Abstract: 

Rising environmental and ethical concerns associated with conventional livestock production have driven interest in cultivated meat (CM). CM involves growing animal cells under controlled conditions to produce meat without slaughtering animals. However, expensive cell culture media remains a major barrier to economically viable production. While research has largely focused on serum alternatives, free amino acids contribute significantly to media costs. This study investigated whether DMEM, a common nutrient-rich culture medium, could be diluted while maintaining the growth of C2C12 myoblast cells.

Cells were cultured in serum-containing media incrementally diluted with buffer (PBS) between 0-100% (v/v) while glucose and osmolality were controlled. 50% DMEM supported greater short-term cell proliferation than conventional 100% DMEM (1000 cells.cm^-2, n=1). However, beyond 48 hours, 100% DMEM showed higher proliferation. This suggests that DMEM nutrients, particularly amino acids, vitamins, and trace minerals, may become limiting with increased cell growth. Additionally, cells grown in 50% DMEM showed a more elongated morphology. Upon repeating the experiment with passage on day 2, no significant difference in cell proliferation was observed between 50% DMEM and 100% DMEM over 4 days (2000 cells.cm^-2, n=4 biological replicates). Hence, DMEM can be diluted by up to 50% without compromising cell growth, reducing media costs substantially.

Yeast lysate and hydrolysate were also investigated as DMEM replacements due to their high protein content and the established large-scale production of yeast. Both formulations resulted in cell death at 50% and 100% v/v replacement. This may be due to the release of harmful cellular components during homogenisation or the hydrolysis conditions. Future work should therefore investigate the composition of yeast extracts and evaluate their cytotoxicity. Additionally, analysis of spent-media metabolites, morphological changes, and cellular stress could further clarify the observed effects. Overall, this study demonstrates the potential of DMEM dilution for developing cost-effective culture media.