Impact of donor pool size on the variability of platelet lysate-derived extracellular vesicles for regenerative medicine.

Aim: The objective of the present study was to determine the variability of platelet lysate-derived extracellular vesicles (pEV), in terms of characteristics and functionality through wound healing assays, when isolated either from platelet concentrates (PC, obtained from 5 donors) or from multiple PC (MPC, that is 50 donors).  Methods: pEV were isolated, under GMP-like conditions in a clean room, using Size Exclusion Chromatography (SEC). The differential characteristics between pEV obtained from PC (PC-EV) or from MPC (MPC-EV) were evaluated by means of protein concentration, Nanoparticle Tracking Analysis (NTA), Transmission Electron Microscopy (TEM), and flow cytometry using the MACSPlex™ arrays for surface analysis profiling of EVs. The functionality of the isolated pEV was determined in cell culture by metabolic activity and LDH activity determination and through a wound healing assay after 24h treatment.  Results: No significant differences were obtained in the pEV characteristics evaluated either isolated from PC or from MPC. As regards to functionality, a higher wound closure percentage was obtained in those pEV pools isolated from PC (5 donors). No differences on the coefficient of variation (CV) were found when comparing all the evaluated variables of pEV derived either from PC (5 donors) or from MPC (50 donors).  Conclusion: Our findings challenge the necessity of a high number donor pool for pEV isolation, revealing no significant variations in the analyzed variables of MPC-EV and PC-EV. Notably, our results suggest that, unlike platelet concentrates, a high number of donors is not required to reduce the variability of pEV; showing that the pool of only 5 donors can provide a consistent and reliable therapeutic product.