Chicken Embryo Extract Remodeling of Extracellular Matrix Sustains Self-Renewal and Differentiation for Scaffold-Free Cell Sheet Formation

Fetal bovine serum (FBS) is commonly used in cell culture but can make up to 60% of total production costs, limiting the scalability of cultured meat (CM). Here, we investigated chicken embryo extract (CEE) as a functional and cost-effective substitute for FBS in culturing porcine muscle satellite cells and generating scaffold-free CM constructs. The 20% CEE + 5% horse serum (HS) medium enhanced myogenic cell growth and development while maintaining paired box 7 expression and up-regulating Myogenin, supporting the coexistence of self-renewing and differentiating states. Oxygen consumption and gene expression analyses revealed reduced oxidative metabolism alongside activation of self-renewal pathways. Transcriptomic analysis showed a specific increase in growth-factor-related genes in 20% CEE + 5% HS group, including CXCL12, TGFB3, and FGF1. Furthermore, 20% CEE + 5% HS differentiation media promoted the extracellular matrix and stable cell sheet organization. Stacked CEE-derived sheets yielded CM constructs with hardness and chewiness levels comparable to those of conventional pork cuts, while maintaining similar springiness and cohesiveness. Our findings show that 20% CEE + 5% HS is a feasible and cost-effective alternative to FBS, allowing for dual cell fate regulation and facilitates structured CM.