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.
Key wordsSkeletal muscle Myoblasts Myotubes Gelatin Hydrogels Micromolding
This project was supported by the USC Viterbi School of Engineering, The Eli and Edythe Broad Foundation, USC Undergraduate Research Associates Program, USC Women in Science and Engineering, USC Provost’s Undergraduate Research Fellowship, and USC Provost’s PhD Fellowship. We acknowledge the W.M. Keck Foundation Photonics Center Cleanroom for photolithography equipment and facilities.