Sustained release of canine mesenchymal stem/stromal cell-derived extracellular vesicles rescues motor function in rodent spinal cord injury models

Introduction: Extracellular vesicles (EVs) are expected to be a novel promotes in regenerative therapy. EVs derived from canine mesenchymal stromal/stem cells (MSCs) have been shown to play an important role in tissue repair. For clinical application of EV therapy, a major challenge is to find scalable methods for EV collection and to facilitate continuing local EV action. The aim of this study is to develop collection methods for EVs derived from canine MSCs using anion exchange chromatography (AEX), and to determine the therapeutic effects of sustained release of EVs from cationized gelatin hydrogels in rats with spinal cord injury (SCI). Methods: Canine MSC-derived EVs were isolated from the culture supernatant by AEX. Lipopolysaccharide-stimulated BV-2 cells were used to determine the immunomodulatory effect of EVs. Distinct cross-linked cationized gelatin hydrogels were created by thermal dehydration. The amount of EVs captured by and released from hydrogels were measured using ELISA for CD63. The effects of sustained release of EVs on SCI in rats were evaluated via motor function and cavity volume. Results: EVs were collected from the culture supernatant of canine MSCs using AEX. Canine MSC derived EVs collected by AEX significantly reduced the expression levels of IL-1β and IL-6 in BV-2 cells induced by LPS. Sustained release of EVs from cationized hydrogels led to significant improvement of locomotion in SCI rats relative to the control group at 14- and 28-days post-injury. Cavity formation in the spinal cord with sustained release of EVs was significantly less than in the control group. Conclusions: Canine MSC derived EVs cultured in serum-free medium using AEX were successfully collected. Sustained release of canine MSC derived EVs suppresses cavity formation in the spinal cord after SCI and promotes improvement in motor function.