Native proteins from Galdieria sulphuraria to replace fetal bovine serum in mammalian cell culture

The use of fetal bovine serum (FBS) in cell culture applications causes high costs and unacceptable animal suffering when FBS is extracted from fetal calves. Despite efforts, the exact composition of FBS still remains partially unresolved. Native proteins in FBS, such as growth factors, and their binding to cell receptors seem to be crucial for cell proliferation and differentiation. Recently, algal extracts with high protein content were considered to reduce the FBS demand. Algae extracts yielded promising results as growth serum in mammalian cell culture. Nevertheless, the dependence on residual FBS and the undefined composition of algae extracts are challenges. In this study, we aimed to yield highly concentrated extracts of native proteins from mixotrophically grown Galdieria sulphuraria to replace FBS in mammalian cell culture. Crude extracts and native proteins were concentrated by ammonium sulfate precipitation, and all extracts underwent heat inactivation (HI) for selective protein inactivation. The remaining proteins' native conformation was verified by enzyme activity assays. All extracts were used to replace FBS during the cultivation of Chinese hamster ovary (CHO) cells, and proliferation was tested. We found that G. sulphuraria crude and protein extracts depended on HI to promote CHO cell growth to a similar extent as FBS. CHO cells grown with 5% or 10% heat-treated algal extracts had a relative proliferation of 260 to 230% compared to FBS controls with 210% and 300%, respectively. We anticipate our findings will help replace FBS in mammalian cell culture, increasing sustainability and consumer acceptance. KEY POINTS: Reproducible production of FBS substitutes from microalgae is a key to sustainable mammalian cell culture. Heat-treated native protein extracts of G. sulphuraria lead the way to new media additives. Identification of effective molecules is mandatory for the composition of a new culture medium.