Comparative Studies of Different Preservation Methods and Relative Freeze-Drying Formulations for Extracellular Vesicle Pharmaceutical Applications

[Image: see text] Extracellular vesicles (EVs) have been studied for years for their role as effectors and mediators of cell-to-cell communication and their potential application to develop new and increasingly performing nanotechnological systems for the diagnosis and/or treatment of many diseases. Given all the EVs applications as just isolated, functionalized, or even engineered cellular-derived pharmaceuticals, the standardization of reliable and reproducible methods for their preservation is urgently needed. In this study, we isolated EVs from a healthy blood cell line, B lymphocytes, and compared the effectiveness of different storage methods and relative freeze-drying formulations to preserve some of the most important EVs’ key features, i.e., concentration, mean size, protein content, and surface antigen’s expression. To develop a preservation method that minimally affects the EVs’ integrity and functionality, we applied the freeze-drying process in combination with different excipients. Since EVs are isolated not only from body fluids but also from culture media conditioned by the cells growing there, we decided to test both the effects of the traditional pharmaceutical excipient and of biological media to develop EVs solidified products with desirable appearance and performance properties. Results showed that some of the tested excipients, i.e., sugars in combination with dextran and glycine, successfully maintained the stability and integrity of EVs upon lyophilization. In addition, to evaluate the preservation of the EVs’ biological activity, we assessed the cytotoxicity and internalization ability of the reconstituted EVs in healthy (B lymphocytes) and tumoral (Burkitt’s lymphoma) cells. Reconstituted EVs demonstrated toxicity only toward the cancerous cells, opening new therapeutic opportunities for the oncological field. Furthermore, our study showed how some biological or cellular-conditioned fluids, commonly used in the field of cell cultures, can act not only as cryoprotectants but also as active pharmaceutical ingredients, significantly tuning the therapeutic effect of EVs, even increasing their cellular internalization.