The production of cultured meat hinges on the effective isolation, expansion, and differentiation of animal stem cells into consumable tissues. Central to this process are muscle stem cells, or satellite cells, due to their regenerative capabilities and ability to form muscle fibres. Historically, fetal bovine serum (FBS) has been utilised for its provision of nutrients and growth factors essential during cell culture. However, FBS presents significant drawbacks — it is costly, chemically undefined, subject to batch variability, and raises ethical and safety issues. These factors have impeded the industrialisation of cultured meat, necessitating the development of serum-free media that can sustain both the proliferation and differentiation of satellite cells over time.
In a pivotal study published in Food Materials Research on 25 June 2025, researchers Shijie Ding, Chunbao Li, and Guanghong Zhou from Nanjing Agricultural University unveiled a serum-free and genetically engineered satellite cell system. This innovation enables stable proliferation and efficient differentiation, laying a scalable foundation for sustainable cultured meat production.
The research team employed an iterative optimisation strategy to design a proliferation medium for porcine satellite cells. Starting with a basal DMEM/F12 medium supplemented with essential factors like ITS-X, BSA, Y-27632, and growth factors (bFGF, EGF, IGF-1, LIF), they assessed cell survival using high-content analysis. Formula 1 supported minimal vitality, leading to the development of Formula 2, which incorporated lipids, non-essential amino acids, and antioxidants, enhancing cell viability. Further refinement through single-factor testing with hydrocortisone, forskolin, HGF, dexamethasone, and LPA resulted in Formula 3, significantly boosting short-term proliferation.
The final optimisation yielded Formula 4, later named A19, composed of 19 components. A19 facilitated robust proliferation of primary satellite cells with over 90% viability across passages, maintaining high expression of myogenic regulators (PAX7, MYOD, MYOG) compared to serum controls. To counteract senescence, CRISPR/Cas9 technology was employed to create CDKN2A−/− satellite cell lines, which demonstrated markedly enhanced proliferation over 18 passages and upregulated myogenic gene expression compared with wild-type cells. CDKN2A−/− clones retained differentiation capacity into mature myotubes at early passages, unlike controls. When cultured in A19, these cells proliferated stably for at least 15 passages while maintaining stemness markers, confirming their compatibility with serum-free conditions.
The differentiation efficiency was further improved through stepwise media testing, culminating in a Version 4.0 medium that enabled elongated, MyHC-positive myotube formation from long-term cultured CDKN2A−/− cells. By seeding these engineered cells onto a plant-based 3D edible scaffold, the team generated meat-like constructs with superior texture parameters, such as chewiness and gumminess, compared to scaffolds alone. This confirmed that the combined serum-free proliferation and differentiation system with CDKN2A−/− satellite cells supports preliminary cultured meat production.
This dual strategy — developing serum-free media and engineering immortalised cell lines — tackles two major challenges in cultured meat production: cost reduction and stable scalability. By eliminating the requirement for animal-derived serum, the approach improves food safety, ethical acceptance, and manufacturing consistency. Furthermore, the CRISPR-based CDKN2A knockout cells provide a renewable source of muscle progenitors, diminishing reliance on repeated animal biopsies. These advancements mark a significant step toward commercially viable cultured pork, with potential applicability to other livestock species.
