Generation of Bioactive Stem Cell-Derived Secretome in 3D Bioreactor System: Towards Cell-Free Therapy in Veterinary Medicine

Canine adipose-derived mesenchymal stem cells (cASC) are promising for regenerative veterinary medicine due to their immunomodulatory and reparative capacities. Three-dimensional (3D) culturing provides a more physiologically relevant environment than conventional two-dimensional (2D) monolayers, enhancing paracrine activity and therapeutic potential of mesenchymal stem cells (MSC). This study investigates the production and biological characterization of cASC secretome generated under hypoxic conditions with platelet lysate (PLT) supplementation, either in a 2D culture or in a stirred-tank 3D culture. Secretomes obtained from 3D cultures were compared with those from 2D cultures prepared under identical hypoxic and PLT-supplemented conditions. Quantitative analyses revealed enhanced secretion of key factors, including monocyte chemoattractant protein-1 (MCP-1) and vascular endothelial growth factor (VEGF), in 3D-derived secretomes. Functional in vitro assays demonstrated superior anti-inflammatory, pro-migratory, and antifibrotic effects of the 3D secretome, evidenced by nuclear factor kappa B (NF-κB) inhibition, increased fibroblast migration, and modulation of extracellular matrix gene expression. Additionally, the bioreactor system enabled consistent secretome production with reproducible biological activity. These findings indicate that 3D bioreactor cultivation under hypoxia with PLT supplementation can generate a biologically active secretome from canine adipose-derived stem cells, providing a promising basis for further exploration in veterinary regenerative applications.