yanlian Yang Journal Small methods Abstract Blood‐based detection and molecular phenotyping are highly desired for the early diagnosis and dyn (show more...)Blood‐based detection and molecular phenotyping are highly desired for the early diagnosis and dynamic monitoring of cancer. Extracellular vesicles (EVs) carry molecular information from the cells of origin and are biomarkers of cancer. However, the detection and molecular analysis of EVs has been challenging due to their nanoscaled size. Here, an assessment of the detection and molecular phenotyping of serum EVs based on microbead‐assisted flow cytometry is established. The clinical utility of this method is validated in the diagnosis and human epidermal growth factor receptor 2 (HER2) phenotyping of breast cancer. Good correlation between the status of epithelial cell adhesion molecule (EpCAM) and HER2 expression in EVs and in the cells of origin is found. Both EpCAM+ and HER2+ EVs are demonstrated to be effective diagnostic markers of breast cancer with high sensitivity and specificity. EV‐based HER2 phenotyping is consistent with tissue‐based HER2 phenotyping by immunohistochemistry and can be used as a surrogate for the invasive tissue assessments. The microbead‐assisted flow cytometry assessment of EVs enables rapid and noninvasive detection and molecular phenotyping of cancer and would help to personalized treatment and cancer survival. (hide) EV-METRIC 77% (99th 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 (d)(U)C Adj. k-factor 120.7 (pelleting) / 120.7 (washing) Protein markers EV: CD81/ Flotillin-1/ CD63/ HER2/ EpCAM non-EV: Calnexin/ Albumin Proteomics no Show all info Study aim Function, Biomarker, New methodological development Sample Species Homo sapiens Sample Type Serum 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) 1200 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 130000 Pelleting: adjusted k-factor 120.7 Wash: time (min) 120 Wash: Rotor Type Type 70 Ti Wash: speed (g) 130000 Wash: adjusted k-factor 120.7 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) 30-120 Western Blot Detected EV-associated proteins CD63, CD81, Flotillin-1 Not detected contaminants Albumin, Calnexin Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 110.2 ± 12.95 nm NTA Report type Size range/distribution Reported size (nm) 124.4 ± 53.3 nm EV concentration Yes Particle yield 3290000000000 EM EM-type Transmission-EM/ Immune-EM EM protein EpCAM,HER2 Image type Close-up, Wide-field EV concentration Yes

	EV170020	1/2	Trypanosoma cruzi	Trypomastigote CL-Brener, Trypomastigote Sylvio, Trypomastigote Y, Trypomastigote RA	DG (d)(U)C Filtration	Caeiro, Lucas	2018	77%
Study summary Full title The protein family TcTASV-C is a novel Trypanosoma cruzi virulence factor secreted in extracellular vesicles by trypomastigotes and highly expressed in bloodstream forms. All authors Caeiro LD, Alba-Soto CD, Rizzi M, Solana ME, Rodriguez G, Chidichimo AM, Rodriguez ME, Sánchez DO, Levy GV, Tekiel V. Journal PLoS Negl Trop Dis Abstract TcTASV-C is a protein family of about 15 members that is expressed only in the trypomastigote stage (show more...)TcTASV-C is a protein family of about 15 members that is expressed only in the trypomastigote stage of Trypanosoma cruzi. We have previously shown that TcTASV-C is located at the parasite surface and secreted to the medium. Here we report that the expression of different TcTASV-C genes occurs simultaneously at the trypomastigote stage and while some secreted and parasite-associated products are found in both fractions, others are different. Secreted TcTASV-C are mainly shedded through trypomastigote extracellular vesicles, of which they are an abundant constituent, despite its scarce expression on culture-derived trypomastigotes. In contrast, TcTASV-C is highly expressed in bloodstream trypomastigotes; its upregulation in bloodstream parasites was observed in different T. cruzi strains and was specific for TcTASV-C, suggesting that some host-molecules trigger TcTASV-C expression. TcTASV-C is also strongly secreted by bloodstream parasites. A DNA prime-protein boost immunization scheme with TcTASV-C was only partially effective to control the infection in mice challenged with a highly virulent T. cruzi strain. Vaccination triggered a strong humoral response that delayed the appearance of bloodstream trypomastigotes at the early phase of the infection. Linear epitopes recognized by vaccinated mice were mapped within the TcTASV-C family motif, suggesting that blockade of secreted TcTASV-C impacts on the settlement of infection. Furthermore, although experimental and naturally T. cruzi-infected hosts did not react with antigens from extracellular vesicles, vaccinated and challenged mice recognized not only TcTASV-C but also other vesicle-antigens. We hypothesize that TcTASV-C is involved in the establishment of the initial T. cruzi infection in the mammalian host. Altogether, these results point towards TcTASV-C as a novel secreted virulence factor of T. cruzi trypomastigotes. (hide) EV-METRIC 77% (97th 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 Filtration Adj. k-factor 156.9 (pelleting) / 156.9 (washing) Protein markers EV: TcTASV-C/ HSP70/ HSP70,TcTASV-C non-EV: TcSR-62 Proteomics yes Show all info Study aim Biomarker, Identification of content (omics approaches) Sample Species Trypanosoma cruzi Sample Type Cell culture supernatant EV-producing cells Trypomastigote CL-Brener, Trypomastigote Sylvio, Trypomastigote Y, Trypomastigote RA EV-harvesting Medium Serum free medium Cell viability (%) NA 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 Type 70 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 156.9 Wash: time (min) 120 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Wash: adjusted k-factor 156.9 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 0.05 Highest density fraction 0.4 Sample volume (mL) 2 Orientation Top-down (sample migrates downwards) Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing None Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) 0.1 Western Blot Detected EV-associated proteins HSP70,TcTASV-C Not detected contaminants TcSR-62 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 50-150

	EV170020	2/2	Trypanosoma cruzi	Trypomastigote CL-Brener, Trypomastigote Sylvio, Trypomastigote Y, Trypomastigote RA	DG (d)(U)C Filtration	Caeiro, Lucas	2018	77%
Study summary Full title The protein family TcTASV-C is a novel Trypanosoma cruzi virulence factor secreted in extracellular vesicles by trypomastigotes and highly expressed in bloodstream forms. All authors Caeiro LD, Alba-Soto CD, Rizzi M, Solana ME, Rodriguez G, Chidichimo AM, Rodriguez ME, Sánchez DO, Levy GV, Tekiel V. Journal PLoS Negl Trop Dis Abstract TcTASV-C is a protein family of about 15 members that is expressed only in the trypomastigote stage (show more...)TcTASV-C is a protein family of about 15 members that is expressed only in the trypomastigote stage of Trypanosoma cruzi. We have previously shown that TcTASV-C is located at the parasite surface and secreted to the medium. Here we report that the expression of different TcTASV-C genes occurs simultaneously at the trypomastigote stage and while some secreted and parasite-associated products are found in both fractions, others are different. Secreted TcTASV-C are mainly shedded through trypomastigote extracellular vesicles, of which they are an abundant constituent, despite its scarce expression on culture-derived trypomastigotes. In contrast, TcTASV-C is highly expressed in bloodstream trypomastigotes; its upregulation in bloodstream parasites was observed in different T. cruzi strains and was specific for TcTASV-C, suggesting that some host-molecules trigger TcTASV-C expression. TcTASV-C is also strongly secreted by bloodstream parasites. A DNA prime-protein boost immunization scheme with TcTASV-C was only partially effective to control the infection in mice challenged with a highly virulent T. cruzi strain. Vaccination triggered a strong humoral response that delayed the appearance of bloodstream trypomastigotes at the early phase of the infection. Linear epitopes recognized by vaccinated mice were mapped within the TcTASV-C family motif, suggesting that blockade of secreted TcTASV-C impacts on the settlement of infection. Furthermore, although experimental and naturally T. cruzi-infected hosts did not react with antigens from extracellular vesicles, vaccinated and challenged mice recognized not only TcTASV-C but also other vesicle-antigens. We hypothesize that TcTASV-C is involved in the establishment of the initial T. cruzi infection in the mammalian host. Altogether, these results point towards TcTASV-C as a novel secreted virulence factor of T. cruzi trypomastigotes. (hide) EV-METRIC 77% (97th 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 Filtration Adj. k-factor 156.9 (pelleting) / 156.9 (washing) Protein markers EV: TcTASV-C/ HSP70/ HSP70,TcTASV-C non-EV: TcSR-62 Proteomics yes Show all info Study aim Biomarker, Identification of content (omics approaches) Sample Species Trypanosoma cruzi Sample Type Cell culture supernatant EV-producing cells Trypomastigote CL-Brener, Trypomastigote Sylvio, Trypomastigote Y, Trypomastigote RA EV-harvesting Medium Serum free medium Cell viability (%) NA 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) 1080 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 156.9 Wash: time (min) 1080 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Wash: adjusted k-factor 156.9 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 0.05 Highest density fraction 0.4 Sample volume (mL) 2 Orientation Top-down (sample migrates downwards) Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing None Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) 0.15 Western Blot Detected EV-associated proteins HSP70,TcTASV-C Not detected contaminants TcSR-62 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 50-150

