Medium Volume Modulates C2C12 Myoblasts Differentiation Efficiency and Expression of Myod1 and Myog

Background: Differentiation of C2C12 myoblasts into multinucleated myofibers is a well-established model for studying skeletal muscle biology. While numerous factors influencing myogenesis have been thoroughly investigated, the effects of medium volume during differentiation of myoblasts have not been extensively explored, and most studies do not report the specific medium volumes used during differentiation. In this study, we investigated how medium volume affects myogenic differentiation, metabolic activity, and gene expression in C2C12 cultures.Materials and methods: The impact of different medium volumes (100, 250, and 400 µL/cm²) on C2C12 differentiation was evaluated over a five-day period, with medium replacement every two days. Morphological assessments and metabolic activity assays were performed at the end of differentiation. Gene expression analyses for Myod1, Myog, Myf6, Myh1 and Myh7 were conducted through RT-qPCR on initial cultures and on the final day of differentiation for comparison.Results: The expression levels of the early myogenic regulatory factors Myod1 and Myog were inversely correlated with medium volume, whereas the late differentiation markers Myh1 (type II) and Myh7 (type I) remained unaffected. Cultures differentiated with higher medium volume presented both increased metabolic activity and a higher differentiation index at the end of differentiation period.Conclusion: Medium volume significantly affects the differentiation of C2C12 myoblasts. Higher medium volumes increased myofiber thickness, differentiation index, and metabolic activity, and reduced the expression of the early myogenic markers Myod1 and Myog. These findings highlight medium volume as an important experimental parameter that should be reported alongside medium composition to improve reproducibility and physiological relevance of in vitro skeletal muscle differentiation studies. Keywords: C2C12 differentiation, myogenesis, myogenic regulatory factors, skeletal muscle in vitro model