Lyophilized Extracellular Vesicles Retain Regenerative Activity and Accelerate Wound Healing

Extracellular vesicles (EVs) are promising therapeutic agents for tissue regeneration because they regulate intercellular communication and modulate inflammatory responses. However, preserving EV bioactivity during long-term storage remains a major barrier to clinical application. We examined whether lyophilized EVs stored at −80 °C maintain their structural integrity and therapeutic efficacy in an in vivo wound-healing model. Mesenchymal stem cell-derived EVs were isolated and freeze-dried before storage at −80 °C. We evaluated EV physicochemical characteristics before and after lyophilization using nanoparticle tracking analysis, transmission electron microscopy, and EV marker-expression analysis. To assess regenerative efficacy, lyophilized EVs were applied topically to full-thickness ear wounds in CCR2-GFP mice. Wound-healing progression and CCR2-positive cell infiltration were monitored during tissue recovery using intravital microscopy. Lyophilized EVs preserved their characteristic morphology, particle-size distribution, and EV surface marker expression after storage. In vivo analysis showed that EV-treated wounds closed significantly faster than phosphate-buffered saline-treated controls. Additionally, lyophilized EV treatment reduced CCR2-positive inflammatory cell recruitment during healing, suggesting an immunomodulatory role in tissue regeneration. These findings show that EVs lyophilized and stored at −80 °C retain biological function and therapeutic potential in vivo. Lyophilized EVs may, therefore, provide a practical strategy for long-term storage and delivery of EV-based regenerative therapeutics.