Small extracellular vesicles derived from sequential stimulation of canine adipose-derived mesenchymal stem cells enhance anti-infammatory activity

Background Small extracellular vesicles (sEVs) derived from mesenchymal stem cells (MSCs) are recognized for their
therapeutic potential in immune modulation and tissue repair, especially in veterinary medicine. This study introduces an innovative sequential stimulation (IVES) technique, involving low-oxygen gas mixture preconditioning using in vitro fertilization gas (IVFG) and direct current electrical stimulation (ES20), to enhance the anti-infammatory properties of sEVs from canine adipose-derived MSCs (cAD-MSCs). Initial steps involved isolation and comprehensive characterization of cAD-MSCs, including morphology, gene expression, and diferentiation potentials, alongside validation of the electrical stimulation protocol. IVFG, ES20, and IVES were applied simultaneously with a control condition. Stimulated cAD-MSCs were evaluated for morphological changes, cell viability, and gene expressions. Conditioned media were collected and purifed for sEV isolation on Day1, Day2, and Day3. To validate the efcacy of IVES for sEV production, various analyses were conducted, including microscopic examination, surface marker assessment, zetapotential measurement, protein quantifcation, nanoparticle tracking analysis, and determination of anti-infammatory activity.

Results We found that IVES demonstrated non-cytotoxicity and induced crucial genotypic changes associated with sEV production in cAD-MSCs. Interestingly, IVFG infuenced cellular adaptation, while ES20 induced hypoxia activation. By merging these stimulations, IVES enhanced sEV stability and quality profles. The cAD-MSC-derived sEVs exhibited anti-infammatory activity in lipopolysaccharide-induced RAW264.7 macrophages, emphasizing their improved efectiveness without cytotoxicity or immunogenicity. These efects were consistent across day 3 collection,
indicating the establishment of an efective protocol for sEV production.

Conclusions This research established an innovative sequential stimulation method with positive impact
on sEV characteristics including stability, quality, and anti-infammatory activity. This study not only contributes
to the enhancement of sEV production but also sheds light on their functional aspects for therapeutic interventions.