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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV230996	1/5	Homo sapiens	HEK293F	(d)(U)C DC Filtration UF SEC (non-commercial)	Vo N	2024	89%
Study summary Full title A novel multi-stage enrichment workflow and comprehensive characterization for HEK293F-derived extracellular vesicles. All authors Vo N, Tran C, Tran NHB, Nguyen NT, Nguyen T, Ho DTK, Nguyen DDN, Pham T, Nguyen TA, Phan HTN, Nguyen HN, Tu LN Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are emerging as a promising drug delivery vehicle as they are biocompat (show more...)Extracellular vesicles (EVs) are emerging as a promising drug delivery vehicle as they are biocompatible and capable of targeted delivery. However, clinical translation of EVs remains challenging due to the lack of standardized and scalable manufacturing protocols to consistently isolate small EVs (sEVs) with both high yield and high purity. The heterogenous nature of sEVs leading to unknown composition of biocargos causes further pushback due to safety concerns. In order to address these issues, we developed a robust quality-controlled multi-stage process to produce and isolate sEVs from human embryonic kidney HEK293F cells. We then compared different 2-step and 3-step workflows for eliminating protein impurities and cell-free nucleic acids to meet acceptable limits of regulatory authorities. Our results showed that sEV production was maximized when HEK293F cells were grown at high-density stationary phase in semi-continuous culture. The novel 3-step workflow combining tangential flow filtration, sucrose-cushion ultracentrifugation and bind-elute size-exclusion chromatography outperformed other methods in sEV purity while still preserved high yield and particle integrity. The purified HEK293F-derived sEVs were thoroughly characterized for identity including sub-population analysis, content profiling including proteomics and miRNA sequencing, and demonstrated excellent preclinical safety profile in both in-vitro and in-vivo testing. Our rigorous enrichment workflow and comprehensive characterization will help advance the development of EVs, particularly HEK293F-derived sEVs, to be safe and reliable drug carriers for therapeutic applications. (hide) EV-METRIC 89% (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 (Differential) (ultra)centrifugation Density cushion Filtration Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: CD9/ CD81/ TSG101/ CD63 non-EV: CANX Proteomics yes Show all info Study aim New methodological development/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK293F EV-harvesting Medium Serum free medium Cell count 3.00E+06 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 50.2 Ti Pelleting: speed (g) 120000 Filtration steps 0.2 or 0.22 µm Ultra filtration Cut-off size (kDa) 300 Membrane type Polyethersulfone (PES) Density cushion Density medium Sucrose Sample volume 20 Cushion volume 5 Density of the cushion 30% Centrifugation time 240 Centrifugation speed 120,000 Size-exclusion chromatography Total column volume (mL) 5 Sample volume/column (mL) 50 Resin type None of these Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins CD9/ CD81/ TSG101 Not detected contaminants CANX Proteomics database Vesiclepedia #3590 Detected EV-associated proteins CD9/ CD63/ CD81/ TSG101 Not detected contaminants CANX Characterization: RNA analysis RNA analysis Type RNA-sequencing Proteinase treatment No RNAse treatment Yes RNAse type Benzonase RNAse concentration 20U/100ml Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 164.3+/-84.2 EM EM-type Scanning-EM Image type Close-up, Wide-field Report size (nm) 52.6+/-25.8 Other particle analysis name(3) Exoview Report type Size range/distribution EV-concentration Yes Particle yield as number of particles per milliliter of starting sample: 1E13/L

	EV230996	2/5	Homo sapiens	HEK293F	(d)(U)C Filtration	Vo N	2024	44%
Study summary Full title A novel multi-stage enrichment workflow and comprehensive characterization for HEK293F-derived extracellular vesicles. All authors Vo N, Tran C, Tran NHB, Nguyen NT, Nguyen T, Ho DTK, Nguyen DDN, Pham T, Nguyen TA, Phan HTN, Nguyen HN, Tu LN Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are emerging as a promising drug delivery vehicle as they are biocompat (show more...)Extracellular vesicles (EVs) are emerging as a promising drug delivery vehicle as they are biocompatible and capable of targeted delivery. However, clinical translation of EVs remains challenging due to the lack of standardized and scalable manufacturing protocols to consistently isolate small EVs (sEVs) with both high yield and high purity. The heterogenous nature of sEVs leading to unknown composition of biocargos causes further pushback due to safety concerns. In order to address these issues, we developed a robust quality-controlled multi-stage process to produce and isolate sEVs from human embryonic kidney HEK293F cells. We then compared different 2-step and 3-step workflows for eliminating protein impurities and cell-free nucleic acids to meet acceptable limits of regulatory authorities. Our results showed that sEV production was maximized when HEK293F cells were grown at high-density stationary phase in semi-continuous culture. The novel 3-step workflow combining tangential flow filtration, sucrose-cushion ultracentrifugation and bind-elute size-exclusion chromatography outperformed other methods in sEV purity while still preserved high yield and particle integrity. The purified HEK293F-derived sEVs were thoroughly characterized for identity including sub-population analysis, content profiling including proteomics and miRNA sequencing, and demonstrated excellent preclinical safety profile in both in-vitro and in-vivo testing. Our rigorous enrichment workflow and comprehensive characterization will help advance the development of EVs, particularly HEK293F-derived sEVs, to be safe and reliable drug carriers for therapeutic applications. (hide) EV-METRIC 44% (84th 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 Filtration Protein markers EV: CD9/ CD81/ TSG101/ CD63 non-EV: CANX Proteomics no Show all info Study aim New methodological development/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK293F EV-harvesting Medium Serum free medium Cell count 3.00E+06 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 50.2 Ti Pelleting: speed (g) 120000 Filtration steps 0.2 or 0.22 µm Size-exclusion chromatography Resin type Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins CD9/ CD81/ TSG101 Not detected contaminants CANX Detected EV-associated proteins CD9/ CD63/ CD81/ TSG101 Not detected contaminants CANX Characterization: Lipid analysis No Characterization: Particle analysis NTA Particle yield per milliliter of starting sample