	EV180059	1/6	Gram-negative bacteria	Escherichia coli Nissle 1917	DG SEC	Tulkens J	2018	75%
Study summary Full title Increased levels of systemic LPS-positive bacterial extracellular vesicles in patients with intestinal barrier dysfunction. All authors Tulkens J, Vergauwen G, Van Deun J, Geeurickx E, Dhondt B, Lippens L, De Scheerder MA, Miinalainen I, Rappu P, De Geest BG, Vandecasteele K, Laukens D, Vandekerckhove L, Denys H, Vandesompele J, De Wever O, Hendrix A. Journal Gut Abstract (show more...) (hide) EV-METRIC 75% (96th 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 Escherichia coli 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 SEC Protein markers EV: Limulus Amebocyte Lysate assay/ LPS/ OmpA/ Western blot non-EV: Proteomics no EV density (g/ml) 1.141-1.186 Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Gram-negative bacteria Sample Type Cell culture supernatant EV-producing cells Escherichia coli Nissle 1917 EV-harvesting Medium Serum free medium Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 10 Highest density fraction 50 Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 0.667 Orientation Bottom-up Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins OmpA/ LPS Proteomics database No Detected EV-associated proteins LPS Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-300 EV concentration Yes EM EM-type Immuno-EM EM protein LPS Image type Close-up, Wide-field

	EV180053	1/1	Homo sapiens	MKN74, MKN45	(d)(U)C Filtration	Rocha, Sara	2018	75%
Study summary Full title 3D Cellular Architecture Affects MicroRNA and Protein Cargo of Extracellular Vesicles All authors Sara Rocha, Joana Carvalho, Patrícia Oliveira, Maren Voglstaetter, Domitille Schvartz, Andreas R. Thomsen, Nadia Walter, Richa Khanduri, Jean‐Charles Sanchez, Andreas Keller, Carla Oliveira, Irina Nazarenko Journal Advanced Science Abstract The success of malignant tumors is conditioned by the intercellular communication between tumor cell (show more...)The success of malignant tumors is conditioned by the intercellular communication between tumor cells and their microenvironment, with extracellular vesicles (EVs) acting as main mediators. While the value of 3D conditions to study tumor cells is well established, the impact of cellular architecture on EV content and function is not investigated yet. Here, a recently developed 3D cell culture microwell array is adapted for EV production and a comprehensive comparative analysis of biochemical features, RNA and proteomic profiles of EVs secreted by 2D vs 3D cultures of gastric cancer cells, is performed. 3D cultures are significantly more efficient in producing EVs than 2D cultures. Global upregulation of microRNAs and downregulation of proteins in 3D are observed, indicating their dynamic coregulation in response to cellular architecture, with the ADP‐ribosylation factor 6 signaling pathway significantly downregulated in 3D EVs. The data strengthen the biological relevance of cellular architecture for production and cargo of EVs. (hide) EV-METRIC 75% (96th 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 Filtration Protein markers EV: integrin-alpha6/ catenin-beta1/ integrin-alpha2/ integrin-alpha3/ tubulin/ Flotillin-1/ E-cadherin/ GAPDH/ CD81/ integrin-beta1/ catenin-delta1/ CD9 non-EV: CytochromeC Proteomics yes Show all info Study aim New methodological development, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MKN74, MKN45 EV-harvesting Medium Serum free medium Cell viability (%) NA 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 No Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) 0.2 Western Blot Detected EV-associated proteins integrin-alpha2, integrin-alpha3, integrin-alpha6, integrin-beta1, E-cadherin, catenin-beta1, catenin-delta1, GAPDH, tubulin Proteomics database Yes Characterization: RNA analysis Database Yes Proteinase treatment Yes Moment of Proteinase treatment After Proteinase type Proteinase K Proteinase concentration 5 RNAse treatment Yes Moment of RNAse treatment After RNAse type RNase A RNAse concentration 1 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 110 EV concentration Yes Particle yield 1.50E+09 particles/million cells Particle analysis: flow cytometry Flow cytometer type Amnis ImageStream Hardware adjustment Calibration bead size 200 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 110

	EV180033	1/3	Homo sapiens	Lipoaspirate	(d)(U)C Tangential flow filtration	Busatto S	2018	75%
Study summary Full title Tangential Flow Filtration for Highly Efficient Concentration of Extracellular Vesicles from Large Volumes of Fluid. All authors Busatto S, Vilanilam G, Ticer T, Lin WL, Dickson DW, Shapiro S, Bergese P, Wolfram J1. Journal Cells Abstract Concentration of extracellular vesicles (EVs) from biological fluids in a scalable and reproducible (show more...)Concentration of extracellular vesicles (EVs) from biological fluids in a scalable and reproducible manner represents a major challenge. This study reports the use of tangential flow filtration (TFF) for the highly efficient isolation of EVs from large volumes of samples. When compared to ultracentrifugation (UC), which is the most widely used method to concentrate EVs, TFF is a more efficient, scalable, and gentler method. Comparative assessment of TFF and UC of conditioned cell culture media revealed that the former concentrates EVs of comparable physicochemical characteristics, but with higher yield, less single macromolecules and aggregates (<15 nm in size), and improved batch-to-batch consistency in half the processing time (1 h). The TFF protocol was then successfully implemented on fluids derived from patient lipoaspirate. EVs from adipose tissue are of high clinical relevance, as they are expected to mirror the regenerative properties of the parent cells. (hide) EV-METRIC 75% (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 Lipoaspirate 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 Tangential flow filtration Protein markers EV: CD81/ CD63/ CD9 non-EV: Calnexin Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Lipoaspirate Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Pelleting performed No Other Name other separation method Tangential flow filtration Characterization: Protein analysis Protein Concentration Method Not determined Western Blot 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) 170-185 EV concentration Yes Particle yield 20000000000 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV180008	1/4	Homo sapiens	HCCLM3	DG (d)(U)C	Hao, Liu	2018	75%
Study summary Full title Tumor-derived exosomes promote tumor self-seeding in hepatocellular carcinoma by transferring miRNA-25-5p to enhance cell motility All authors Hao Liu, Wei Chen, Xiao Zhi, En-Jiang Chen, Tao Wei, Jian Zhang, Jian Shen, Li-Qiang Hu, Bin Zhao, Xin-Hua Feng, Xue-Li Bai, Ting-Bo Liang Journal Oncogene Abstract Tumor self-seeding occurs when circulating malignant cells reinfiltrate the original tumor. The proc (show more...)Tumor self-seeding occurs when circulating malignant cells reinfiltrate the original tumor. The process may breed more aggressive tumor cells, which may contribute to cancer progression. In this study, we observed tumor self-seeding in mouse xenograft models of hepatocellular carcinoma (HCC) for the first time. We confirmed that circulating tumor cell uptake of tumor-derived exosomes, which are increasingly recognized as key instigators of cancer progression by facilitating cell-cell communication, promoted tumor self-seeding by enhancing the invasive and migration capability of recipient HCC cells. Horizontal transfer of exosomal microRNA-25-5p to anoikis-resistant HCC cells significantly enhanced their migratory and invasive abilities, whereas inhibiting microRNA-25-5p alleviated these effects. Our experiments delineate an exosome-based novel pathway employed by functional microRNA from the original tumor cells that can influence the biological fate of circulating tumor cells. (hide) EV-METRIC 75% (96th 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 DG (d)(U)C Protein markers EV: CD81/ TSG101/ CD63 non-EV: Grp94/ Argonaute-2 Proteomics no Show all info Study aim Function, Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HCCLM3 EV-harvesting Medium EV-depleted serum 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 No Density gradient Only used for validation of main results Yes Density medium 255.8 Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 0.05 Highest density fraction 0.4 Sample volume (mL) 0.5 Orientation Top-down (sample migrates downwards) Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 12 Pelleting: duration (min) 180 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 255.8 Pelleting-wash: volume per pellet (mL) 12 Pelleting-wash: duration (min) 180 Pelleting-wash: rotor type 255.8 Pelleting-wash: speed (g) SW 41 Ti Pelleting-wash: adjusted k-factor 255.8 Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) 0.38 Western Blot Detected EV-associated proteins CD63, CD81, TSG101 Not detected contaminants Grp94 Characterization: RNA analysis Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Wide-field

