Impact of Long-Term Refrigeration, Freezing, and Repeated Freeze-Thaw Processes on the Physicochemical Characteristics of Extracellular Vesicles

Abstract Extracellular vesicles (EVs) are crucial mediators of intercellular communication and have significant diagnostic and therapeutic potential. However, their preservation poses considerable challenges. This study examined the stability of EVs derived from PC3 cells when stored in PBS, HBSS, or HBSS with 5% glucose under long-term storage conditions (4 °C, -30 °C, and -80 °C) and subjected to up to six freeze-thaw cycles. The physicochemical properties of EVs, including their concentration, size, morphology, and protein content, were assessed over 90-day period. The findings indicate that storage at -30 °C significantly compromised EV integrity across all buffers, resulting in particle loss and morphological degradation. Although storage at -80 °C better preserved particle concentrations, morphological instability persisted at this temperature. In contrast, storage at 4 °C in PBS was most effective for maintaining morphology, albeit with a reduction in protein content over time. Repeated freeze-thaw cycles, particularly at -30 °C, caused substantial damage. These results show that storage temperature, duration, and suspension buffer affect EV integrity, providing useful information for developing improved preservation methods.