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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV190090	1/1	Homo sapiens	HCT116	(d)(U)C Filtration	Torres Crigna A	2019	67%
Study summary Full title Inter-Laboratory Comparison of Extracellular Vesicle Isolation Based on Ultracentrifugation All authors Torres Crigna A., Fricke F., Nitschke K., Worst T., Erb U., Karremann M., Buschmann D., Elvers-Hornung S., Tucher C., Schiller M., Hausser I., Gebert J., Bieback K. Journal Transfus Med Hemother Abstract Background/Aims: Extracellular vesicles (EVs), including microvesicles and exosomes, deliver bioacti (show more...)Background/Aims: Extracellular vesicles (EVs), including microvesicles and exosomes, deliver bioactive cargo mediating intercellular communication in physiological and pathological conditions. EVs are increasingly investigated as therapeutic agents and targets, but also as disease biomarkers. However, a definite consensus regarding EV isolation methods is lacking, which makes it intricate to standardize research practices and eventually reach a desirable level of data comparability. In our study, we performed an inter-laboratory comparison of EV isolation based on a differential ultracentrifugation protocol carried out in 4 laboratories in 2 independent rounds of isolation. Methods: Conditioned medium of colorectal cancer cells was prepared and pooled by 1 person and distributed to each of the participating laboratories for isolation according to a pre-defined protocol. After EV isolation in each laboratory, quantification and characterization of isolated EVs was collectively done by 1 person having the highest expertise in the respective test method: Western blot, flow cytometry (fluorescence-activated cell sorting [FACS], nanoparticle tracking analysis (NTA), and transmission electron microscopy (TEM). Results: EVs were visualized with TEM, presenting similar cup-shaped and spherical morphology and sizes ranging from 30 to 150 nm. NTA results showed similar size ranges of particles in both isolation rounds. EV preparations showed high purity by the expression of EV marker proteins CD9, CD63, CD81, Alix, and TSG101, and the lack of calnexin. FACS analysis of EVs revealed intense staining for CD63 and CD81 but lower levels for CD9 and TSG101. Preparations from 1 laboratory presented significantly lower particle numbers (p < 0.0001), most probably related to increased processing time. However, even when standardizing processing time, particle yields still differed significantly between groups, indicating inter-laboratory differences in the efficiency of EV isolation. Importantly, no relation was observed between centrifugation speed/k-factor and EV yield. Conclusions: Our findings demonstrate that quantitative differences in EV yield might be due to equipment- and operator-dependent technical variability in ultracentrifugation-based EV isolation. Furthermore, our study emphasizes the need to standardize technical parameters such as the exact run speed and k-factor in order to transfer protocols between different laboratories. This hints at substantial inter-laboratory biases that should be assessed in multi-centric studies. (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 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 Filtration Protein markers EV: TSG101/ CD81/ CD63/ CD9/ Alix non-EV: calnexin Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HCT116 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) 95 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g 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: time(min) 120 Pelleting: rotor type Surespin 630 (17 ml) Pelleting: speed (g) 23000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD9/ CD63/ TSG101/ Alix/ CD81 Not detected contaminants calnexin Flow cytometry specific beads Detected EV-associated proteins CD9/ CD63/ TSG101/ CD81 Not detected EV-associated proteins Alix Not detected contaminants calnexin Characterization: Lipid analysis No EM EM-type Transmission-EM Image type Close-up Report size (nm) 20-180

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EV-TRACK ID	EV190090
species	Homo sapiens
sample type	Cell culture
cell type	HCT116
condition	Control condition
separation protocol	(d)(U)C Filtration
Exp. nr.	1
EV-METRIC %	67