Engineering muscle stem cells for sustainable cultured meat production

The production of cultured meat depends on the isolation, expansion, and differentiation of animal stem cells into edible tissues. Muscle stem cells, or satellite cells, are central to this process due to their ability to regenerate and form muscle fibers. Traditionally, fetal bovine serum (FBS) has been used to supply nutrients and growth factors during cell culture. However, serum is expensive, chemically undefined, prone to batch variability, and raises ethical and safety concerns. These challenges have hindered the industrialization of cultured meat. Addressing them requires serum-free media that can support both the proliferation and differentiation of satellite cells while maintaining their functionality over time.

A study (DOI: 10.48130/fmr-0025-0006) published in Food Materials Research on 25 June 2025 by Shijie Ding, Chunbao Li & Guanghong Zhou's team, Nanjing Agricultural University, establishes a serum-free and genetically engineered satellite cell system that enables stable proliferation and efficient differentiation, providing a scalable foundation for sustainable cultured meat production.

To systematically establish a serum-free system for cultured meat production, the researchers first employed an iterative optimization strategy to design a proliferation medium for porcine satellite cells (SCs). Beginning with a basal DMEM/F12 medium supplemented with essential factors such as ITS-X, BSA, Y-27632, and growth factors (bFGF, EGF, IGF-1, LIF), they tested survival using high-content analysis. While Formula 1 supported minimal vitality, Formula 2 was improved by adding lipids, non-essential amino acids, and antioxidants, which enhanced cell viability. Further single-factor testing with hydrocortisone, forskolin, HGF, dexamethasone, and LPA led to Formula 3, which significantly boosted short-term proliferation.

Final optimization of factor concentrations produced Formula 4, later named A19, containing 19 components. A19 supported robust proliferation of primary SCs with >90% viability across passages and maintained high expression of myogenic regulators (PAX7, MYOD, MYOG) compared to serum controls. To address senescence, CRISPR/Cas9 was used to generate CDKN2A^−/− SC lines, which displayed markedly enhanced proliferation over 18 passages and upregulated myogenic gene expression compared with wild-type cells. Notably, CDKN2A^−/− clones retained differentiation capacity into mature myotubes at early passages, unlike controls. When cultured in A19, CDKN2A^−/− cells proliferated stably for at least 15 passages while maintaining stemness markers, demonstrating compatibility with serum-free conditions.

Differentiation efficiency was further improved through stepwise testing of media, culminating in a Version 4.0 medium that enabled elongated, MyHC-positive myotube formation from long-term cultured CDKN2A^−/− cells. Finally, by seeding these engineered cells onto a plant-based 3D edible scaffold, the team generated meat-like constructs. These exhibited improved texture parameters such as chewiness and gumminess compared to scaffolds alone, confirming that the combined serum-free proliferation/differentiation system with CDKN2A^−/− SCs can support preliminary cultured meat production.

This dual strategy—developing serum-free media and engineering immortalized cell lines—addresses two of the most pressing challenges in cultured meat production: cost reduction and stable scalability. By eliminating the need for animal-derived serum, the approach enhances food safety, ethical acceptance, and consistency in manufacturing. Moreover, the CRISPR-based CDKN2A knockout cells provide a renewable source of muscle progenitors, reducing dependence on repeated animal biopsies. Together, these innovations mark a critical step toward commercially viable cultured pork and potentially extendable to other livestock species.