Protocol for in vitro differentiation of C2C12 myoblasts into mature myotubes

Background: Mouse C2C12 myoblast cell line is widely used as a good in vitro model to study muscle physiology, metabolism, and regeneration. The in vitro model is capable of replicating the main steps of skeletal muscle development, which may help in investigating the molecular mechanism behind myogenesis, muscle degeneration, especially in cancer patients, and in the atrophy of muscles in older people. Objective: Considering the in vitro muscle differentiation study, there is a need to develop an ideal protocol for C2C12 myoblast differentiation. Results: C2C12 myoblasts were differentiated into myotubes within 4 days when cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 2% horse serum, which represents the key feature of myogenesis. Before differentiation, C2C12 cells were cultured up to 90–100% confluence, then the medium was replaced with differentiation media. Then, the cells started to differentiate, on day 2 (D2); the cells were fused with neighboring cells to form long cells. On day 3, the cells became more elongated, and on day 4, myotubes were clearly detected, having a cylindrical and elongated shape. The cells can be maintained at this stage for more than a week with regular media changes. Conclusion: This protocol could be helpful for translational research related to cancer, genetic disorders, and age-related muscle-wasting diseases.