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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV190075	1/3	Homo sapiens	mesenchymal stem cells	Filtration Total Exosome Isolation UF	Thomas E. Whittaker	2020	50%
Study summary Full title Experimental artefacts can lead to misattribution of bioactivity from soluble mesenchymal stem cell paracrine factors to extracellular vesicles All authors Thomas E. Whittaker, Anika Nagelkerke , Valeria Nele , Ulrike Kauscher & Molly M. Stevens Journal J Extracell Vesicles Abstract It has been demonstrated that some commonly used Extracellular Vesicle (EV) isolation techniques can (show more...)It has been demonstrated that some commonly used Extracellular Vesicle (EV) isolation techniques can lead to substantial contamination with non-EV factors. Whilst it has been established that this impacts the identification of biomarkers, the impact on apparent EV bioactivity has not been explored. Extracellular vesicles have been implicated as critical mediators of therapeutic human mesenchymal stem cell (hMSC) paracrine signalling. Isolated hMSC-EVs have been used to treat multiple in vitro and in vivo models of tissue damage. However, the relative contributions of EVs and non-EV factors have not been directly compared. The dependence of hMSC paracrine signalling on EVs was first established by ultrafiltration of hMSC-conditioned medium to deplete EVs, which led to a loss of signalling activity. Here, we show that this method also causes depletion of non-EV factors, and that when this is prevented proangiogenic signalling activity is fully restored in vitro. Subsequently, we used size-exclusion chromatography (SEC) to separate EVs and soluble proteins to directly and quantitatively compare their relative contributions to signalling. Non-EV factors were found to be necessary and sufficient for the stimulation of angiogenesis and wound healing in vitro. EVs in isolation were found to be capable of potentiating signalling only when isolated by a low-purity method, or when used at comparatively high concentrations. These results indicate a potential for contaminating soluble factors to artefactually increase the apparent bioactivity of EV isolates and could have implications for future studies on the biological roles of EVs. (hide) EV-METRIC 50% (87th 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 Total Exosome Isolation UF Protein markers EV: / CD81/ CD63/ CD9 non-EV: VEGF Proteomics no Show all info Study aim Function/New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells mesenchymal stem cells EV-harvesting Medium Serum free medium Separation Method Filtration steps 0.45µm > x > 0.22µm, Ultra filtration Cut-off size (kDa) 3 Membrane type Regenerated cellulose Commercial kit Total Exosome Isolation Other Name other separation method Total Exosome Isolation Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) ELISA Antibody details provided? No Detected EV-associated proteins Not detected EV-associated proteins Detected contaminants VEGF Not detected contaminants CD63/ CD81/ CD9 Other 1 Dot Blot Detected EV-associated proteins CD63/ CD81/ CD9 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 115.8 EV concentration Yes

	EV190075	2/3	Homo sapiens	mesenchymal stem cells	Filtration SEC SEC (non-commercial) UF	Thomas E. Whittaker	2020	50%
Study summary Full title Experimental artefacts can lead to misattribution of bioactivity from soluble mesenchymal stem cell paracrine factors to extracellular vesicles All authors Thomas E. Whittaker, Anika Nagelkerke , Valeria Nele , Ulrike Kauscher & Molly M. Stevens Journal J Extracell Vesicles Abstract It has been demonstrated that some commonly used Extracellular Vesicle (EV) isolation techniques can (show more...)It has been demonstrated that some commonly used Extracellular Vesicle (EV) isolation techniques can lead to substantial contamination with non-EV factors. Whilst it has been established that this impacts the identification of biomarkers, the impact on apparent EV bioactivity has not been explored. Extracellular vesicles have been implicated as critical mediators of therapeutic human mesenchymal stem cell (hMSC) paracrine signalling. Isolated hMSC-EVs have been used to treat multiple in vitro and in vivo models of tissue damage. However, the relative contributions of EVs and non-EV factors have not been directly compared. The dependence of hMSC paracrine signalling on EVs was first established by ultrafiltration of hMSC-conditioned medium to deplete EVs, which led to a loss of signalling activity. Here, we show that this method also causes depletion of non-EV factors, and that when this is prevented proangiogenic signalling activity is fully restored in vitro. Subsequently, we used size-exclusion chromatography (SEC) to separate EVs and soluble proteins to directly and quantitatively compare their relative contributions to signalling. Non-EV factors were found to be necessary and sufficient for the stimulation of angiogenesis and wound healing in vitro. EVs in isolation were found to be capable of potentiating signalling only when isolated by a low-purity method, or when used at comparatively high concentrations. These results indicate a potential for contaminating soluble factors to artefactually increase the apparent bioactivity of EV isolates and could have implications for future studies on the biological roles of EVs. (hide) EV-METRIC 50% (87th 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 SEC Size-exclusion chromatography (non-commercial) UF Protein markers EV: / CD81/ CD63/ CD9 non-EV: VEGF Proteomics no Show all info Study aim Function/New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells mesenchymal stem cells EV-harvesting Medium Serum free medium Separation Method Filtration steps 0.45µm > x > 0.22µm, Ultra filtration Cut-off size (kDa) 3 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 22 Sample volume/column (mL) 0.5 Resin type Sepharose CL-2B Other Name other separation method Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) ELISA Antibody details provided? No Detected EV-associated proteins Not detected EV-associated proteins Not detected contaminants CD63/ CD81/ CD9 Other 1 Dot Blot Detected EV-associated proteins CD63/ CD81/ CD9 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 125 EV concentration Yes

