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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200183	1/2	Homo sapiens	mesenchymal stem cells derived from human Wharton's jelly	(d)(U)C Filtration Other;exoEasy Maxi Kit, Exo2D	Kim, Eun Seo	2021	75%
Study summary Full title Cellhesion VP enhances the immunomodulating potential of human mesenchymal stem cell-derived extracellular vesicles All authors Eun Seo Kim, Katsuhiko Kida, Jongsoo Mok, Yeonwoo Seong, Seo Yeon Jo, Tatsuro Kanaki, Masato Horikawa, Kyung-Hee Kim, Tae Min Kim, Tae Sub Park, Joonghoon Park Journal Biomaterials Abstract Mesenchymal stem cell (MSC) transplantation is a promising therapy for regenerative medicine. Howeve (show more...)Mesenchymal stem cell (MSC) transplantation is a promising therapy for regenerative medicine. However, MSCs grown under two-dimensional (2D) culture conditions differ significantly in cell shape from those in the body, with downregulated stemness genes and secretion of paracrine factors. Here, we evaluated the effect of 3D culture using Cellhesion VP, a water-insoluble material composed of chitin-based polysaccharide fibers, on the characteristics of human Wharton's jelly-derived MSCs (hMSCs). Cellhesion VP significantly increased cell proliferation after retrieval. Transcriptome analyses suggested that genes involved in cell stemness, migration ability, and extracellular vesicle (EV) production were enhanced by 3D culture. Subsequent biochemical analyses showed that the expression levels of stemness genes including OCT4, NANOG, and SSEA4 were upregulated and migration capacity was elevated in 3D-cultured hMSCs. In addition, EV production was significantly elevated in 3D cells, which contained a distinct protein profile from 2D cells. Gene and drug connectivity analyses revealed that the 2D and 3D EVs had similar functions as immunomodulators; however, 3D EVs had completely distinct therapeutic profiles for various infectious and metabolic diseases based on activation of disease-associated signaling pathways. Therefore, EVs from Cellhesion VP-primed hMSCs offer a new treatment for immune and metabolic diseases. (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 2D culture 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 Commercial method Protein markers EV: CD81/ TSG101/ CD9 non-EV: Calnexin 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 mesenchymal stem cells derived from human Wharton's jelly EV-harvesting Medium EV-depleted medium Preparation of EDS 8 hrs at 100,000 g;Other preparation Cell viability (%) 95 Cell count 6.00E+06 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Commercial kit Other;exoEasy Maxi Kit, Exo2D Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD9/ TSG101/ CD81 Not detected contaminants Calnexin Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 171 Used for determining EV concentration? Yes NTA Report type Mean Reported size (nm) 168 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 100

	EV200183	2/2	Homo sapiens	mesenchymal stem cells derived from human Wharton's jelly	(d)(U)C Filtration Other;exoEasy Maxi Kit, Exo2D	Kim, Eun Seo	2021	75%
Study summary Full title Cellhesion VP enhances the immunomodulating potential of human mesenchymal stem cell-derived extracellular vesicles All authors Eun Seo Kim, Katsuhiko Kida, Jongsoo Mok, Yeonwoo Seong, Seo Yeon Jo, Tatsuro Kanaki, Masato Horikawa, Kyung-Hee Kim, Tae Min Kim, Tae Sub Park, Joonghoon Park Journal Biomaterials Abstract Mesenchymal stem cell (MSC) transplantation is a promising therapy for regenerative medicine. Howeve (show more...)Mesenchymal stem cell (MSC) transplantation is a promising therapy for regenerative medicine. However, MSCs grown under two-dimensional (2D) culture conditions differ significantly in cell shape from those in the body, with downregulated stemness genes and secretion of paracrine factors. Here, we evaluated the effect of 3D culture using Cellhesion VP, a water-insoluble material composed of chitin-based polysaccharide fibers, on the characteristics of human Wharton's jelly-derived MSCs (hMSCs). Cellhesion VP significantly increased cell proliferation after retrieval. Transcriptome analyses suggested that genes involved in cell stemness, migration ability, and extracellular vesicle (EV) production were enhanced by 3D culture. Subsequent biochemical analyses showed that the expression levels of stemness genes including OCT4, NANOG, and SSEA4 were upregulated and migration capacity was elevated in 3D-cultured hMSCs. In addition, EV production was significantly elevated in 3D cells, which contained a distinct protein profile from 2D cells. Gene and drug connectivity analyses revealed that the 2D and 3D EVs had similar functions as immunomodulators; however, 3D EVs had completely distinct therapeutic profiles for various infectious and metabolic diseases based on activation of disease-associated signaling pathways. Therefore, EVs from Cellhesion VP-primed hMSCs offer a new treatment for immune and metabolic diseases. (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 3D culture 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 Commercial method Protein markers EV: CD81/ TSG101/ CD9 non-EV: Calnexin 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 mesenchymal stem cells derived from human Wharton's jelly EV-harvesting Medium EV-depleted medium Preparation of EDS 8 hrs at 100,000 g;Other preparation Cell viability (%) 95 Cell count 6.00E+06 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Commercial kit Other;exoEasy Maxi Kit, Exo2D Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD9/ TSG101/ CD81 Not detected contaminants Calnexin Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 153 Used for determining EV concentration? Yes NTA Report type Mean Reported size (nm) 182 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 100

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EV-TRACK ID	EV200183
species	Homo sapiens
sample type	Cell culture
cell type	mesenchymal stem cells derived from human Wharton's jelly
condition	2D culture	3D culture
separation protocol	dUC Filtration Other exoEasy Maxi Kit Exo2D	dUC Filtration Other exoEasy Maxi Kit Exo2D
Exp. nr.	1	2
EV-METRIC %	75	75