Lyophilization Maintains the Storage Stability and Bioactivity of Mesenchymal Stem Cell–Derived Extracellular Vesicles

BackgroundExtracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) are emerging therapeutic candidates for bone regeneration, but their long-term stability remains a barrier to clinical translation. This study evaluated whether lyophilization supports the stable storage of extracellular vesicles derived from human epidural fat MSCs (hEF-MSCs) cell line. Research Design and MethodsEVs were isolated, lyophilized with 8.5 % sucrose and HEPES(4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)), and stored for 3 months at -80, -20, 4, and room temperature (20). After reconstitution in phosphate-buffered saline (PBS), structural characteristics were assessed using transmission electron microscopy, nanoparticle tracking analysis, and flow cytometry. Functional activity was evaluated using MC3T3-E1 osteogenic differentiation assays. The main outcome measures included morphology, particle counts, marker expression, cytotoxicity, and sequencing profiles. ResultsLyophilization maintained EV morphology, structural integrity, and particle distribution across all storage temperatures. Marker expression remained comparable among the conditions. Reconstituted EVs promoted osteogenic differentiation of MC3T3-E1 cells without evidence of cytotoxicity. Sequencing profiles revealed no significant differences among storage conditions. ConclusionsLyophilized EVs from hEF-MSCs cell lines exhibited stable structural and functional properties across a range of storage temperatures, supporting their suitability for further development in bone regeneration applications.