	EV180008	4/4	Homo sapiens	HuH7	DG (d)(U)C	Hao, Liu	2018	75%
Study summary Full title Tumor-derived exosomes promote tumor self-seeding in hepatocellular carcinoma by transferring miRNA-25-5p to enhance cell motility All authors Hao Liu, Wei Chen, Xiao Zhi, En-Jiang Chen, Tao Wei, Jian Zhang, Jian Shen, Li-Qiang Hu, Bin Zhao, Xin-Hua Feng, Xue-Li Bai, Ting-Bo Liang Journal Oncogene Abstract Tumor self-seeding occurs when circulating malignant cells reinfiltrate the original tumor. The proc (show more...)Tumor self-seeding occurs when circulating malignant cells reinfiltrate the original tumor. The process may breed more aggressive tumor cells, which may contribute to cancer progression. In this study, we observed tumor self-seeding in mouse xenograft models of hepatocellular carcinoma (HCC) for the first time. We confirmed that circulating tumor cell uptake of tumor-derived exosomes, which are increasingly recognized as key instigators of cancer progression by facilitating cell-cell communication, promoted tumor self-seeding by enhancing the invasive and migration capability of recipient HCC cells. Horizontal transfer of exosomal microRNA-25-5p to anoikis-resistant HCC cells significantly enhanced their migratory and invasive abilities, whereas inhibiting microRNA-25-5p alleviated these effects. Our experiments delineate an exosome-based novel pathway employed by functional microRNA from the original tumor cells that can influence the biological fate of circulating tumor cells. (hide) EV-METRIC 75% (96th 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 DG (d)(U)C Protein markers EV: CD81/ TSG101/ CD63 non-EV: Grp94/ Argonaute-2 Proteomics no Show all info Study aim Function, Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HuH7 EV-harvesting Medium EV-depleted serum 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 No Density gradient Only used for validation of main results Yes Density medium 255.8 Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 0.05 Highest density fraction 0.4 Sample volume (mL) 0.5 Orientation Top-down (sample migrates downwards) Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 12 Pelleting: duration (min) 180 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 255.8 Pelleting-wash: volume per pellet (mL) 12 Pelleting-wash: duration (min) 180 Pelleting-wash: rotor type 255.8 Pelleting-wash: speed (g) SW 41 Ti Pelleting-wash: adjusted k-factor 255.8 Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) 0.12 Western Blot Detected EV-associated proteins CD63, CD81, TSG101 Not detected contaminants Grp94 Characterization: RNA analysis Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Wide-field

	YJ7428AP	1/2	Homo sapiens	Unconditioned serum-containing medium	(d)(U)C Filtration qEV UF	Sjöqvist S	2018	75%
Study summary Full title Exosomes derived from clinical-grade oral mucosal epithelial cell sheets promote wound healing. All authors Sjöqvist S, Ishikawa T, Shimura D, Kasai Y, Imafuku A, Bou-Ghannam S, Iwata T, Kanai N Journal J Extracell Vesicles Abstract The oral mucosa exhibits unique regenerative properties, sometimes referred to as foetal-like wound (show more...)The oral mucosa exhibits unique regenerative properties, sometimes referred to as foetal-like wound healing. Researchers from our institute have used sheets of oral mucosa epithelial cells (OMECs) for regenerative medicine applications including cornea replacement and oesophageal epithelial regeneration for stricture prevention. Here, we have isolated exosomes from clinical-grade production of OMEC sheets from healthy human donors (n = 8), aiming to evaluate the clinical potential of the exosomes to stimulate epithelial regeneration and to improve understanding of the mode-of-action of the cells. Exosomes were isolated from conditioned (cExo) and non-conditioned (ncExo) media. Characterization was performed using Western blot for common exosomal-markers: CD9 and flotillin were positive while annexin V, EpCam and contaminating marker GRP94 were negative. Nanoparticle tracking analysis revealed a diameter of ~120 nm and transmission electron microscopy showed a corresponding size and spherical appearance. Human skin fibroblasts exposed to exosomes showed dose-dependent reduction of proliferation and a considerable increase of growth factor gene expression (HGF, VEGFA, FGF2 and CTGF). The results were similar for both groups, but with a trend towards a larger effect from cExo. To study adhesion, fluorescently labelled exosomes were topically applied to pig oesophageal wound-beds ex vivo and subsequently washed. Positive signal could be detected after as little as 1 min of adhesion, but increased adhesion time produced a stronger signal. Next, labelled exosomes were added to full-thickness skin wounds in rats and signal was detected up to 5 days after application. cExo significantly reduced the wound size at days 6 and 17. In conclusion, exosomes from OMEC sheets showed pro-regenerative effects on skin wound healing. This is the first time that the healing capacity of the oral mucosa is studied from an exosome perspective. These findings might lead to a combinational therapy of cell sheets and exosomes for future patients with early oesophageal cancer. (hide) EV-METRIC 75% (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 Unconditioned serum-containing medium 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 (d)(U)C Filtration qEV UF Protein markers EV: Flotillin-1/ CD9 non-EV: GRP94 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Unconditioned serum-containing medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10, 100 Membrane type Regenerated cellulose Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) 1.905 Western Blot Detected EV-associated proteins CD9, Flotillin-1 Not detected contaminants GRP94 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 119.2±4.5 EV concentration Yes Particle yield 2.035E09 ± 3.735E08 EM EM-type Transmission-EM Image type Close-up, Wide-field

