Matrix-Dependent Enhancement of Proliferation and Myogenesis in Chicken Muscle Stem Cell Monolayers and Their Taste-Related Metabolites

The choice of extracellular matrix (ECM) coating can profoundly affect the behavior of muscle stem cells (MuSCs), yet optimal substrates for cultured chicken meat remain unclear. Here, we compared collagen, fibronectin, fish gelatin, porcine gelatin, Geltrex, and laminin coatings for their ability to support proliferation and differentiation of MuSCs isolated from 18-day-old chicken embryos. Cells were expanded over passages 4–6 (three-day intervals) or induced to differentiate for four days at each passage. Serial passaging led to a consistent increase in the differentiating PAX7⁻/MYOD⁺ population and a decrease in the self-renewing PAX7⁺/MYOD⁺ cohort, regardless of coating, with only a late-passage difference between porcine and fish gelatin. During proliferation, <italic>PAX7</italic> mRNA remained stable across all substrates, whereas <italic>MYOD</italic> levels rose significantly on laminin and Geltrex at passages 5 and 6. Upon differentiation, laminin, followed closely by Geltrex, supported the thickest myotubes and the highest fusion indices, accompanied by elevated <italic>MYOG</italic> and <italic>MYH1</italic> expression. In contrast, analyses of free amino acids and nucleotides revealed no substantial coating-dependent differences, except for glycine, which was significantly higher in differentiated cells on laminin than on fish gelatin coatings. Nonetheless, cultured cells, regardless of substrate, retained profiles distinct from those of embryonic and adult chicken tissues. These data identify laminin as the most effective single-component coating for enhancing chicken MuSC expansion and myogenic maturation. However, the persistent gap in flavor-related metabolite content underscores the need to further optimize culture conditions—such as medium composition and differentiation protocols—to more closely recapitulate native muscle tissue.