Optimizing extracellular vesicle yield for clinical application

Abstract

Background & Aim Regenerative medicine has seen great expansion in the last decade, with more than one thousand current clinical trials involving mesenchymal stem/stromal cell (MSC) treatment. In recent years, multiple groups have shown that MSCs exert their beneficial effects by the release of paracrine factors, including growth factors, cytokines, and extracellular vesicles (EVs). EVs are contained within multi-vesicular bodies and released to the cytoplasm by exocytosis. EV cargo includes free fatty acids, miRNA, and proteins. The therapeutic advantages of cell-free EVs include their small size, low immunogenicity, inability to form tumors, and ability to reach target sites by systemic delivery. The goal of this project is to develop a GMP protocol for the manufacturing of clinical-grade EVs that meet regulatory requirements. Methods, Results & Conclusion Condition media from clinical-grade MSCs (cultured in an automated bioreactor) were used, and EVs were isolated by tangential flow filtration (TFF). The primary independent variables assessed in this project were 1) filter pore size, 2) storage solution, and 3) freeze-thaw. Particle size and counts were analyzed by nanoparticle tracking analysis and protein content by bicinchoninic acid (BCA) protein BCA analysis. Other optimizations included filtration settings, filtration flow rate, pre-centrifugation, sample volume:filter surface ratio, and fibronectin contamination. A filter size of 500kD resulted in a 53.5% increase in particle size compared to a 300kD filter. More particles were detected in phosphate buffered saline(PBS)-isolated EVs compared to lactated ringers solution (LRS)-isolated EVs; however, EVs stored in PBS were 27% larger than EVs stored in LRS. EV particle counts post-freezing also consistently increased compared to freshly-isolated EVs. These results show that TFF is a promising closed system for manufacturing clinical-grade EVs.