	YJ7428AP	2/2	Homo sapiens	primary mucosal epithelial cells	(d)(U)C Filtration qEV UF	Sjöqvist S	2018	75%
Study summary Full title Exosomes derived from clinical-grade oral mucosal epithelial cell sheets promote wound healing. All authors Sjöqvist S, Ishikawa T, Shimura D, Kasai Y, Imafuku A, Bou-Ghannam S, Iwata T, Kanai N Journal J Extracell Vesicles Abstract The oral mucosa exhibits unique regenerative properties, sometimes referred to as foetal-like wound (show more...)The oral mucosa exhibits unique regenerative properties, sometimes referred to as foetal-like wound healing. Researchers from our institute have used sheets of oral mucosa epithelial cells (OMECs) for regenerative medicine applications including cornea replacement and oesophageal epithelial regeneration for stricture prevention. Here, we have isolated exosomes from clinical-grade production of OMEC sheets from healthy human donors (n = 8), aiming to evaluate the clinical potential of the exosomes to stimulate epithelial regeneration and to improve understanding of the mode-of-action of the cells. Exosomes were isolated from conditioned (cExo) and non-conditioned (ncExo) media. Characterization was performed using Western blot for common exosomal-markers: CD9 and flotillin were positive while annexin V, EpCam and contaminating marker GRP94 were negative. Nanoparticle tracking analysis revealed a diameter of ~120 nm and transmission electron microscopy showed a corresponding size and spherical appearance. Human skin fibroblasts exposed to exosomes showed dose-dependent reduction of proliferation and a considerable increase of growth factor gene expression (HGF, VEGFA, FGF2 and CTGF). The results were similar for both groups, but with a trend towards a larger effect from cExo. To study adhesion, fluorescently labelled exosomes were topically applied to pig oesophageal wound-beds ex vivo and subsequently washed. Positive signal could be detected after as little as 1 min of adhesion, but increased adhesion time produced a stronger signal. Next, labelled exosomes were added to full-thickness skin wounds in rats and signal was detected up to 5 days after application. cExo significantly reduced the wound size at days 6 and 17. In conclusion, exosomes from OMEC sheets showed pro-regenerative effects on skin wound healing. This is the first time that the healing capacity of the oral mucosa is studied from an exosome perspective. These findings might lead to a combinational therapy of cell sheets and exosomes for future patients with early oesophageal cancer. (hide) EV-METRIC 75% (96th 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 (d)(U)C Filtration qEV UF Protein markers EV: Flotillin-1/ CD9 non-EV: GRP94 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary mucosal epithelial cells EV-harvesting Medium Serum-containing medium Cell viability (%) NA Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 10, 100 Membrane type Regenerated cellulose Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) 0.97 Western Blot Detected EV-associated proteins CD9, Flotillin-1 Not detected contaminants GRP94 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 124.8±4.1 EV concentration Yes Particle yield 1.027E09 ± 9.279E08 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV170014	1/4	Homo sapiens	HEK293	(d)(U)C Filtration SEC Tangential flow filtration (50 nm pore size)	Dionysios C Watson	2018	75%
Study summary Full title Scalable, cGMP-compatible purification of extracellular vesicles carrying bioactive human heterodimeric IL-15/lactadherin complexes All authors Dionysios C Watson, Bryant C Yung, Cristina Bergamaschi, Bhabadeb Chowdhury, Jenifer Bear, Dimitris Stellas, Aizea Morales-Kastresana, Jennifer C Jones, Barbara K Felber, Xiaoyuan Chen, George N Pavlakis Journal J Extracell Vesicles Abstract The development of extracellular vesicles (EV) for therapeutic applications is contingent upon the e (show more...)The development of extracellular vesicles (EV) for therapeutic applications is contingent upon the establishment of reproducible, scalable, and high-throughput methods for the production and purification of clinical grade EV. Methods including ultracentrifugation (U/C), ultrafiltration, immunoprecipitation, and size-exclusion chromatography (SEC) have been employed to isolate EV, each facing limitations such as efficiency, particle purity, lengthy processing time, and/or sample volume. We developed a cGMP-compatible method for the scalable production, concentration, and isolation of EV through a strategy involving bioreactor culture, tangential flow filtration (TFF), and preparative SEC. We applied this purification method for the isolation of engineered EV carrying multiple complexes of a novel human immunostimulatory cytokine-fusion protein, heterodimeric IL-15 (hetIL-15)/lactadherin. HEK293 cells stably expressing the fusion cytokine were cultured in a hollow-fibre bioreactor. Conditioned medium was collected and EV were isolated comparing three procedures: U/C, SEC, or TFF + SEC. SEC demonstrated comparable particle recovery, size distribution, and hetIL-15 density as U/C purification. Relative to U/C, SEC preparations achieved a 100-fold reduction in ferritin concentration, a major protein-complex contaminant. Comparative proteomics suggested that SEC additionally decreased the abundance of cytoplasmic proteins not associated with EV. Combination of TFF and SEC allowed for bulk processing of large starting volumes, and resulted in bioactive EV, without significant loss in particle yield or changes in size, morphology, and hetIL-15/lactadherin density. Taken together, the combination of bioreactor culture with TFF + SEC comprises a scalable, efficient method for the production of highly purified, bioactive EV carrying hetIL-15/lactadherin, which may be useful in targeted cancer immunotherapy approaches. (hide) EV-METRIC 75% (96th 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 human hetIL-15 stably transfected,human hetIL-15/lactadherin stably transfected 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 SEC Tangential flow filtration (50 nm pore size) Protein markers EV: IL-15/ hetIL-15/lactadherin/ CD63 non-EV: calnexin/ ferritin Proteomics yes Show all info Study aim New methodological development, Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK293 EV-harvesting Medium Serum free medium 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 Size-exclusion chromatography Total column volume (mL) 120 Sample volume/column (mL) 5 Resin type Superdex 200 Other Name other separation method Tangential flow filtration (50 nm pore size) Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Detected EV-associated proteins CD63 Not detected contaminants calnexin Flow cytometry specific beads Selected surface protein(s) CD63 Proteomics database Yes Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-200 EV concentration Yes Particle yield 480000000000 EM EM-type Immune-EM EM protein IL-15 Image type Close-up, Wide-field

	EV230894	1/1	Staphylococcus aureus	Newbould 305 (ATCC 29740)	(d)(U)C DG Filtration UF	Tartaglia NR	2018	71%
Study summary Full title Extracellular Vesicles Elicit an Immunostimulatory Response on the Murine Mammary Gland. All authors Tartaglia NR, Breyne K, Meyer E, Cauty C, Jardin J, Chrétien D, Dupont A, Demeyere K, Berkova N, Azevedo V, Guédon E, Le Loir Y Journal Front Cell Infect Microbiol Abstract is a major pathogen responsible for bovine mastitis, the most common and costly disease affecting da (show more...)is a major pathogen responsible for bovine mastitis, the most common and costly disease affecting dairy cattle. naturally releases extracellular vesicles (EVs) during its growth. EVs play an important role in the bacteria-bacteria and bacteria-host interactions and are notably considered as nanocarriers that deliver virulence factors to the host tissues. Whether EVs play a role in a mastitis context is still unknown. In this work, we showed that Newbould 305 (N305), a bovine mastitis isolate, has the ability to generate EVs with a designated protein content. Purified N305-secreted EVs were not cytotoxic when tested on MAC-T and PS, two bovine mammary epithelial cell lines. However, they induced the gene expression of inflammatory cytokines at levels similar to those induced by live N305. The immune response to purified N305-secreted EVs was tested in a mouse model for bovine mastitis and their immunogenic effect was compared to that of live N305, heat-killed N305 and to lipoteichoic acid (LTA). Clinical and histopathological signs were evaluated and pro-inflammatory and chemotactic cytokine levels were measured in the mammary gland 24 h post-inoculation. Live induced a significantly stronger inflammatory response than that of any other condition tested. Nevertheless, N305-secreted EVs induced a dose-dependent neutrophil recruitment and the production of a selected set of pro-inflammatory mediators as well as chemokines. This immune response elicited by intramammary N305-secreted EVs was comparable to that of heat-killed N305 and, partly, by LTA. These results demonstrated that N305-secreted EVs induce a mild inflammatory response distinct from the live pathogen after intramammary injection. Overall, our combined and data suggest that EVs are worth to be investigated to better understand the pathogenesis and are relevant tools to develop strategies against bovine mastitis. (hide) EV-METRIC 71% (96th 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 Filtration Ultrafiltration Protein markers EV: None non-EV: None Proteomics yes EV density (g/ml) 1.08–1.13 Show all info Study aim Function/Identification of content (omics approaches) Sample Species Staphylococcus aureus Sample Type Cell culture supernatant EV-producing cells Newbould 305 (ATCC 29740) EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: speed (g) 150000 Density gradient Type Discontinuous Lowest density fraction 8% Highest density fraction 68% Speed (g) 100000 Duration (min) 150 Fraction processing Centrifugation Pelleting: speed (g) 150000 Pelleting: adjusted k-factor TDB Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type NS Characterization: Protein analysis Protein Concentration Method Bradford Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 126 EV concentration Yes Particle yield particles per milliliter of starting sample: 4.00E+09 TRPS Report type Mean Reported size (nm) 67 EV concentration Yes Particle yield particles per milliliter of starting sample: 4.00E+09 EM EM-type Transmission-EM/ Cryo-EM Image type Close-up, Wide-field Report size (nm) 91