	EV230996	3/5	Homo sapiens	HEK293F	(d)(U)C Filtration UF	Vo N	2024	44%
Study summary Full title A novel multi-stage enrichment workflow and comprehensive characterization for HEK293F-derived extracellular vesicles. All authors Vo N, Tran C, Tran NHB, Nguyen NT, Nguyen T, Ho DTK, Nguyen DDN, Pham T, Nguyen TA, Phan HTN, Nguyen HN, Tu LN Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are emerging as a promising drug delivery vehicle as they are biocompat (show more...)Extracellular vesicles (EVs) are emerging as a promising drug delivery vehicle as they are biocompatible and capable of targeted delivery. However, clinical translation of EVs remains challenging due to the lack of standardized and scalable manufacturing protocols to consistently isolate small EVs (sEVs) with both high yield and high purity. The heterogenous nature of sEVs leading to unknown composition of biocargos causes further pushback due to safety concerns. In order to address these issues, we developed a robust quality-controlled multi-stage process to produce and isolate sEVs from human embryonic kidney HEK293F cells. We then compared different 2-step and 3-step workflows for eliminating protein impurities and cell-free nucleic acids to meet acceptable limits of regulatory authorities. Our results showed that sEV production was maximized when HEK293F cells were grown at high-density stationary phase in semi-continuous culture. The novel 3-step workflow combining tangential flow filtration, sucrose-cushion ultracentrifugation and bind-elute size-exclusion chromatography outperformed other methods in sEV purity while still preserved high yield and particle integrity. The purified HEK293F-derived sEVs were thoroughly characterized for identity including sub-population analysis, content profiling including proteomics and miRNA sequencing, and demonstrated excellent preclinical safety profile in both in-vitro and in-vivo testing. Our rigorous enrichment workflow and comprehensive characterization will help advance the development of EVs, particularly HEK293F-derived sEVs, to be safe and reliable drug carriers for therapeutic applications. (hide) EV-METRIC 44% (84th 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 Filtration Ultrafiltration Protein markers EV: CD9/ CD81/ TSG101/ CD63 non-EV: CANX Proteomics no Show all info Study aim New methodological development/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK293F EV-harvesting Medium Serum free medium Cell count 3.00E+06 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 50.2 Ti Pelleting: speed (g) 120000 Filtration steps 0.2 or 0.22 µm Ultra filtration Cut-off size (kDa) 300 Membrane type Polyethersulfone (PES) Size-exclusion chromatography Resin type Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins CD9/ CD81/ TSG101 Not detected contaminants CANX Detected EV-associated proteins CD9/ CD63/ CD81/ TSG101 Not detected contaminants CANX Characterization: Lipid analysis No Characterization: Particle analysis NTA Particle yield per milliliter of starting sample