	EV190075	3/3	Homo sapiens	mesenchymal stem cells	Filtration SEC SEC (non-commercial) UF	Thomas E. Whittaker	2020	33%
Study summary Full title Experimental artefacts can lead to misattribution of bioactivity from soluble mesenchymal stem cell paracrine factors to extracellular vesicles All authors Thomas E. Whittaker, Anika Nagelkerke , Valeria Nele , Ulrike Kauscher & Molly M. Stevens Journal J Extracell Vesicles Abstract It has been demonstrated that some commonly used Extracellular Vesicle (EV) isolation techniques can (show more...)It has been demonstrated that some commonly used Extracellular Vesicle (EV) isolation techniques can lead to substantial contamination with non-EV factors. Whilst it has been established that this impacts the identification of biomarkers, the impact on apparent EV bioactivity has not been explored. Extracellular vesicles have been implicated as critical mediators of therapeutic human mesenchymal stem cell (hMSC) paracrine signalling. Isolated hMSC-EVs have been used to treat multiple in vitro and in vivo models of tissue damage. However, the relative contributions of EVs and non-EV factors have not been directly compared. The dependence of hMSC paracrine signalling on EVs was first established by ultrafiltration of hMSC-conditioned medium to deplete EVs, which led to a loss of signalling activity. Here, we show that this method also causes depletion of non-EV factors, and that when this is prevented proangiogenic signalling activity is fully restored in vitro. Subsequently, we used size-exclusion chromatography (SEC) to separate EVs and soluble proteins to directly and quantitatively compare their relative contributions to signalling. Non-EV factors were found to be necessary and sufficient for the stimulation of angiogenesis and wound healing in vitro. EVs in isolation were found to be capable of potentiating signalling only when isolated by a low-purity method, or when used at comparatively high concentrations. These results indicate a potential for contaminating soluble factors to artefactually increase the apparent bioactivity of EV isolates and could have implications for future studies on the biological roles of EVs. (hide) EV-METRIC 33% (75th 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 SEC Size-exclusion chromatography (non-commercial) UF Protein markers EV: / CD81/ CD63/ CD9 non-EV: VEGF Proteomics no Show all info Study aim Function/New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells mesenchymal stem cells EV-harvesting Medium Serum free medium Separation Method Filtration steps 0.45µm > x > 0.22µm, Ultra filtration Cut-off size (kDa) 3 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 22 Sample volume/column (mL) 0.5 Resin type Sepharose CL-2B Other Name other separation method Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) ELISA Antibody details provided? No Detected EV-associated proteins Not detected EV-associated proteins Not detected contaminants CD63/ CD81/ CD9 Other 1 Detected EV-associated proteins Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 116 EV concentration Yes

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EV-TRACK ID	EV190075
species	Homo sapiens
sample type	Cell culture
cell type	mesenchymal stem cells
condition	Control condition
separation protocol	Filtration Total Exosome Isolation UF	Filtration SEC Size-exclusion chromatography (non-commercial) UF	Filtration SEC Size-exclusion chromatography (non-commercial) UF
Exp. nr.	1	2	3
EV-METRIC %	50	50	33