Serum-free medium modulates the immunomodulatory and anabolic function of equine bone marrow-derived mesenchymal...

Adult bone marrow-derived mesenchymal stromal cells (BM-MSCs) hold significant therapeutic potential in human and veterinary medicine. Traditionally, BM-MSC cultures use fetal bovine serum (FBS) to promote growth, but this practice introduces batch variability and xenogeneic contamination that complicate clinical translation. While serum-free medium (SFM) alternatives have been optimized for human MSCs, limited options have been explored for alternative or veterinary species. Here, we compared a chemically defined, customizable SFM formulation with conventional serum-containing medium (SCM) for isolation and expansion of equine bone marrow mesenchymal cells (eqBM-MSCs). Both conditions supported adherence and trilineage differentiation. However, SFM cultures maintained a colony-based growth pattern across passages and showed up to 3-fold higher colony-forming output. SFM cells were enriched for a CD29⁺ population relative to SCM, despite slower proliferation. At early passage, SFM cells displayed a two-fold longer population doubling time than SCM with reduced cumulative expansion across serial passaging. Quantitative proteomics indicated that SFM expansion was associated with increased abundance of proteins mapping to immune signaling and repair-related pathways, including neutrophil degranulation, interferon-associated signaling, and hemostasis/angiogenesis, whereas SCM was enriched for pathways linked to proliferation and metabolism. Cytokine profiling further showed elevated KC-GRO, IL-8, and FGF in SFM relative to SCM. Together, these findings demonstrate the potential of a defined SFM platform to modulate eqBM-MSC growth behavior and protein/cytokine signatures, while highlighting a scalability trade-off between yield and phenotype that will require further optimization and functional validation.