A serum-free media formulation for cultured meat production supports bovine satellite cell differentiation in the absence of serum starvation [SF_Diff]

Production of cultured meat requires the robust differentiation of satellite cells into mature muscle fibers without the use of animal-derived components. Current protocols induce myogenic differentiation in vitro through serum starvation, an abrupt reduction in serum concentration. Here, we used RNA sequencing to investigate the transcriptomic remodelling of bovine satellite cells during myogenic differentiation induced by serum starvation. We characterized canonical myogenic gene expression, and identified surface receptors upregulated during the early phase of differentiation. Supplementation of ligands to these receptors enabled the formulation of a chemically defined media that induced differentiation in the absence of serum starvation and/or transgene expression. Serum-free myogenic differentiation was of similar extent to that induced by serum starvation, as evaluated by transcriptome analysis, protein expression and the presence of a functional contractile apparatus. Moreover, the serum-free differentiation media supported the fabrication of mature three-dimensional bioartificial muscle constructs, demonstrating its suitability for cultured beef production. Satellite cells were cultured in either FBS-containing growth media (GM) containing 20% FBS or in serum-free growth media (SFGM). For the 72h myogenic differentiation, cells in GM were switched to differentiation media (DM) containing 2% FBS while cells in SFGM were either switched to serum-free base media (SFB), serum-free differentiation media with a DMEM/F-12 base (SFDM) or with a DMEM base (DMEM). RNA samples were taken from at 0 h from GM and SFGM, and at 72 h from DM, SFDM and DMEM in biological triplicates (A, B, C).