Functional replacement of fetal bovine serum by extracts from Galdieria sulphuraria in muscle cell culture

The development of serum-free culture systems is a major challenge in the large-scale production of cultured meat, where myoblasts must be expanded and differentiated without the use of fetal bovine serum (FBS). FBS remains the gold standard in myoblast culture, yet it poses ethical, economic, and environmental concerns. In this study, we tested the hypothesis, that protein-rich extracts derived from the red microalga Galdieria sulphuraria can functionally replace FBS during proliferation and differentiation of murine C2C12 skeletal muscle myoblasts. Native algal protein extracts were prepared, heat-treated, and fractionated by ammonium sulfate precipitation. The effects of these extracts on cell viability, metabolic activity, proliferation kinetics and terminal myogenic differentiation were systematically assessed under serum-reduced and serum-free conditions. Crude extracts exhibited cytotoxicity, whereas heat-treated and fractionated extracts enhanced cell viability and metabolic activity during short-term culture. During long-term cultivation, gradual replacement of FBS with heat-treated algal extracts supported sustained myoblast growth, albeit with reduced proliferation rates, and preserved the capacity for differentiation into multinucleated, myosin heavy chain-positive myotubes. Comparative proteomic analysis revealed distinct protein profiles between crude and heat-treated extracts, suggesting that thermal processing removes inhibitory components while enriching bioactive protein fractions. Collectively, these results demonstrate that G. sulphuraria-derived protein extracts can support long-term myoblast proliferation and differentiation following appropriate adaption, highlighting their potential as a sustainable and animal-free alternative to serum-derived supplements in muscle cell culture systems.