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 high-throughput screening through a Design of Experiments (DoE) approach to develop an animal-free, serum-free medium (MMM1) specifically for the C2C12 murine myoblasts model cell line with applicability in cultivated meat research including for pet food. Low cost, food-grade inputs such as methylcellulose and spirulina extract resulted in significant cell growth improvements. The optimised MMM1 formulation containing low cost, food-grade inputs, achieved cumulative population doublings comparable to 10% (v/v) fetal bovine serum over four consecutive passages. Furthermore, MMM1 supported scalable cell expansion on commercially available dextran-based microcarriers (Cytodex-3) in both static and agitated conditions 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 the pharmaceutical-grade DMEM/F12, but at a significantly lower cost, thus offering a viable strategy to substantially reduce biomanufacturing costs which is a critical challenge in cultivated meat production. HighlightsO_LIA serum-free medium formulation for C2C12 murine myoblasts (MMM1) C_LIO_LIC2C12 growth in MMM1 comparable to serum-based medium C_LIO_LIC2C12 growth in MMM1 in microcarrier culture as effective as serum-based medium C_LIO_LIMMM1 can be translated to be animal-free and fully food-grade C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/736713v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@18daf49org.highwire.dtl.DTLVardef@ef1ef0org.highwire.dtl.DTLVardef@d85b90org.highwire.dtl.DTLVardef@63725a_HPS_FORMAT_FIGEXP M_FIG C_FIG