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

## TL;DR

Chicken embryo extract can replace expensive fetal bovine serum in cultured meat production, supporting cell growth and forming meat-like structures.

## Contribution

CEE is shown to be a cost-effective alternative to FBS for culturing muscle cells and forming scaffold-free meat constructs.

## Key findings

- 20% CEE + 5% HS medium supports myogenic cell growth and maintains self-renewal and differentiation.
- CEE promotes extracellular matrix remodeling and stable cell sheet organization.
- CEE-derived meat constructs have texture similar to conventional pork.

## Abstract

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.