The ensured proliferative capacity of myoblast in serum-reduced conditions with Methyl-β-cyclodextrin

Abstract

To produce muscle fibers for cultured meat on a large scale, it is important to expand myoblasts in a serum-reduced or serum-free medium to avoid cost, ethical, and environmental issues. Myoblasts such as C2C12 cells differentiate quickly into myotubes and lose their ability to proliferate when the serum-rich medium is replaced with a serum-reduced medium. This study demonstrates that Methyl-β-cyclodextrin (MβCD), a starch-derived agent that depletes cholesterol, can inhibit further differentiation of myoblasts at the MyoD-positive stage by reducing plasma membrane cholesterol on C2C12 cells and primary cultured chick muscle cells. Furthermore, MβCD efficiently blocks cholesterol-dependent apoptotic cell death of myoblasts, which is one of the mechanisms by which it inhibits the differentiation of C2C12 myoblast cells, as dead cells of myoblast are necessary for the fusion of adjacent myoblasts during the differentiation process into myotubes. Importantly, MβCD maintains the proliferative capacity of myoblasts only under differentiation conditions with a serum-reduced medium, suggesting that its mitogenic effect is due to its inhibitory effect on myoblast differentiation into myotube. In conclusion, this study provides significant insights into ensuring the proliferative capacity of myoblasts in a future serum-free condition for cultured meat production.

Aligned Collagen Sponges with Tunable Pore Size for Skeletal Muscle Tissue Regeneration

“…The lack of any increase in metabolic activity between days 3-10 of culture suggests that the myoblasts switched from a proliferation phase to a differentiation phase, as cell number is not expected to increase as myoblasts fuse to form myofibers. This was also likely because we transitioned the culture to a differentiation medium at this time point, which has been shown to limit myoblast proliferation while supporting differentiation [61]. The conclusion that differentiation was occurring in these scaffolds is supported by immunostaining images such as that found in Figure 7A, which reveals myofiber formation.…”