Fabrication of Micromolded Gelatin Hydrogels for Long-Term Culture of Aligned Skeletal Myotubes.

Cultured skeletal myotubes are a powerful in vitro system for identifying mechanisms of skeletal muscle development and disease. However, skeletal myotubes routinely delaminate from conventional culture substrates after approximately 1 week, which significantly hampers their utility for in vitro disease modeling and drug screening. To address this problem, we fabricated micromolded gelatin hydrogels as culture substrates that are more biomimetic than conventional substrates. On micromolded gelatin hydrogels, C2C12 skeletal myoblasts align and differentiate into skeletal myotubes that are stable in culture for multiple weeks. With this protocol, we detail three key steps: (1) Fabrication of micromolded gelatin hydrogels; (2) Culture of mouse C2C12 myoblasts and differentiation into myotubes; and (3) Quantification of myotube morphology. These substrates have many applications for skeletal muscle disease modeling and drug screening over longer time scales.