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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	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

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EV-TRACK ID	YJ7428AP
species	Homo sapiens
sample type	Unconditioned serum-containing medium	Cell culture
cell type	NA	primary mucosal epithelial cells
medium	NA	Serum-containing medium
condition	Control condition	Control condition
separation protocol	(d)(U)C Filtration qEV UF	(d)(U)C Filtration qEV UF
Exp. nr.	1	2
EV-METRIC %	75	75