Stability and Function of Extracellular Vesicles Derived from Immortalized Human Corneal Stromal Stem Cells: A Proof of Concept Study

Abstract With significant advancement and development of extracellular vesicle (EV)-based therapies, there is a growing need to understand how their storage affects their physical and functional characteristics. EVs were isolated from the conditioned medium of a corneal stromal stem cell line (imCSSC) using Total Exosome isolation kit (TEI) and ultracentrifugation (UC) combined protocol. Purified EVs were stored at 4°C, − 80°C, room temperature (RT) after lyophilization with or without trehalose for 4 weeks. EVs stored at − 80°C and RT (lyophilization with trehalose) demonstrated a comparable morphology, while the freeze-dried samples without trehalose showed aggregation and degradation under a transmission electron microscope (TEM). Lyophilized samples without trehalose demonstrated a decreased particle concentration, recovery rate and protein concentration, which was remediated by the addition of trehalose. EVs stored at − 80℃ showed no change in the protein expression of CD9, CD63, and CD81. Regardless of the storage condition, all EV samples investigated reduced inflammation, as well as inhibited expression of fibrotic markers in vitro . Lyophilization of EVs with trehalose was a feasible storage method that retained the physical property and in vitro biological activities of EVs after 4 weeks of storage, while − 80°C offered the best retention of imCSSC-derived EV physical properties. For the first time, this data demonstrated a practical and translatable method for the storage of CSSC-derived EVs for clinical use. Graphical Abstract