Storage-associated physicochemical changes in extracellular vesicles derived from human mesenchymal stromal cells

Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) are considered a promising therapeutic tool due to their involvement in intercellular signaling and their ability to carry a wide range of biologically active molecules, including proteins, lipids, and nucleic acids. Standardized protocols for EV manufacturing and evaluation of EV properties under different storage conditions are important to ensure the quality, reproducibility, safety, and therapeutic efficacy of EV-based products. This study evaluated the physicochemical stability of EVs derived from MSCs of various tissue origins under different storage temperatures.

MSC populations were isolated from adipose tissue (ADSCs), umbilical cord (UC-MSCs), and dental pulp (DPSCs). The isolated cells met the minimal criteria for defining MSCs. EVs were isolated from conditioned media by differential ultracentrifugation and characterized by nanoparticle tracking analysis (NTA) and ELISA detection of the EV-specific markers CD63, CD81, and TSG101. Total protein concentration was determined using the Pierce BCA colorimetric assay. The physicochemical stability of MSC-derived EVs diluted in Hank's solution was evaluated at predefined intervals during storage at +4 °C to +6 °C, -20 °C, and -80 °C.

EV particle concentration and total protein concentration remained stable for at least 14 days at +4 °C to +6 °C and for at least 6 months at -20 °C. EV samples preserved their characteristics for at least 10 months when stored at -80 °C. The assessment of bFGF complemented conventional physicochemical characterization by providing information on the preservation of a selected biologically relevant quality attribute of EV preparations.

MSC-derived EVs diluted in saline solution retain physicochemical stability during long-term storage under clinically relevant conditions, supporting the further development of EV-based therapeutic products.