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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV220293	1/1	Homo sapiens	Primary mesenchymal stromal cells	(d)(U)C DG	Nguyen, Vivian	2022	67%
Study summary Full title A human kidney and liver organoid-based multi-organ-on-a-chip model to study the therapeutic effects and biodistribution of mesenchymal stromal cell-derived extracellular vesicles All authors Vivian V T Nguyen, Shicheng Ye, Vasiliki Gkouzioti, Monique E van Wolferen, Fjodor Yousef Yengej, Dennis Melkert, Sofia Siti, Bart de Jong, Paul J Besseling, Bart Spee, Luc J W van der Laan, Reyk Horland, Marianne C Verhaar, Bas W M van Balkom Journal J Extracell Vesicles Abstract Mesenchymal stromal cell (MSC)-derived small extracellular vesicles (sEVs) show therapeutic potentia (show more...)Mesenchymal stromal cell (MSC)-derived small extracellular vesicles (sEVs) show therapeutic potential in multiple disease models, including kidney injury. Clinical translation of sEVs requires further preclinical and regulatory developments, including elucidation of the biodistribution and mode of action (MoA). Biodistribution can be determined using labelled sEVs in animal models which come with ethical concerns, are time-consuming and expensive, and may not well represent human physiology. We hypothesised that, based on developments in microfluidics and human organoid technology, in vitro multi-organ-on-a-chip (MOC) models allow us to study effects of sEVs in modelled human organs like kidney and liver in a semi-systemic manner. Human kidney- and liver organoids combined by microfluidic channels maintained physiological functions, and a kidney injury model was established using hydrogenperoxide. MSC-sEVs were isolated, and their size, density and potential contamination were analysed. These sEVs stimulated recovery of the renal epithelium after injury. Microscopic analysis shows increased accumulation of PKH67-labelled sEVs not only in injured kidney cells, but also in the unharmed liver organoids, compared to healthy control conditions. In conclusion, this new MOC model recapitulates therapeutic efficacy and biodistribution of MSC-sEVs as observed in animal models. Its human background allows for in-depth analysis of the MoA and identification of potential side effects. (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 Control condition Focus vesicles small 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 Density gradient Protein markers EV: Flotillin-1/ GAPDH non-EV: ATP5a/ Lamin A/C/ TOM20 Proteomics no EV density (g/ml) 1.13-1.14 Show all info Study aim Function/New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Primary mesenchymal stromal cells EV-harvesting Medium Serum free medium Cell count 10000000 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 SW 32 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 4 Wash: time (min) 60 Wash: Rotor Type SW 60 Ti Wash: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Continuous Lowest density fraction 0.25M Highest density fraction 2M Orientation Bottom-up Rotor type SW 60 Ti Speed (g) 190000 Duration (min) 960 Fraction volume (mL) 0.25 Fraction processing None Characterization: Protein analysis Protein Concentration Method NTA Western Blot Detected EV-associated proteins Flotillin-1/ GAPDH Not detected contaminants ATP5a/ Lamin A/C/ TOM20 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 149 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.00E+10

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EV-TRACK ID	EV220293
species	Homo sapiens
sample type	Cell culture
cell type	Primary mesenchymal stromal cells
condition	Control condition
separation protocol	dUC/ Density gradient
Exp. nr.	1
EV-METRIC %	67