Serum-free long-term expansion and functional maintenance of pig satellite cells via targeted genetic engineering for cultured meat

Cultured meat represents a promising alternative for future meat consumption. However, the progress of research and industrialization has been hindered by the absence of serum-free media capable of supporting the long-term expansion of specific cells, such as satellite cells (SCs). In this study, we developed a serum-free proliferation medium (A19), which supported cell expansion of porcine SCs at early passages. Subsequently, using CRISPR/Cas-mediated genetic editing, we established <italic>CDKN2A</italic>^{<italic>−/−</italic>} SC lines that exhibited enhanced proliferation and differentiation capacity in serum conditions compared to <italic>CDKN2A</italic>^+/+ SCs. Notably, the serum-free medium allowed for the vigorous expansion of <italic>CDKN2A</italic>^{<italic>−/−</italic>}cells over 15 passages, while maintaining the expression of key myogenic factors. Further, we optimized the formula of serum-free differentiation medium, which effectively induced myogenic differentiation of serum-free cultivated <italic>CDKN2A</italic>^{<italic>−/−</italic>} SCs. Moreover, the serum-free media supported the generation of <italic>CDKN2A</italic>^{<italic>−/−</italic>} SCs-derived cultured meat constructs using three-dimensional plant-based edible scaffold. Collectively, our study establishes a comprehensive serum-free system for SCs-based cultured meat production through targeted genetic engineering.