Scalable Production of Human Mesenchymal Stromal Cell (MSC)-Derived Extracellular Vesicles in Microcarrier-based Bioreactors under Xeno(geneic)-free Conditions

Background & Aim Mesenchymal stromal cells (MSC) show a great potential for tissue engineering and cell-based therapies due to their multilineage differentiation potential and their inherent immunomodulatory and trophic activities. Recently, increasing evidence suggests that several MSC-associated therapeutic features are mediated by extracellular vesicles (EVs). EVs display a small size, ideal to cross biological barriers, while resembling the cell membrane, providing high biocompatibility to target cells. Despite the promising potential of EVs for therapeutic applications, robust manufacturing processes that would increase the consistency and scalability of EV production are still lacking. In this work, a scalable bioreactor-based manufacturing system has been established for the production of MSC-derived EVs, aiming at their use for therapeutic purposes. Methods, Results & Conclusion In this work, EVs were produced by MSC isolated from different human tissue sources (bone marrow, adipose tissue and umbilical cord matrix). A xeno(geneic)-free microcarrier-based culture system was implemented in a Vertical-Wheel™ bioreactor, employing a human platelet lysate culture supplement (UltraGRO™-PURE), for the production of MSC-derived EVs and compared with traditional static culture systems (t-flasks). The microcarrier-based bioreactor system allowed to obtain EVs at higher concentration (6.5-fold increase) and productivity (3-fold increase) when compared to traditional static systems, yielding an average of 3.5 ± 0.3 × 1011 particles in a 60 mL final volume. The bioreactor system also allowed to obtain EVs with more homogeneous size distribution profiles, as determined by nanoparticle tracking analysis. The morphology and structure of the manufactured EVs were assessed by atomic force microscopy and specific protein markers (e.g. synthenin and CD63) were successfully identified in the EVs by Western blot. Overall, we demonstrate that this culture system is able to robustly manufacture human MSC-derived EVs in a scalable manner, which can be used as delivery vehicles or as intrinsic medicines in Regenerative Medicine settings.