	EV230996	4/5	Homo sapiens	HEK293F	(d)(U)C DC Filtration UF	Vo N	2024	44%
Study summary Full title A novel multi-stage enrichment workflow and comprehensive characterization for HEK293F-derived extracellular vesicles. All authors Vo N, Tran C, Tran NHB, Nguyen NT, Nguyen T, Ho DTK, Nguyen DDN, Pham T, Nguyen TA, Phan HTN, Nguyen HN, Tu LN Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are emerging as a promising drug delivery vehicle as they are biocompat (show more...)Extracellular vesicles (EVs) are emerging as a promising drug delivery vehicle as they are biocompatible and capable of targeted delivery. However, clinical translation of EVs remains challenging due to the lack of standardized and scalable manufacturing protocols to consistently isolate small EVs (sEVs) with both high yield and high purity. The heterogenous nature of sEVs leading to unknown composition of biocargos causes further pushback due to safety concerns. In order to address these issues, we developed a robust quality-controlled multi-stage process to produce and isolate sEVs from human embryonic kidney HEK293F cells. We then compared different 2-step and 3-step workflows for eliminating protein impurities and cell-free nucleic acids to meet acceptable limits of regulatory authorities. Our results showed that sEV production was maximized when HEK293F cells were grown at high-density stationary phase in semi-continuous culture. The novel 3-step workflow combining tangential flow filtration, sucrose-cushion ultracentrifugation and bind-elute size-exclusion chromatography outperformed other methods in sEV purity while still preserved high yield and particle integrity. The purified HEK293F-derived sEVs were thoroughly characterized for identity including sub-population analysis, content profiling including proteomics and miRNA sequencing, and demonstrated excellent preclinical safety profile in both in-vitro and in-vivo testing. Our rigorous enrichment workflow and comprehensive characterization will help advance the development of EVs, particularly HEK293F-derived sEVs, to be safe and reliable drug carriers for therapeutic applications. (hide) EV-METRIC 44% (84th 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 cushion Filtration Ultrafiltration Protein markers EV: CD9/ CD81/ TSG101/ CD63 non-EV: CANX Proteomics no Show all info Study aim New methodological development/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK293F EV-harvesting Medium Serum free medium Cell count 3.00E+06 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 50.2 Ti Pelleting: speed (g) 120000 Filtration steps 0.2 or 0.22 µm Ultra filtration Cut-off size (kDa) 300 Membrane type Polyethersulfone (PES) Density cushion Density medium Sucrose Sample volume 20 Cushion volume 5 Density of the cushion 30% Centrifugation time 240 Centrifugation speed 120,000 Size-exclusion chromatography Resin type Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins CD9/ CD81/ TSG101 Not detected contaminants CANX Detected EV-associated proteins CD9/ CD63/ CD81/ TSG101 Not detected contaminants CANX Characterization: Lipid analysis No Characterization: Particle analysis NTA Particle yield per milliliter of starting sample

	EV230996	5/5	Homo sapiens	HEK293F	(d)(U)C DC Filtration UF	Vo N	2024	44%
Study summary Full title A novel multi-stage enrichment workflow and comprehensive characterization for HEK293F-derived extracellular vesicles. All authors Vo N, Tran C, Tran NHB, Nguyen NT, Nguyen T, Ho DTK, Nguyen DDN, Pham T, Nguyen TA, Phan HTN, Nguyen HN, Tu LN Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are emerging as a promising drug delivery vehicle as they are biocompat (show more...)Extracellular vesicles (EVs) are emerging as a promising drug delivery vehicle as they are biocompatible and capable of targeted delivery. However, clinical translation of EVs remains challenging due to the lack of standardized and scalable manufacturing protocols to consistently isolate small EVs (sEVs) with both high yield and high purity. The heterogenous nature of sEVs leading to unknown composition of biocargos causes further pushback due to safety concerns. In order to address these issues, we developed a robust quality-controlled multi-stage process to produce and isolate sEVs from human embryonic kidney HEK293F cells. We then compared different 2-step and 3-step workflows for eliminating protein impurities and cell-free nucleic acids to meet acceptable limits of regulatory authorities. Our results showed that sEV production was maximized when HEK293F cells were grown at high-density stationary phase in semi-continuous culture. The novel 3-step workflow combining tangential flow filtration, sucrose-cushion ultracentrifugation and bind-elute size-exclusion chromatography outperformed other methods in sEV purity while still preserved high yield and particle integrity. The purified HEK293F-derived sEVs were thoroughly characterized for identity including sub-population analysis, content profiling including proteomics and miRNA sequencing, and demonstrated excellent preclinical safety profile in both in-vitro and in-vivo testing. Our rigorous enrichment workflow and comprehensive characterization will help advance the development of EVs, particularly HEK293F-derived sEVs, to be safe and reliable drug carriers for therapeutic applications. (hide) EV-METRIC 44% (84th 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 cushion Filtration Ultrafiltration Protein markers EV: CD9/ CD81/ TSG101/ CD63 non-EV: CANX Proteomics no Show all info Study aim New methodological development/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK293F EV-harvesting Medium Serum free medium Cell count 3.00E+06 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 50.2 Ti Pelleting: speed (g) 120000 Filtration steps 0.2 or 0.22 µm Ultra filtration Cut-off size (kDa) 300 Membrane type Polyethersulfone (PES) Density cushion Density of the cushion 30% Centrifugation speed 120,000 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins CD9/ CD81/ TSG101 Not detected contaminants CANX Detected EV-associated proteins CD9/ CD63/ CD81/ TSG101 Not detected contaminants CANX Characterization: Lipid analysis No Characterization: Particle analysis NTA

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EV-TRACK ID	EV230996
species	Homo sapiens
sample type	Cell culture
cell type	HEK293F
condition	Control condition
separation protocol	dUC/ DC/ Filtration/ Ultrafiltration/ Size-exclusion chromatography (non-commercial)	dUC/ Filtration	dUC/ Filtration/ Ultrafiltration	dUC/ DC/ Filtration/ Ultrafiltration	dUC/ DC/ Filtration/ Ultrafiltration
Exp. nr.	1	2	3	4	5
EV-METRIC %	89	44	44	44	44