	EV220208	1/2	Homo sapiens	HUVEC	Filtration dUC	Hosseinkhani B	2018	67%
Study summary Full title Extracellular Vesicles Work as a Functional Inflammatory Mediator Between Vascular Endothelial Cells and Immune Cells. All authors Hosseinkhani B, Kuypers S, van den Akker NMS, Molin DGM, Michiels L Journal Front Immunol Abstract Extracellular vesicles (EV) mediated intercellular communication between monocytes and endothelial c (show more...)Extracellular vesicles (EV) mediated intercellular communication between monocytes and endothelial cells (EC) might play a major role in vascular inflammation and atherosclerotic plaque formation during cardiovascular diseases (CVD). While critical involvement of small (exosomes) and large EV (microvesicles) in CVD has recently been appreciated, the pro- and/or anti-inflammatory impact of a bulk EV (exosomes + microvesicles) on vascular cell function as well as their inflammatory capacity are poorly defined. This study aims to unravel the immunomodulatory content of EV bulk derived from control (uEV) and TNF-α induced inflamed endothelial cells (tEV) and to define their capacity to affect the inflammatory status of recipients monocytes (THP-1) and endothelial cells (HUVEC) . Here, we show that EV derived from inflamed vascular EC were readily taken up by THP-1 and HUVEC. Human inflammation antibody array together with ELISA revealed that tEV contain a pro-inflammatory profile with chemotactic mediators, including intercellular adhesion molecule (ICAM)-1, CCL-2, IL-6, IL-8, CXCL-10, CCL-5, and TNF-α as compared to uEV. In addition, EV may mediate a selective transfer of functional inflammatory mediators to their target cells and modulate them toward either pro-inflammatory (HUVEC) or anti/pro-inflammatory (THP-1) mode. Accordingly, the expression of pro-inflammatory markers (IL-6, IL-8, and ICAM-1) in tEV-treated HUVEC was increased. In the case of THP-1, EC-EV do induce a mixed of pro- and anti-inflammatory response as indicated by the elevated expression of ICAM-1, CCL-4, CCL-5, and CXCL-10 proteins. At the functional level, EC-EV mediated inflammation and promoted the adhesion and migration of THP-1. Taken together, our findings proved that the EV released from inflamed EC were enriched with a cocktail of inflammatory markers, chemokines, and cytokines which are able to establish a targeted cross-talk between EC and monocytes and reprogramming them toward a pro- or anti-inflammatory phenotypes. (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 Filtration dUC Protein markers EV: CD63/ ICAM-1/ IL-6, IL-8, ICAM-1, CCL-2/ CD9/ IL-8/ TIMP2/ ICAM-1/ IL6-R/ CXCL10/ CCL-2/ PDGF-BB non-EV: GM130 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HUVEC EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS 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: rotor type Type 70 Ti Pelleting: speed (g) 110000 Filtration steps 0.22µm or 0.2µm Other Name other separation method dUC Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD9/ CD63/ ICAM-1/ ?-Actin Not detected contaminants GM130 ELISA Detected EV-associated proteins IL-6, IL-8, ICAM-1, CCL-2 Other 1 ELISA Detected EV-associated proteins IL-8/ TIMP2/ ICAM-1/ IL6-R/ CXCL10/ CCL-2/ PDGF-BB Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes EM EM-type Transmission-EM Image type Wide-field

	EV210198	3/7	Homo sapiens	A172	(d)(U)C Filtration	Hu, Guoku	2018	67%
Study summary Full title Astrocyte EV-Induced lincRNA-Cox2 Regulates Microglial Phagocytosis: Implications for Morphine-Mediated Neurodegeneration All authors Guoku Hu, Ke Liao, Fang Niu, Lu Yang, Blake W Dallon, Shannon Callen, Changhai Tian, Jiang Shu, Juan Cui, Zhiqiang Sun, Yuri L Lyubchenko, Minhan Ka, Xian-Ming Chen, Shilpa Buch Journal Mol Ther Nucleic Acids Abstract Impairment of microglial functions, such as phagocytosis and/or dysregulation of immune responses, h (show more...)Impairment of microglial functions, such as phagocytosis and/or dysregulation of immune responses, has been implicated as an underlying factor involved in the pathogenesis of various neurodegenerative disorders. Our previous studies have demonstrated that long intergenic noncoding RNA (lincRNA)-Cox2 expression is influenced by nuclear factor κB (NF-κB) signaling and serves as a coactivator of transcriptional factors to regulate the expression of a vast array of immune-related genes in microglia. Extracellular vesicles (EVs) have been recognized as primary facilitators of cell-to-cell communication and cellular regulation. Herein, we show that EVs derived from astrocytes exposed to morphine can be taken up by microglial endosomes, leading, in turn, to activation of Toll-like receptor 7 (TLR7) with a subsequent upregulation of lincRNA-Cox2 expression, ultimately resulting in impaired microglial phagocytosis. This was further validated in vivo, wherein inhibition of microglial phagocytic activity was also observed in brain slices isolated from morphine-administrated mice compared with control mice. Additionally, we also showed that intranasal delivery of EVs containing lincRNA-Cox2 siRNA (small interfering RNA) was able to restore microglial phagocytic activity in mice administered morphine. These findings have ramifications for the development of EV-loaded RNA-based therapeutics for the treatment of various disorders involving functional impairment of microglia. (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 Filtration Protein markers EV: Alix/ TSG101/ CD63 non-EV: Calnexin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells A172 EV-harvesting Medium Not specified 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) 70 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins Alix/ CD63/ 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 Modus Reported size (nm) 80 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field

	EV200136	2/7	Homo sapiens	Blood plasma	DG (d)(U)C Filtration	Miranda, Jezid	2018	67%
Study summary Full title Placental exosomes profile in maternal and fetal circulation in intrauterine growth restriction - Liquid biopsies to monitoring fetal growth All authors Jezid Miranda, Cristina Paules, Soumyalekshmi Nair, Andrew Lai, Carlos Palma, Katherin Scholz-Romero, Gregory E. Rice, Eduard Gratacos, Fatima Crispi, Carlos Salomon Journal Placenta Abstract Introduction: Placenta-derived exosomes may represent an additional pathway by which the placenta co (show more...)Introduction: Placenta-derived exosomes may represent an additional pathway by which the placenta communicates with the maternal system to induce maternal vascular adaptations to pregnancy and it may be affected during Fetal growth restriction (FGR). The objective of this study was to quantify the concentration of total and placenta-derived exosomes in maternal and fetal circulation in small fetuses classified as FGR or small for gestational age (SGA). Methods: Prospective cohort study in singleton term gestations including 10 normally grown fetuses and 20 small fetuses, sub-classified into SGA and FGR accordingly to birth weight (BW) percentile and fetoplacental Doppler. Exosomes were isolated from maternal and fetal plasma and characterized by morphology, enrichment of exosomal proteins, and size distribution by electron microscopy, western blot, and nanoparticle tracking analysis, respectively. Total and specific placenta-derived exosomes were determined using quantum dots coupled with CD63þve and placental-type alkaline phosphatase (PLAP)þve antibodies, respectively. Results: Maternal concentrations of CD63þve and PLAPþve exosomes were similar between the groups (all p > 0.05). However, there was a significant positive correlation between the ratio of placental-derived to total exosomes (PLAPþve ratio) and BW percentile, [rho ¼ 0.77 (95% CI: 0.57 to 0.89); p ¼ 0.0001]. The contribution of placental exosomes to the total exosome concentration in maternal and fetal circulation showed a significant decrease among cases, with lower PLAPþve ratios in FGR compared to controls and SGA cases. Discussion: Quantification of placental exosomes in maternal plasma reflects fetal growth and it may be a useful indicator of placental function. (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 Maternal; Normal pregnancy 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 Density gradient (Differential) (ultra)centrifugation Filtration Protein markers EV: TSG101/ Flotillin1/ PLAP/ CD63 non-EV: Grp94 Proteomics no EV density (g/ml) 1.12-1.188g/ml Show all info Study aim Function/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 T-8100 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 10 Wash: time (min) 120 Wash: Rotor Type T-8100 Wash: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Sample volume (mL) 0.5mL Orientation Bottom-up Rotor type T-8100 Speed (g) 100000 Duration (min) 1200 Fraction processing Centrifugation Pelleting: volume per fraction 0.05 Pelleting: duration (min) 120 Pelleting: rotor type T-8100 Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Flotillin1/ CD63/ TSG101 Not detected contaminants Grp94 ELISA Detected EV-associated proteins PLAP Fluorescent NTA Relevant measurements variables specified? NA Antibody details provided? No Detected EV-associated proteins PLAP/ CD63 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 87+/-23 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field

