Mesenchymal Stromal Cell-derived Extracellular Vesicles (MSC-EVs) hold great promise for treating a wide array of diseases, notably due to their immunomodulatory properties. MSC-EVs are increasingly studied as a cell-free therapeutic option that overcomes the limitations associated with cell-based therapies. However, achieving robust and scalable manufacturing of potent MSC-EV products remains challenging. This is largely due to various factors throughout the manufacturing process, particularly cell culture conditions and the choice of cellular source. Regarding cell culture conditions, human platelet lysate (hPL) has increasingly been used as a replacement for fetal bovine serum (FBS) in the development of therapeutics. However, the presence of coagulation factors in hPL poses challenges during the in vitro culture of MSCs and subsequent EV preparations from conditioned media. To address this issue, we compared various strategies to effectively deplete coagulation factors from hPL. We found that the most effective method was a CaCl2 declotting procedure, which did not affect the characteristics of MSCs and enabled the preparation of potent MSC-EV products from bone marrow-derived MSCs. Concerning the cellular source, bone marrow has historically been used as the gold standard to obtain MSCs for MSC-EV production. However, we observed that the potency of bone marrow-derived MSC-EVs varies significantly among different donors and batches, both in vitro and in vivo. Therefore, we investigated whether other sources, such as perinatal derivatives like umbilical cord and placental-derived MSCs, could overcome the limitations of bone marrow-derived MSCs. We found that the potency of MSC-EV products prepared from umbilical cord and placental tissues varied significantly, similar to bone marrow-derived MSCs. Thus, we concluded that developing a robust manufacturing process for MSC-EV products based on primary MSCs is hardly achievable. To overcome this challenge, we developed a method to immortalize primary MSCs to create cell lines and expand them at the clonal level (ciMSCs). ciMSCs retained their bona fide characteristics and, importantly, potent ciMSC-EVs can be prepared reproducibly, demonstrating beneficial effects in vitro and in vivo in animal models, including ischemic stroke, hypoxic-ischemic encephalopathy, and Alzheimer's disease.

Furthermore, we identified an important parameter in the raw material influencing the manufacturing of potent ciMSC-EVs: the hPL batches used during cell expansion. In summary, this doctoral dissertation aimed to address several limitations of MSC-EV manufacturing for therapeutics. Firstly, we optimized cell culture conditions by developing a new declotted CaCl2-hPL, which facilitated cell culture and EV preparation. Secondly, we addressed the heterogeneity among donors and batches of different tissue sources for MSCs. Finally, we designed ciMSCs for the robust manufacturing of ciMSC-EVs, which showed beneficial effects in vitro and in vivo in several animal models, bringing closer the successful translation of MSC-EVs into therapy.