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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV170002	1/1	Homo sapiens	Umbilical cord mesenchymal stem cells	(d)(U)C SEC UF	Monguió-Tortajada M	2017	50%
Study summary Full title Nanosized UCMSC-derived extracellular vesicles but not conditioned medium exclusively inhibit the inflammatory response of stimulated T cells: implications for nanomedicine. All authors Monguió-Tortajada M, Roura S, Gálvez-Montón C, Pujal JM, Aran G, Sanjurjo L, Franquesa M, Sarrias MR, Bayes-Genis A, Borràs FE Journal Theranostics Abstract Undesired immune responses have drastically hampered outcomes after allogeneic organ transplantation (show more...)Undesired immune responses have drastically hampered outcomes after allogeneic organ transplantation and cell therapy, and also lead to inflammatory diseases and autoimmunity. Umbilical cord mesenchymal stem cells (UCMSCs) have powerful regenerative and immunomodulatory potential, and their secreted extracellular vesicles (EVs) are envisaged as a promising natural source of nanoparticles to increase outcomes in organ transplantation and control inflammatory diseases. However, poor EV preparations containing highly-abundant soluble proteins may mask genuine vesicular-associated functions and provide misleading data. Here, we used Size-Exclusion Chromatography (SEC) to successfully isolate EVs from UCMSCs-conditioned medium. These vesicles were defined as positive for CD9, CD63, CD73 and CD90, and their size and morphology characterized by NTA and cryo-EM. Their immunomodulatory potential was determined in polyclonal T cell proliferation assays, analysis of cytokine profiles and in the skewing of monocyte polarization. In sharp contrast to the non-EV containing fractions, to the complete conditioned medium and to ultracentrifuged pellet, SEC-purified EVs from UCMSCs inhibited T cell proliferation, resembling the effect of parental UCMSCs. Moreover, while SEC-EVs did not induce cytokine response, the non-EV fractions, conditioned medium and ultracentrifuged pellet promoted the secretion of pro-inflammatory cytokines by polyclonally stimulated T cells and supported Th17 polarization. In contrast, EVs did not induce monocyte polarization, but the non-EV fraction induced CD163 and CD206 expression and TNF-α production in monocytes. These findings increase the growing evidence confirming that EVs are an active component of MSC's paracrine immunosuppressive function and affirm their potential for therapeutics in nanomedicine. In addition, our results highlight the importance of well-purified and defined preparations of MSC-derived EVs to achieve the immunosuppressive effect. (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 (d)(U)C SEC UF Adj. k-factor 138.6 (pelleting) Protein markers EV: CD63/ CD73/ CD90/ MHC2/ CD9/ MHC1 non-EV: None Proteomics no Show all info Study aim Function, Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Umbilical cord mesenchymal stem cells EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g 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: time(min) 120 Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 138.6 Ultra filtration Cut-off size (kDa) 100 Membrane type NS Size-exclusion chromatography Total column volume (mL) 1 Sample volume/column (mL) 0.1 Resin type Sepharose CL-2B Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean;Median and size distribution Reported size (nm) 160-230 EV concentration Yes EM EM-type Cryo-EM Image type Close-up, Wide-field

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EV-TRACK ID	EV170002
species	Homo sapiens
sample type	Cell culture
cell type	Umbilical cord mesenchymal stem cells
condition	Control condition
separation protocol	(d)(U)C SEC UF
Exp. nr.	1
EV-METRIC %	50