	EV200136	5/7	Homo sapiens	Blood plasma	DG (d)(U)C Filtration	Miranda, Jezid	2018	67%
Study summary Full title Placental exosomes profile in maternal and fetal circulation in intrauterine growth restriction - Liquid biopsies to monitoring fetal growth All authors Jezid Miranda, Cristina Paules, Soumyalekshmi Nair, Andrew Lai, Carlos Palma, Katherin Scholz-Romero, Gregory E. Rice, Eduard Gratacos, Fatima Crispi, Carlos Salomon Journal Placenta Abstract Introduction: Placenta-derived exosomes may represent an additional pathway by which the placenta co (show more...)Introduction: Placenta-derived exosomes may represent an additional pathway by which the placenta communicates with the maternal system to induce maternal vascular adaptations to pregnancy and it may be affected during Fetal growth restriction (FGR). The objective of this study was to quantify the concentration of total and placenta-derived exosomes in maternal and fetal circulation in small fetuses classified as FGR or small for gestational age (SGA). Methods: Prospective cohort study in singleton term gestations including 10 normally grown fetuses and 20 small fetuses, sub-classified into SGA and FGR accordingly to birth weight (BW) percentile and fetoplacental Doppler. Exosomes were isolated from maternal and fetal plasma and characterized by morphology, enrichment of exosomal proteins, and size distribution by electron microscopy, western blot, and nanoparticle tracking analysis, respectively. Total and specific placenta-derived exosomes were determined using quantum dots coupled with CD63þve and placental-type alkaline phosphatase (PLAP)þve antibodies, respectively. Results: Maternal concentrations of CD63þve and PLAPþve exosomes were similar between the groups (all p > 0.05). However, there was a significant positive correlation between the ratio of placental-derived to total exosomes (PLAPþve ratio) and BW percentile, [rho ¼ 0.77 (95% CI: 0.57 to 0.89); p ¼ 0.0001]. The contribution of placental exosomes to the total exosome concentration in maternal and fetal circulation showed a significant decrease among cases, with lower PLAPþve ratios in FGR compared to controls and SGA cases. Discussion: Quantification of placental exosomes in maternal plasma reflects fetal growth and it may be a useful indicator of placental function. (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 Fetal; Normal pregnancy 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 Density gradient (Differential) (ultra)centrifugation Filtration Protein markers EV: TSG101/ Flotillin1/ PLAP/ CD63 non-EV: Grp94 Proteomics no EV density (g/ml) 1.12-1.188g/ml Show all info Study aim Function/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 T-8100 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 10 Wash: time (min) 120 Wash: Rotor Type T-8100 Wash: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Sample volume (mL) 0.5mL Orientation Bottom-up Rotor type T-8100 Speed (g) 100000 Duration (min) 1200 Fraction processing Centrifugation Pelleting: volume per fraction 0.05 Pelleting: duration (min) 120 Pelleting: rotor type T-8100 Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Flotillin1/ CD63/ TSG101 Not detected contaminants Grp94 ELISA Detected EV-associated proteins PLAP Fluorescent NTA Relevant measurements variables specified? NA Antibody details provided? No Detected EV-associated proteins PLAP/ CD63 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 90 ± 17 nm EV concentration Yes EM EM-type Transmission-EM Image type Wide-field

	EV190002	5/6	Homo sapiens	HT-29	DG (d)(U)C Filtration	Sachiko Matsumura	2018	67%
Study summary Full title Subtypes of tumour cell-derived small extracellular vesicles having differently externalized phosphatidylserine All authors Sachiko Matsumura, Tamiko Minamisawa, Kanako Suga, Hiromi Kishita, Takanori Akagi, Takanori Ichiki, Yuki Ichikawa & Kiyotaka Shiba Journal J Extracell Vesicles Abstract Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially locate (show more...)Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially located in the inner leaflet of normal cells. Tumour cells, however, expose PS at the outer leaflet of cell surfaces, thereby potentially modulating the bio-signalling of cells. Interestingly, exosomes – or, more properly, small extracellular vesicles (sEVs) – which are secreted from tumour cells, are enriched with externalized PS, have been proposed as being involved in the progression of cancers, and could be used as a marker for tumour diagnostics. However, the sEV fractions prepared from various methods are composed of different subtypes of vesicles, and knowledge about the subtypes enriched with exposed PS is still limited. Here, we differentiated sEVs from cancer cell lines by density gradient centrifugation and characterized the separated fractions by using gold-labelling of PS in atomic force microscopy, thrombin generation assay, size and zeta potential measurements, and western blot analysis. These analyses revealed a previously unreported PS+-enriched sEV subtype, which is characterized by a lower density than that of canonical exosomes (1.06 g/ml vs. 1.08 g/ml), larger size (122 nm vs. 105 nm), more negative zeta potential (−28 mV vs. −21 mV), and lower abundance of canonical exosomal markers. The identification of the PS-exposed subtype of sEVs will provide deeper insight into the role of EVs in tumour biology and enhance the development of EV-based tumour diagnosis and therapy. (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 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 Filtration Protein markers EV: CD81/ EpCAM/ CD63/ CD9 non-EV: Proteomics no EV density (g/ml) 1.05-1.1 Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HT-29 EV-harvesting Medium Serum free medium Cell viability (%) 95 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 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 Density gradient Type Continuous Lowest density fraction 8% Highest density fraction 40% Total gradient volume, incl. sample (mL) 32 Sample volume (mL) 2 Orientation Bottom-up Rotor type SW 32 Ti Speed (g) 100000 Duration (min) 1020 Fraction volume (mL) 3.2 Fraction processing Centrifugation Pelleting: volume per fraction 33.2 Pelleting: duration (min) 120 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 160000 Filtration steps 0.22µm or 0.2µm EV-subtype Distinction between multiple subtypes Density Used subtypes < 1.06 g/ml Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins EpCAM/ CD9 Not detected EV-associated proteins CD81/ CD63 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 162 EV concentration Yes EM EM-type Atomic force-EM Image type Wide-field

