Optimizing 2D in vitro differentiation conditions for C2C12 murine myoblasts on gelatin hydrogel.

Optimizing in vitro differentiation protocols for skeletal muscle cells is essential for producing mature, functional myotubes suitable for disease modeling and therapeutic screening. While C2C12 murine myoblasts are a widely used model, achieving consistent and advanced differentiation remains challenging. In this study, we systematically evaluated conditions that improve myotube formation and maturation in a 2D culture system using ultra-compliant gelatin hydrogels. We compared standard and commercial differentiation media and identified that supplementation of DMEM with 2% horse serum and 10% Opti-MEM (DMO) supported robust myotube formation, with thin and aligned fibers. Insulin supplementation significantly increased expression of myosin heavy chain (MyHC) and calsequestrin, while pyruvate provided additional benefit by further enhancing myotube maturation. Media change frequency was also critical: daily replacement was necessary to maintain optimal differentiation, although the addition of insulin and pyruvate partly mitigated the effects of less frequent changes. Application of electrical pulse stimulation (EPS) improved sarcomeric α-actinin organization without significantly altering MyHC isoform expression. RNA sequencing confirmed transcriptional reprogramming consistent with myogenic progression, including early upregulation of key muscle-specific genes. Our findings present a cost-effective, reproducible protocol that supports advanced C2C12 differentiation in a scalable 2D system, offering practical guidance for generating mature, functional myotubes in vitro for both basic and translational muscle research.