The Impact of Collection Protocol on the Yield and Purity of Mesenchymal Stem Cell‐Derived Extracellular Vesicles Isolated From Serum‐Free Media

The immunomodulatory and regenerative effects of mesenchymal stem cell (MSC) extracellular vesicles (EVs) have spurred the development of strategies to manufacture these EVs as therapeutics. Clinical-grade EV manufacturing requires defined serum-free media (SFM) to reduce heterogeneity and improve batch reproducibility, yet standard protocols for collecting EVs from SFM are lacking. A primary concern is the co-isolation of SFM-derived proteins with EVs during separation, which can interfere with the characterization of the collected EVs, and their subsequent application. In this study, we evaluated how removing proteins from SFM affects EV yield and purity, and examined how collection timing and cell confluence influence EV yield and proteomic profile. A defined SFM (PPRF-msc6) was compared to (i) an ultracentrifuged medium (PPRF-msc6 after overnight ultracentrifugation), and (ii) a starvation medium (PPRF-msc6 without albumin and fetuin). MSC growth and viability were reduced in starvation medium, but were not adversely impacted when using ultracentrifuged medium. Compared to unmodified PPRF-msc6, ultracentrifuged medium improved EV purity but lowered EV yield. MSC confluence impacted the proteomic profile of EV fractions, demonstrating the importance of determining when EVs are collected during the culture period. Defining protocols for MSC-EV collection contributes to standardization within the rapidly growing EV field, and informs the development of clinically relevant bioprocesses.