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

Abstract

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.

Biomaterials in Tendon and Skeletal Muscle Tissue Engineering: Current Trends and Challenges

“…Of the materials used, collagen [ 173 , 174 , 176 , 188 , 199 , 200 , 203 , 213 , 243 ], fibrin [ 175 , 202 , 204 , 246 , 266 ], gelatin [ 182 , 183 , 194 , 267 ], alginate [ 177 , 245 ], and polymers, such as PLLA [ 180 ], PDMS [ 178 , 220 ], or PEG [ 196 , 268 ] generally functionalized or coated with adhesion peptides, are the most commonly found. To compensate for the mechanical weakness of hydrogels and their lack of conductive properties, which are useful in muscle tissue engineering [ 269 , 270 ], nanomaterials have often been added to the initial polymer.…”

Microenvironmental Modulation of Calcium Wave Propagation Velocity in Engineered Cardiac Tissues

“…To fabricate micromolded gelatin hydrogels on coverslips, we adapted previously published protocols. 6,29,46 Briefly, glass coverslips (25 mm diameter) were covered with low-adhesive tape. A 17-mm diameter circle was laser-cut (Epilog Mini 24 Laser Engraver, 30 Watt) into the tape and removed from the center.…”

A real-time monitoring platform of myogenesis regulators using double fluorescent labeling

“…The double fluorescent tagging allowed us to monitor both nuclei count and fusion levels over time on a collagen coated scaffold without the need for sacrificial staining. Similar applications are possible in a patterned substrate, which can allow for longer myotube stability in vitro [ 71 ].…”