Eliminating serum dependency of bovine mesenchymal stem cell expansion via media composition and genetic screen

The advancement of cultured fat toward viable industrial production is currently hindered by economic and technical constraints, including the formulation of cost-effective, chemically defined culture media. This study addresses serum dependency in bovine mesenchymal stem cells (bMSCs), a common cell source for cellular agriculture, and particularly for cultured fat given their well-characterized adipogenic potential. We show that while supplementing reduced-serum media with insulin-transferrin-selenium-BSA (ITS+) and fibroblast growth factor 2 (FGF2) mitigates short-term viability loss, these factors fail to sustain prolonged proliferation in serum-free media. To uncover the molecular barriers to serum-free adaptation, we performed a targeted CRISPR-Cas9 screen, identifying TP53 as the primary checkpoint restricting survival. Targeted TP53 disruption conferred a robust survival advantage, enabling long-term expansion in serum-free conditions. By contrast, PTEN knockout displayed an intermediate phenotype, improving viability relative to wild-type cells but falling short of the proliferative capacity achieved by TP53 ablation. Furthermore, we demonstrated that mitigating proteolytic stress using soybean trypsin inhibitor is essential for effective serum-free subculturing. These findings establish that p53 pathway disruption decouples adipose-derived bMSCs (Ad-bMSCs) expansion from serum dependency, offering a scalable strategy for chemically defined fat production.