Reproducing the hierarchical structures of skeletal muscles is a potent strategy to produce cultured meats with the native texture of meats. In this study, to develop a method for reproducing the hierarchical structures, we investigated the effects of modifying the rheological properties of MCCGs on the morphologies of engineered muscles (EMs). The viscoelastic properties of MCCGs were modified using different chemical crosslinking methods: genipin crosslinking and riboflavin-assisted UV-irradiation. Dynamic viscoelasticity measurements of MCCGs showed a transition from physical to chemical gels due to chemical cross-links. We constructed EMs by seeding C2C12 into MCCGs. Comparison of the shrinkage ratios of the EMs indicated that the shape and size of EMs are determined by the balance between the viscoelasticity of the MCCGs and the cell-generated contractile forces. Fluorescence microscopy revealed that the formation of myotubes was promoted by chemical crosslinking. The myotubes aligned perpendicular to the longitudinal axis of the channel for EM-UV, whereas they oriented parallel for EM-GE. Anisotropic alignment of myotubes was not observed for the sample prepared using MCCG without chemical crosslinking. The results suggest that the morphologies and myo-differentiation of EMs can be regulated by modifying the rheological properties of MCCG.
Kazuya Furusawa (Wed,) studied this question.