Osteoarthritis (OA) is the most common degenerative joint disease in horses and humans, but no effective treatment exists yet. Mesenchymal stromal cells (MSCs) are promising therapies as they secrete bioactive molecules, collectively known as the secretome. However, the translation of these therapies has been limited due to a poor understanding of MSC mechanisms of action. To bridge the gap between experimental and clinical results, representative in vitro models are needed. To this end, spheroids of different cell types (chondrocytes, synovial fibroblasts, and osteoblasts) will be cultured in a hydrogel containing cell-derived extracellular matrix, crosslinked with chemically modified alginate, and subjected to media perfusion and mechanical loading. To develop a biomimetic model for OA, mechanical overloading and perfusion with conditioned medium from pro-inflammatory macrophages will be added. Various MSC sources will be evaluated to identify the most effective source for OA treatment. To assess their full cartilage regeneration potential, MSC from the best MSC source will be cultured within the OA microsystem. Finally, biologically active factors involved in the observed beneficial effects will be identified using metabolomics and lipidomics. This approach focuses on advancing the translation of MSC-based therapies from the bench to the clinic, ultimately benefiting both equine and human patients.