Fetal bovine serum concentration-dependent differentiation of multi-layered myoblasts

Skeletal muscle tissue engineering has gained significant attention for applications in drug screening and regenerative medicine. Among the available approaches for engineering skeletal muscle tissue, lamination of cell sheets is particularly promising because it enables the fabrication of tissues with defined size and shape. Myoblast differentiation into multinucleated myotubes is typically induced by reducing serum concentration. However, the optimal conditions for multi-layered skeletal myoblasts remain poorly characterized. In this study, we investigated the optimal concentration of fetal bovine serum (FBS) to induce multi-layered myoblast differentiation. Differentiation was evaluated by Western blotting, immunofluorescence, assessment of contractile capability, and analysis of multi-layered muscle construct formation. We observed that higher serum concentrations promoted multi-layered myoblast differentiation, compared to lower concentrations. To further explore the mechanism underlying this effect, we measured cell migration speed at different FBS concentrations and found that migration increased with FBS concentration, a change that may have facilitated differentiation.