	EV190002	6/6	Homo sapiens	HT-29	DG (d)(U)C Filtration	Sachiko Matsumura	2018	67%
Study summary Full title Subtypes of tumour cell-derived small extracellular vesicles having differently externalized phosphatidylserine All authors Sachiko Matsumura, Tamiko Minamisawa, Kanako Suga, Hiromi Kishita, Takanori Akagi, Takanori Ichiki, Yuki Ichikawa & Kiyotaka Shiba Journal J Extracell Vesicles Abstract Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially locate (show more...)Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially located in the inner leaflet of normal cells. Tumour cells, however, expose PS at the outer leaflet of cell surfaces, thereby potentially modulating the bio-signalling of cells. Interestingly, exosomes – or, more properly, small extracellular vesicles (sEVs) – which are secreted from tumour cells, are enriched with externalized PS, have been proposed as being involved in the progression of cancers, and could be used as a marker for tumour diagnostics. However, the sEV fractions prepared from various methods are composed of different subtypes of vesicles, and knowledge about the subtypes enriched with exposed PS is still limited. Here, we differentiated sEVs from cancer cell lines by density gradient centrifugation and characterized the separated fractions by using gold-labelling of PS in atomic force microscopy, thrombin generation assay, size and zeta potential measurements, and western blot analysis. These analyses revealed a previously unreported PS+-enriched sEV subtype, which is characterized by a lower density than that of canonical exosomes (1.06 g/ml vs. 1.08 g/ml), larger size (122 nm vs. 105 nm), more negative zeta potential (−28 mV vs. −21 mV), and lower abundance of canonical exosomal markers. The identification of the PS-exposed subtype of sEVs will provide deeper insight into the role of EVs in tumour biology and enhance the development of EV-based tumour diagnosis and therapy. (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 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 Filtration Protein markers EV: CD81/ EpCAM/ CD63/ CD9 non-EV: Proteomics no EV density (g/ml) 1.05-1.1 Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HT-29 EV-harvesting Medium Serum free medium Cell viability (%) 95 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 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 Density gradient Type Continuous Lowest density fraction 8% Highest density fraction 40% Total gradient volume, incl. sample (mL) 32 Sample volume (mL) 2 Orientation Bottom-up Rotor type SW 32 Ti Speed (g) 100000 Duration (min) 1020 Fraction volume (mL) 3.2 Fraction processing Centrifugation Pelleting: volume per fraction 33.2 Pelleting: duration (min) 120 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 160000 Filtration steps 0.22µm or 0.2µm EV-subtype Distinction between multiple subtypes Density Used subtypes ca. 1.1 g/ml Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ CD63/ EpCAM/ CD81 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 142 EV concentration Yes EM EM-type Atomic force-EM Image type Wide-field

	EV180030	3/7	Homo sapiens	PM1	DG (d)(U)C	Zhaohao Liao	2018	67%
Study summary Full title Acetylcholinesterase is not a generic marker of extracellular vesicles. All authors Zhaohao Liao, Lorena Martin Jaular ORCID Icon, Estelle Soueidi, Mabel Jouve, Dillon C. Muth, Tine H. Schøyen, Tessa Seale, Norman J. Haughey, Matias Ostrowski, Clotilde Théry ORCID Icon & Kenneth W. Witwer Journal J Extracell Vesicles Abstract Acetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was deve (show more...)Acetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was developed as a marker of reticulocyte EVs in the 1970s. Easily, quickly, and cheaply assayed, AChE activity has more recently been proposed as a generic marker for small extracellular vesicles (sEV) or exosomes, and as a negative marker of HIV-1 virions. To evaluate these proposed uses of AChE activity, we examined data from different EV and virus isolation methods using T-lymphocytic (H9, PM1 and Jurkat) and promonocytic (U937) cell lines grown in culture conditions that differed by serum content. When EVs were isolated by differential ultracentrifugation, no correlation between AChE activity and particle count was observed. AChE activity was detected in non-conditioned medium when serum was added, and most of this activity resided in soluble fractions and could not be pelleted by centrifugation. The serum-derived pelletable AChE protein was not completely eliminated from culture medium by overnight ultracentrifugation; however, a serum “extra-depletion” protocol, in which a portion of the supernatant was left undisturbed during harvesting, achieved near-complete depletion. In conditioned medium also, only small percentages of AChE activity could be pelleted together with particles. Furthermore, no consistent enrichment of AChE activity in sEV fractions was observed. Little if any AChE activity is produced by the cells we examined, and this activity was mainly present in non-vesicular structures, as shown by electron microscopy. Size-exclusion chromatography and iodixanol gradient separation showed that AChE activity overlaps only minimally with EV-enriched fractions. AChE activity likely betrays exposure to blood products and not EV abundance, echoing the MISEV 2014 and 2018 guidelines and other publications. Additional experiments may be merited to validate these results for other cell types and biological fluids other than blood. (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 HIV-BaL infected 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: / CD81/ CD63/ CD9 non-EV: AChE Proteomics no EV density (g/ml) Not specified Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells PM1 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 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 50 Wash: time (min) 90 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 11 Lowest density fraction 6% Highest density fraction 18% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1 Orientation Top-down Rotor type TH-641 Speed (g) 210000 Duration (min) 120 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 35 Pelleting: duration (min) 40 Pelleting: rotor type AH-629 (36 ml) Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ CD63/ CD81 Other 1 Acetylcholinesterase assay Detected EV-associated proteins Not detected contaminants AChE Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes

	EV180022	1/4	Homo sapiens	pluripotent stem cells	(d)(U)C	Kaur S	2018	67%
Study summary Full title Small non-coding RNA landscape of extracellular vesicles from human stem cells. All authors Kaur S, Abu-Shahba AG, Paananen RO, Hongisto H, Hiidenmaa H, Skottman H, Seppänen-Kaijansinkko R, Mannerström B Journal J Cell Sci Abstract Extracellular vesicles (EVs) are reported to be involved in stem cell maintenance, self-renewal, and (show more...)Extracellular vesicles (EVs) are reported to be involved in stem cell maintenance, self-renewal, and differentiation. Due to their bioactive cargoes influencing cell fate and function, interest in EVs in regenerative medicine has rapidly increased. EV-derived small non-coding RNA mimic the functions of the parent stem cells, regulating the maintenance and differentiation of stem cells, controlling the intercellular regulation of gene expression, and eventually affecting the cell fate. In this study, we used RNA sequencing to provide a comprehensive overview of the expression profiles of small non-coding transcripts carried by the EVs derived from human adipose tissue stromal/stem cells (AT-MSCs) and human pluripotent stem cells (hPSCs), both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSC). Both hPSCs and AT-MSCs were characterized and their EVs were extracted using standard protocols. Small non-coding RNA sequencing from EVs showed that hPSCs and AT-MSCs showed distinct profiles, unique for each stem cell source. Interestingly, in hPSCs, most abundant miRNAs were from specific miRNA families regulating pluripotency, reprogramming and differentiation (miR-17-92, mir-200, miR-302/367, miR-371/373, CM19 microRNA cluster). For the AT-MSCs, the highly expressed miRNAs were found to be regulating osteogenesis (let-7/98, miR-10/100, miR-125, miR-196, miR-199, miR-615-3p, mir-22-3p, mir-24-3p, mir-27a-3p, mir-193b-5p, mir-195-3p). Additionally, abundant small nuclear and nucleolar RNA were detected in hPSCs, whereas Y- and tRNA were found in AT-MSCs. Identification of EV-miRNA and non-coding RNA signatures released by these stem cells will provide clues towards understanding their role in intracellular communication, and well as their roles in maintaining the stem cell niche. (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 Adj. k-factor 208.3 (pelleting) / 208.3 (washing) Protein markers EV: TSG101/ HSP70/ CD63/ CD90 non-EV: Calnexin Proteomics no Show all info Study aim Identification of content (omics approaches), Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells pluripotent stem cells EV-harvesting Medium Serum free medium Cell viability (%) NA Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps 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) 121896 Pelleting: adjusted k-factor 208.3 Wash: time (min) 120 Wash: Rotor Type SW 28 Wash: speed (g) 121896 Wash: adjusted k-factor 208.3 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63, HSP70, TSG101, CD90 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 101-500 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field

