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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210264	1/4	Homo sapiens	HPV-16 E6/E7 transformed bone marrow mesenchymal stromal cells	(d)(U)C Filtration	Hettich BF	2020	67%
Study summary Full title Exosomes for Wound Healing: Purification Optimization and Identification of Bioactive Components. All authors Hettich BF, Ben-Yehuda Greenwald M, Werner S, Leroux JC Journal Adv Sci (Weinh) Abstract Human mesenchymal stem cell exosomes have been shown to promote cutaneous wound healing. Their bioac (show more...)Human mesenchymal stem cell exosomes have been shown to promote cutaneous wound healing. Their bioactivity is most often attributed to their protein and nucleic acid components, while the function of exosomal lipids remains comparatively unexplored. This work specifically assesses the involvement of lipids and the transmembrane enzyme CD73 in the exosomes' biological activity in stimulating the cutaneous wound healing process. Since exosome preparation processes are not harmonized yet, certain production and purification parameters are first systematically investigated, enabling the optimization of a standardized protocol delivering high exosome integrity, yield, and purity. An in situ enzymatic assay is introduced to specifically assess the vesicle functionality, and quantitative proteomics is employed to establish the cell culture conditions yielding a stable exosome protein profile. Using a combination of in vitro approaches, CD73 and constitutional lipids of HPV-16 E6/E7 transformed human bone marrow stromal cell-derived exosomes are identified as key bioactive components promoting the exosome-driven acceleration of processes required for wound repair. A pilot wound healing study in mice indeed suggests a role of lipids in the exosomes' biological activity. Strikingly, the extent of the bioactivity of these exosomal components is found to be dependent on the target cell type. (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 24h conditioned medium Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Protein markers EV: CD73/ TSG101/ CD9/ GAPDH/ CD63 non-EV: Calregulin Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HPV-16 E6/E7 transformed bone marrow mesenchymal stromal 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 Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 10 Wash: time (min) 70 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Micro BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD73/ TSG101/ CD9/ GAPDH/ CD63 Detected contaminants Calregulin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mode Reported size (nm) 104 EV concentration Yes EM EM-type Transmission EM Image type Close-up, Wide-field Report size (nm) 25-300

	EV210264	2/4	Homo sapiens	HPV-16 E6/E7 transformed bone marrow mesenchymal stromal cells	(d)(U)C Filtration	Hettich BF	2020	67%
Study summary Full title Exosomes for Wound Healing: Purification Optimization and Identification of Bioactive Components. All authors Hettich BF, Ben-Yehuda Greenwald M, Werner S, Leroux JC Journal Adv Sci (Weinh) Abstract Human mesenchymal stem cell exosomes have been shown to promote cutaneous wound healing. Their bioac (show more...)Human mesenchymal stem cell exosomes have been shown to promote cutaneous wound healing. Their bioactivity is most often attributed to their protein and nucleic acid components, while the function of exosomal lipids remains comparatively unexplored. This work specifically assesses the involvement of lipids and the transmembrane enzyme CD73 in the exosomes' biological activity in stimulating the cutaneous wound healing process. Since exosome preparation processes are not harmonized yet, certain production and purification parameters are first systematically investigated, enabling the optimization of a standardized protocol delivering high exosome integrity, yield, and purity. An in situ enzymatic assay is introduced to specifically assess the vesicle functionality, and quantitative proteomics is employed to establish the cell culture conditions yielding a stable exosome protein profile. Using a combination of in vitro approaches, CD73 and constitutional lipids of HPV-16 E6/E7 transformed human bone marrow stromal cell-derived exosomes are identified as key bioactive components promoting the exosome-driven acceleration of processes required for wound repair. A pilot wound healing study in mice indeed suggests a role of lipids in the exosomes' biological activity. Strikingly, the extent of the bioactivity of these exosomal components is found to be dependent on the target cell type. (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 48h conditioned medium Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Protein markers EV: CD73/ TSG101/ CD9/ GAPDH/ CD63 non-EV: Calregulin Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HPV-16 E6/E7 transformed bone marrow mesenchymal stromal 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 Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 10 Wash: time (min) 70 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Micro BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD73/ TSG101/ CD9/ GAPDH/ CD63 Detected contaminants Calregulin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mode Reported size (nm) 89 EV concentration Yes EM EM-type Transmission EM/ Cryo-EM Image type Close-up, Wide-field Report size (nm) 25-250

