Designing the MyoFusion Media: A Serum-Free Medium Optimized for Bovine Satellite Cell Differentiation

Cultivated meat production requires efficient differentiation of muscle progenitor cells into myotubes without relying on animal-derived serum, which poses ethical and scalability challenges. This study aimed to develop a chemically defined, serum-free medium optimized for bovine satellite cell differentiation. Using a data-driven design of experiments approach, we systematically screened 13 growth factors and supplements previously associated with myogenesis. Fusion index served as the primary metric for differentiation efficiency, complemented by additional morphological traits. Initial screening identified platelet-derived growth factor BB (PDGF-BB), cytosine arabinoside, and linoleic acid as key contributors, with transforming growth factor beta (TGF-β) included for its biological relevance. Subsequent optimization employed full factorial and central composite designs combined with response surface modeling to refine concentration ranges and evaluate interactions. The resulting formulation, termed MyoFusion, consists of DMEM supplemented with 4.5 × 10⁻⁶ mg/mL TGF-β1, 4.66 × 10⁻⁶ mg/mL PDGF-BB, 0.73 × 10⁻⁵ mg/mL cytosine arabinoside, and 2.07 × 10⁻⁴ mg/mL linoleic acid. Experimental validation demonstrated that MyoFusion achieved a fusion index of 65.08 ± 4.97, representing an improvement of up to 52.7% compared to serum-based controls (2% FBS). Correlation analyses confirmed strong associations between fusion index and other differentiation traits under serum-free conditions, indicating robust myotube formation. These findings establish MyoFusion as a promising serum-free alternative for cultivated meat applications, supporting ethical and scalable production while maintaining differentiation efficiency comparable to or exceeding traditional serum-containing media.