The ensured proliferative potential of myoblast in a serum-reduced condition with Methyl-b-cyclodextrin (MbCD)

Muscle fiber as a source of meat is a bundle of mature myotubes, whereas myoblasts are the only "proliferative cells" in the myogenic differentiation process in embryogenesis. To demonstrate the innovative concept of "cultured meat," it is crucial to amplify myoblasts at a low cost and without any ethical issues or environmental burden. To that end, it is necessary to realize the expansion of myoblasts in a serum-reduced or -free medium to produce myotubes and further myofibers (cultured meat) on a large scale. A critical challenge is the proliferation of myoblasts with inhibition of differentiation into myotubes even in a serum-reduced medium since myoblasts such as C2C12 cells, mouse skeletal muscle myoblast cells, rapidly differentiate into myotubes and lose proliferative capacity by switching a serum-rich (10%) medium into the serum-reduced (2%) medium. This study will show that Methyl-b-cyclodextrin (MbCD), a cholesterol-depleting agent, significantly inhibits myoblast differentiation into myotubes by lowering plasma membrane cholesterol on C2C12 cells and primary cultured chicken muscle cells. In addition, MbCD was found to efficiently block cholesterol-depending apoptotic cell death of myoblast, which suggests one of the mechanisms by which MbCD has potent competency to inhibit the differentiation of C2C12 myoblast cells since dead cells are required for cell fusion of neighboring myoblasts in the process of differentiation into myotubes. More importantly, MbCD consistently maintains the proliferation capacity of myoblasts only under differentiation conditions with a serum-reduced medium, but not under cell-growing conditions with a serum-rich medium. In conclusion, our findings could be considered a significant technology in ensuring myoblasts' proliferation capacity over the long term for a future serum-free condition in the production of cultured meat.