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. 2022 Aug 16;13:960909. doi: 10.3389/fimmu.2022.960909

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Copyright © 2022 Papait, Ragni, Cargnoni, Vertua, Romele, Masserdotti, Perucca Orfei, Signoroni, Magatti, Silini, De Girolamo and Parolini

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Definition of hAMSC-CM fractionation protocol and EV characterization. (A) Workflow of the protocol used to identify and validate the most efficient centrifugation procedure to obtain EV-depleted and EV-enriched fractions from hAMSC-CM. (B) Particle size and concentration profiles from NTA data for both fractions (EV free and EVs) after centrifugation for the indicated times (1, 3, 9, and 24 h). CM and PBS were used as starting condition and background, respectively (N = 3, values indicated as mean ± SD). (C) EV recovery or contamination in the EV-free fraction for the tested centrifugation times (1, 3, 9, and 24 h) calculated from the NTA data with respect to EVs in the CM set as 100% (N = 3, mean ± SD). (D) Size analysis from NTA data for the EVs identified in the CM, EVs, or EV-free fractions obtained after centrifugation for the indicated times (1, 3, 9, and 24 h) (N = 3, mean ± SD). (E) Transmission electron micrographs of EVs in the 3-h centrifugation pellets showing characteristic cup-shaped morphology and size compatible with NTA data. Black arrows indicate the EVs. (F) Visualization of EVs (in the unprocessed CM or in EV-fraction) after a 3-h centrifugation), after CFSE staining and comparison with nanometric FITC-fluorescent beads of indicated sizes. A representative cytogram is presented. (G) % of EVs recovery after 3 h of centrifugation.