Multi-response optimization of defined and undefined serum-free media for enhanced proliferation of immortalized lamb muscle cells

The development of serum-free media (SFM) is critical for advancing the scalability and ethical viability of cultivated meat. To our knowledge, this study represents the first systematic, species-specific optimization of serum-free media for immortalized lamb muscle cells (ILMCs), introducing two novel formulations: LM7, a chemically defined medium comprising seven essential components for lamb muscle (LM), and LM8, a chemically undefined medium incorporating eight ingredients. Using design of experiments (DOE), response surface methodology (RSM), we systematically optimized both LM7 and LM8 to maximize ILMC proliferation, preserve cell morphology, and minimize media cost. A custom MATLAB-based image analysis pipeline enabled high-throughput viability classification from cell morphology. Several LM8 formulations achieved proliferation rates statistically comparable to 10% and even 20% fetal bovine serum (FBS). Both LM7 and LM8 sustained ILMC stemness (Pax 7 and Actin) and differentiation potential, as demonstrated by Desmin and myosin heavy chain staining. Finally, both media supported scalable culture in 3D microcarrier systems using CellBIND microcarriers, with significantly better performance observed for LM8 compared to LM7. These findings establish LM7 and LM8 as effective, serum-free platforms for cultivated lamb meat production, enabling both cost-efficiency and regulatory flexibility in cellular agriculture.