A Good Manufacturing Practice-grade standard protocol for exclusively human mesenchymal stromal cell-derived extracellular vesicles

Background aims. Extracellular vesicles (EVs) released by mesenchymal stromal cells (MSCs) may contribute to biological processes such as tissue regeneration, immunomodulation and neuroprotection. Evaluation of their therapeutic potential and application in future clinical trials demands thorough characterization of EV content and production under defined medium conditions, devoid of xenogenic substances and serum-derived vesicles. Addressing the apparent need for such a growth medium, we have developed a medium formulation based on pooled human platelet lysate (pHPL), free from animal derived xenogenic additives and depleted of EVs. Methods. Depletion of EVs from complete growth medium was achieved by centrifugation at 120 000 g for 3 h, which reduced RNA-containing pHPL EVs to below the detection limit. Results. Bone marrow (BM)-derived MSCs propagated in this medium retained the characteristic surface marker expression, cell morphology, viability and in vitro osteogenic and adipogenic differentiation potential. The proliferation rate was not significantly affected after 48 h but was decreased by 13% after 96 h. EVs collected from BM-MSCs cultured in EV-depleted medium revealed a similar RNA pattern as EVs generated in standard pHPL EV-containing medium but displayed a more clearly defined pattern of proteins characteristic for EVs. Reduction of pHPL content from 10% to 2% or serum-/pHPL-free conditions strongly altered MSC characteristics and RNA content of released EV. Conclusions. The 10% pHPL-based EV-depleted medium is appropriate for purification of exclusively human. MSC-derived EVs. With this Good Manufacturing Practice grade protocol, characterization and establishment of protein and RNA profiles from MSC-derived EVs can now be achieved to identify active components in therapeutic EVs for future clinical application.