	EV180022	4/4	Homo sapiens	pluripotent stem cells	miRCURY Exosome Isolation Kit (Exiqon A/S, Vedbaek, Denmark)	Kaur S	2018	67%
Study summary Full title Small non-coding RNA landscape of extracellular vesicles from human stem cells. All authors Kaur S, Abu-Shahba AG, Paananen RO, Hongisto H, Hiidenmaa H, Skottman H, Seppänen-Kaijansinkko R, Mannerström B Journal J Cell Sci Abstract Extracellular vesicles (EVs) are reported to be involved in stem cell maintenance, self-renewal, and (show more...)Extracellular vesicles (EVs) are reported to be involved in stem cell maintenance, self-renewal, and differentiation. Due to their bioactive cargoes influencing cell fate and function, interest in EVs in regenerative medicine has rapidly increased. EV-derived small non-coding RNA mimic the functions of the parent stem cells, regulating the maintenance and differentiation of stem cells, controlling the intercellular regulation of gene expression, and eventually affecting the cell fate. In this study, we used RNA sequencing to provide a comprehensive overview of the expression profiles of small non-coding transcripts carried by the EVs derived from human adipose tissue stromal/stem cells (AT-MSCs) and human pluripotent stem cells (hPSCs), both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSC). Both hPSCs and AT-MSCs were characterized and their EVs were extracted using standard protocols. Small non-coding RNA sequencing from EVs showed that hPSCs and AT-MSCs showed distinct profiles, unique for each stem cell source. Interestingly, in hPSCs, most abundant miRNAs were from specific miRNA families regulating pluripotency, reprogramming and differentiation (miR-17-92, mir-200, miR-302/367, miR-371/373, CM19 microRNA cluster). For the AT-MSCs, the highly expressed miRNAs were found to be regulating osteogenesis (let-7/98, miR-10/100, miR-125, miR-196, miR-199, miR-615-3p, mir-22-3p, mir-24-3p, mir-27a-3p, mir-193b-5p, mir-195-3p). Additionally, abundant small nuclear and nucleolar RNA were detected in hPSCs, whereas Y- and tRNA were found in AT-MSCs. Identification of EV-miRNA and non-coding RNA signatures released by these stem cells will provide clues towards understanding their role in intracellular communication, and well as their roles in maintaining the stem cell niche. (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 miRCURY Exosome Isolation Kit (Exiqon A/S, Vedbaek, Denmark) Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Identification of content (omics approaches), Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells pluripotent stem cells EV-harvesting Medium Serum free medium Cell viability (%) NA Separation Method Commercial kit miRCURY Exosome Isolation Kit (Exiqon A/S, Vedbaek, Denmark) Other Name other separation method miRCURY Exosome Isolation Kit (Exiqon A/S, Vedbaek, Denmark) Characterization: Protein analysis None Protein Concentration Method Not determined Characterization: RNA analysis Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No

	EV180051	2/2	Rattus norvegicus	Blood plasma	(d)(U)C	Takov K	2018	66%
Study summary Full title Comparison of small extracellular vesicles isolated from plasma by ultracentrifugation or size-exclusion chromatography: yield, purity and functional potential. All authors Takov K, Yellon DM, Davidson SM Journal J Extracell Vesicles Abstract Interest in small extracellular vesicles (sEVs) as functional carriers of proteins and nucleic acids (show more...)Interest in small extracellular vesicles (sEVs) as functional carriers of proteins and nucleic acids is growing continuously. There are large numbers of sEVs in the blood, but lack of standardised methods for sEV isolation greatly limits our ability to study them. In this report, we use rat plasma to systematically compare two commonly used techniques for isolation of sEVs: ultracentrifugation (UC-sEVs) and size-exclusion chromatography (SEC-sEVs). SEC-sEVs had higher particle number, protein content, particle/protein ratios and sEV marker signal than UC-sEVs. However, SEC-sEVs also contained greater amounts of APOB⁺ lipoproteins and large quantities of non-sEV protein. sEV marker signal correlated very well with both particle number and protein content in UC-sEVs but not in all of the SEC-sEV fractions. Functionally, both UC-sEVs and SEC-sEVs isolates contained a variety of proangiogenic factors (with endothelin-1 being the most abundant) and stimulated migration of endothelial cells. However, there was no evident correlation between the promigratory potential and the quantity of sEVs added, indicating that non-vesicular co-isolates may contribute to the promigratory effects. Overall, our findings suggest that UC provides plasma sEVs of lower yields, but markedly higher purity compared to SEC. Furthermore, we show that the functional activity of sEVs can depend on the isolation method used and does not solely reflect the sEV quantity. These findings are of importance when working with sEVs isolated from plasma- or serum-containing conditioned medium. (hide) EV-METRIC 66% (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 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 (d)(U)C Adj. k-factor 115.3 (pelleting) / 115.3 (washing) Protein markers EV: CD81/ HSP70/ Endothelin-1/ CD9 non-EV: ApoB Proteomics no Show all info Study aim Function, Technical analysis comparing/optimizing EV-related methods, Identification of content (omics approaches) Sample Species Rattus norvegicus Sample Type Blood 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) 70 Pelleting: rotor type MLA-55 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 115.3 Wash: time (min) 70 Wash: Rotor Type MLA-55 Wash: speed (g) 100000 Wash: adjusted k-factor 115.3 Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) 13.6-14.6 Western Blot Detected EV-associated proteins HSP70 ELISA Other 1 Protein array (ARY007, R&D Systems) Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 96.6 EV concentration Yes Particle yield 20000000000 EM EM-type Transmission-EM Image type Wide-field

	EV180022	3/4	Homo sapiens	adipose tissue-derived mesenchymal stem cells	(d)(U)C Filtration	Kaur S	2018	66%
Study summary Full title Small non-coding RNA landscape of extracellular vesicles from human stem cells. All authors Kaur S, Abu-Shahba AG, Paananen RO, Hongisto H, Hiidenmaa H, Skottman H, Seppänen-Kaijansinkko R, Mannerström B Journal J Cell Sci Abstract Extracellular vesicles (EVs) are reported to be involved in stem cell maintenance, self-renewal, and (show more...)Extracellular vesicles (EVs) are reported to be involved in stem cell maintenance, self-renewal, and differentiation. Due to their bioactive cargoes influencing cell fate and function, interest in EVs in regenerative medicine has rapidly increased. EV-derived small non-coding RNA mimic the functions of the parent stem cells, regulating the maintenance and differentiation of stem cells, controlling the intercellular regulation of gene expression, and eventually affecting the cell fate. In this study, we used RNA sequencing to provide a comprehensive overview of the expression profiles of small non-coding transcripts carried by the EVs derived from human adipose tissue stromal/stem cells (AT-MSCs) and human pluripotent stem cells (hPSCs), both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSC). Both hPSCs and AT-MSCs were characterized and their EVs were extracted using standard protocols. Small non-coding RNA sequencing from EVs showed that hPSCs and AT-MSCs showed distinct profiles, unique for each stem cell source. Interestingly, in hPSCs, most abundant miRNAs were from specific miRNA families regulating pluripotency, reprogramming and differentiation (miR-17-92, mir-200, miR-302/367, miR-371/373, CM19 microRNA cluster). For the AT-MSCs, the highly expressed miRNAs were found to be regulating osteogenesis (let-7/98, miR-10/100, miR-125, miR-196, miR-199, miR-615-3p, mir-22-3p, mir-24-3p, mir-27a-3p, mir-193b-5p, mir-195-3p). Additionally, abundant small nuclear and nucleolar RNA were detected in hPSCs, whereas Y- and tRNA were found in AT-MSCs. Identification of EV-miRNA and non-coding RNA signatures released by these stem cells will provide clues towards understanding their role in intracellular communication, and well as their roles in maintaining the stem cell niche. (hide) EV-METRIC 66% (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 Filtration Adj. k-factor 208.3 (pelleting) / 208.3 (washing) Protein markers EV: TSG101/ CD63/ CD90 non-EV: Calnexin Proteomics no Show all info Study aim Identification of content (omics approaches), Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant

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