The clinical potential of extracellular vesicles (EVs) derived from mesenchymal stromal/stem cells (MSCs) necessitates overcoming formidable production and purification obstacles. Ensuring a smooth transition from research to clinical implementation entails standardized production and optimization, as well as specific medium requirements. Our aim is to identify a low protein content MSC medium that is animal-component-free and lacks primary human components, but still preserves the therapeutic function of obtained EV products.

The establishment of clonally immortalized MSCs (ciMSCs) represents a significant advancement that opens up new avenues for standardized processes and products. In terms of identifying a suitable MSC medium, the growth and EV secretion performance of ciMSCs was performed in different cell culture media, including DMEM media supplemented with differing concentrations of human platelet lysate (hPL) as wells as commercially serum-/xeno-free or chemically defined media. Expansion in hPL-free and chemically defined media turned out to require higher seeding densities, particular detachment agents, and plastic wares with specialized surfaces.

ciMSCs could be expanded in DMEM supplemented with hPL down to 1%. However, compared to DMEM supplemented with 2.5% hPL, the population doubling time was clearly increased. The ciMSCs also exhibited consistent expandability in serum-/xeno-free and in chemically defined media. However, preliminary results imply that in hPL-reduced/-free media significantly less EVs are secreted than in regular media.

Despite the reduced ciMSC-EV secretion, the therapeutic potency of resulting EVs remains to be investigated. After defining an appropriate MSC medium, future work will focus on refining downstream processing procedures for MSC-EV production, aligning with Good Manufacturing Practice standards, to facilitate the clinical application of MSC-EVs.

BG is a scientific advisory board member of Innovex Therapeutics SL, Mursla Ltd, ReNeuron Ltd. and PL BioScience. He is a founding director of Exosla Ltd. All other authors declare that they have no conflicts of interest.