	EV210264	3/4	Homo sapiens	HPV-16 E6/E7 transformed bone marrow mesenchymal stromal cells	(d)(U)C Filtration	Hettich BF	2020	67%
Study summary Full title Exosomes for Wound Healing: Purification Optimization and Identification of Bioactive Components. All authors Hettich BF, Ben-Yehuda Greenwald M, Werner S, Leroux JC Journal Adv Sci (Weinh) Abstract Human mesenchymal stem cell exosomes have been shown to promote cutaneous wound healing. Their bioac (show more...)Human mesenchymal stem cell exosomes have been shown to promote cutaneous wound healing. Their bioactivity is most often attributed to their protein and nucleic acid components, while the function of exosomal lipids remains comparatively unexplored. This work specifically assesses the involvement of lipids and the transmembrane enzyme CD73 in the exosomes' biological activity in stimulating the cutaneous wound healing process. Since exosome preparation processes are not harmonized yet, certain production and purification parameters are first systematically investigated, enabling the optimization of a standardized protocol delivering high exosome integrity, yield, and purity. An in situ enzymatic assay is introduced to specifically assess the vesicle functionality, and quantitative proteomics is employed to establish the cell culture conditions yielding a stable exosome protein profile. Using a combination of in vitro approaches, CD73 and constitutional lipids of HPV-16 E6/E7 transformed human bone marrow stromal cell-derived exosomes are identified as key bioactive components promoting the exosome-driven acceleration of processes required for wound repair. A pilot wound healing study in mice indeed suggests a role of lipids in the exosomes' biological activity. Strikingly, the extent of the bioactivity of these exosomal components is found to be dependent on the target cell type. (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 24h conditioned medium Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Protein markers EV: CD73/ TSG101/ CD9/ GAPDH/ CD63 non-EV: Calregulin Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HPV-16 E6/E7 transformed bone marrow mesenchymal stromal 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: rotor type Type 45 Ti Pelleting: speed (g) 150000 Wash: volume per pellet (ml) 10 Wash: time (min) 70 Wash: Rotor Type Type 45 Ti Wash: speed (g) 150000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Micro BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD73/ TSG101/ CD9/ GAPDH/ CD63 Detected contaminants Calregulin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mode Reported size (nm) 139 EV concentration Yes EM EM-type Transmission EM Image type Close-up, Wide-field Report size (nm) 25-350

	EV210264	4/4	Homo sapiens	HPV-16 E6/E7 transformed bone marrow mesenchymal stromal cells	(d)(U)C qEVoriginal/70nm Filtration UF	Hettich BF	2020	67%
Study summary Full title Exosomes for Wound Healing: Purification Optimization and Identification of Bioactive Components. All authors Hettich BF, Ben-Yehuda Greenwald M, Werner S, Leroux JC Journal Adv Sci (Weinh) Abstract Human mesenchymal stem cell exosomes have been shown to promote cutaneous wound healing. Their bioac (show more...)Human mesenchymal stem cell exosomes have been shown to promote cutaneous wound healing. Their bioactivity is most often attributed to their protein and nucleic acid components, while the function of exosomal lipids remains comparatively unexplored. This work specifically assesses the involvement of lipids and the transmembrane enzyme CD73 in the exosomes' biological activity in stimulating the cutaneous wound healing process. Since exosome preparation processes are not harmonized yet, certain production and purification parameters are first systematically investigated, enabling the optimization of a standardized protocol delivering high exosome integrity, yield, and purity. An in situ enzymatic assay is introduced to specifically assess the vesicle functionality, and quantitative proteomics is employed to establish the cell culture conditions yielding a stable exosome protein profile. Using a combination of in vitro approaches, CD73 and constitutional lipids of HPV-16 E6/E7 transformed human bone marrow stromal cell-derived exosomes are identified as key bioactive components promoting the exosome-driven acceleration of processes required for wound repair. A pilot wound healing study in mice indeed suggests a role of lipids in the exosomes' biological activity. Strikingly, the extent of the bioactivity of these exosomal components is found to be dependent on the target cell type. (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 24h conditioned medium Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Commercial method Filtration Ultrafiltration Protein markers EV: CD73/ TSG101/ CD9/ GAPDH/ CD63 non-EV: Calregulin Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HPV-16 E6/E7 transformed bone marrow mesenchymal stromal 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 Pelleting performed No Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Commercial kit qEVoriginal/70nm Characterization: Protein analysis Protein Concentration Method Micro BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD73/ TSG101/ CD9/ GAPDH/ CD63 Not detected contaminants Calregulin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mode Reported size (nm) 121 EV concentration Yes EM EM-type Transmission EM Image type Close-up, Wide-field Report size (nm) 25-250

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EV-TRACK ID	EV210264
species	Homo sapiens
sample type	Cell culture
cell type	HPV-16 E6/E7 transformed bone marrow mesenchymal stromal cells
condition	24h conditioned medium	48h conditioned medium	24h conditioned medium	24h conditioned medium
separation protocol	dUC/ Filtration	dUC/ Filtration	dUC/ Filtration	dUC/ qEVoriginal/70nm/ Filtration/ Ultrafiltration
Exp. nr.	1	2	3	4
EV-METRIC %	67	67	67	67