Development and optimization of a Defined Serum-Free Medium for Muscle satellite cells towards sustainable meat cultivation

Cultured meat offers a promising alternative to conventional meat but relies heavily on fetal bovine serum (FBS), limiting scalability. Therefore, the creation of scalable, animal-free, and biologically compatible culture systems is essential for both cultured meat bio-manufacturing and muscle tissue engineering. This study addresses this challenge by developing a novel optimized serum-free growth medium, SFGM9, which is based on DMEM/F12 and supplemented with growth factors such as insulin-like growth factor 1 (IGF-1), platelet-derived growth factor (PDGF) and interleukin-6 (IL-6) to activate the PI3K-Akt pathway, thus overcoming the limitations of previous serum-free systems. The optimal composition was determined using response surface methodology, effectively compensating for the lack of proliferative signaling in the absence of serum. Transcriptomic analysis revealed that key pathways, including PI3K-Akt and MAPK, were downregulated under serum-free conditions; however, SFGM9 effectively restored these signaling activities thus improving MuSCs proliferation, delays senescence in later passages, maintained the expression of myogenic markers, mitochondrial function, and cell cycle activity. Further evaluation in a 3D culture system demonstrated that SFGM9 supported high levels of DNA synthesis, robust cell–scaffold adhesion, and morphological extension, indicating excellent biocompatibility and adaptability for tissue construction. Collectively, these findings suggest that SFGM9 not only provides stability and efficiency in 2D cell expansion but also exhibits strong compatibility with 3D culture environments, offering critical technical support for the scalable and animal-free production of cultured meat and tissue engineering with broad implications for biotechnology and regenerative medicine.