Scalable production of anti-inflammatory exosomes from three-dimensional cultures of canine adipose-derived mesenchymal stem cells: production, stability, bioactivity, and safety assessment.

BACKGROUND: The therapeutic potential of exosomes derived from mesenchymal stem cells (MSCs) is increasingly recognized in veterinary medicine. This study explored the feasibility of a microcarrier-based three-dimensional (3D) culture system for producing the exosomes (cEXO). Investigations were conducted to enhance production efficiency, ensure stability, and evaluate the therapeutic potential of cEXO for anti-inflammatory applications while assessing their safety profile. RESULTS: The microcarrier-based 3D culture system improved efficient production of cEXO, yielding exosomes with acceptable profiles, including a size of approximately 81.22 nm, negative surface charge, and high particle concentration (1.32 × 10(9) particles/mL). Confocal imaging proved dynamic changes in cell viability across culture phases, highlighting the challenges of maintaining cell viability during repeated exosome collection cycles. Characterization via transmission electron microscopy, nanoparticle tracking analysis, and zeta-potential measurements confirmed the stability and functionality of cEXO, particularly when stored at -20 °C. Functional assays showed that cEXO exerted significant anti-inflammatory activity in RAW264.7 macrophages in an inverse dose-dependent manner, with no observed cytotoxicity to fibroblasts or macrophages. Acute toxicity testing in rats revealed no adverse effects on clinical parameters, organ health, or body weight, supporting the safety of cEXO for therapeutic use. CONCLUSIONS: This study highlights the potential of a microcarrier-based 3D culture system for scalable cEXO production with robust anti-inflammatory activity, stability, and safety profiles. These findings advance the development of cEXO-based therapies and support their application in veterinary regenerative medicine.