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Producing vesicle-free cell culture additive for human cells extracellular vesicles manufacturing
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International audience. A new paradigm has emerged recently, which consists in shifting from cell therapy to a more flexible acellular“extracellular vesicle (EV) therapy” approach, thereby opening a new and promising field in nanomedicine.Important technical limitations have still to be addressed for the large-scale production of clinical-grade EV.Cells are cultured in media supplemented with human platelet lysate (hPL) (xenogenic-free) or GMP-grade fetalcalf serum (FCS). However, these additives contain high amounts of EV that cannot be separated from cellsecreted-EV. Therefore, cells are generally maintained in additive-free medium during the EV secretionphase, however this can substantially limit their survival. In the present work, we developed a method to preparevesicle-free hPL (EV-free hPL) or vesicle-free FCS (EV-free FCS) using tangential flow filtration (TFF). We show avery efficient EV depletion (>98%) for both pure hPL and FCS, with a highly conserved protein content. Culturemedium containing our EV-free additives supported the survival of human bone marrow MSC (BM-MSC). MSCcould survive at least 216 h, their conditioned medium being collected and changed every 72 h. Both the cellsurvival and the cumulative EV production were substantially higher than in the starving conditions classicallyused for EV production. In EV-free hPL containing medium, we show that purified EV kept their morphologic andmolecular characteristics throughout the production.Finally, we tested our additives with 3 other cell types, human primary Endothelial Colony Forming Cells(ECFC) and two non-adherent human cell lines, Jurkat and THP-1. We confirmed that both EV-free hPL and FCSwere able to maintain cell survival and EV production for at least 216 h.Our method provides therefore a new option to help producing large amounts of EV from virtually anymammalian cells, particularly those that do not tolerate starvation. This method can apply to any animal serumfor research and development purpose. Moreover, EV-free hPL is clinical-grade compatible and allows preparingxenobiotic-free media for massive therapeutic EV production in both 2D (cell plates) and 3D (bioreactor) setting.
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