Serum-free media development and validation for cultivation of C2C12 immortalised murine myosatellite cell line for cultivated meat

Abstract: The development of cost-effective, serum-free media is critical for scalable cultivated meat production. This study used Design of Experiments (DoE) and high-throughput screening to develop "MMM1", an animal-free, serum-free medium for the C2C12 murine myosatellite cell line. Low cost, food-grade inputs such as methylcellulose and spirulina extract proved significant growth improvements, but this was reversed by incorporating food-grade methylcellulose and Spirulina extract. The optimised MMM1 formulation achieved cumulative population doublings comparable to 10% (v/v) foetal bovine serum over four passages. Furthermore, MMM1 supported scalable cell expansion on dextran-based microcarriers (Cytodex-3) in spinner flasks, matching growth rates of serum-based controls. Finally, transitioning to a food-grade DMEM/F12 basal medium maintained cell proliferation equivalent to pharmaceutical-grade media, offering a viable strategy to substantially reduce biomanufacturing costs.

submitted to 'Food Chemistry'

摘要：开发兼具成本效益的无血清培养基，对于规模化培养肉生产至关重要。本研究采用实验设计（Design of Experiments, DoE）与高通量筛选技术，针对C2C12小鼠肌卫星细胞系开发出无动物源、无血清培养基MMM1。研究发现，甲基纤维素、螺旋藻提取物等低成本食品级原料可显著促进细胞生长，但同时添加该两类食品级原料时，促生长效果反而被逆转。经优化的MMM1配方在四代传代过程中，可实现与10%（体积比）胎牛血清相当的累积群体倍增数。此外，MMM1可支持在转瓶中依托葡聚糖基微载体（Cytodex-3）完成规模化细胞扩增，其细胞生长速率与含血清对照组持平。最后，将基础培养基替换为食品级DMEM/F12后，细胞增殖能力仍与药用级培养基相当，为大幅降低生物制造成本提供了可行策略。本研究投稿于《食品化学》。