Whey as cell growth supplement in cultivated meat production

Milk whey is one of the earliest dairy by-products, shown to promote the growth of various mammalian cell types. Milk contains three vital elements of cell culture media as growth factors, adhesion factors, and transport proteins. Presently we demonstrated whey proteins (WPs) as a sustainable alternative to FBS in the cultivation of C2C12 muscle cells, for application in cultivated meat production. Cells were cultured in media with WP mixture (β-lactoglobulin 0.07%, α-lactalbumin 0.15%, bovine serum albumin 0.15%), 10% FBS (positive control), or without (negative control) for 48 h (day-1 and -2) to support proliferation. Subsequently, cells were switched to low-mitogenic 2% horse serum medium, until full differentiation (day-6). Cells cultured in both WPs and FBS maintained cell membrane integrity (lactate dehydrogenase assay) (p<0.05) and substantially improved cell viability (XTT-assay) (p<0.05) and density (microscopic analysis) compared to untreated cells on day-2 of proliferation. On day-6, these cells underwent morphological changes from spindle-shape to fused-elongated myotubes, and exhibited increased levels of myogenesis-specific markers, creatine kinase and citrate synthase (p<0.05). Also, RT-qPCR analysis showed increased expressions of myogenesis-specific genes (MYOG, DES, MRF4, MYH2) compared to untreated cells (p<0.05). Further, preliminary proteomics analysis revealed the positive regulation of cellular pathways corresponding to myogenesis in cells cultured in WP media.