Effect of cell-extracellular matrix interaction on myogenic characteristics and artificial skeletal muscle tissue

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Although various types of artificial skeletal muscle tissue have been reported, the contractile forces generated by tissue-engineered artificial skeletal muscles remain to be improved for biological model and clinical applications. In this study, we investigated the effects of extracellular matrix (ECM) and supplementation of a small molecule, which has been reported to enhance α7β1 integrin expression (SU9516), on cell migration speed, cell fusion rate, myoblast (mouse C2C12 cells) differentiation and contractile force generation of tissue-engineered artificial skeletal muscles. When cells were cultured on varying ECM coated-surfaces, we observed significant enhancement in the migration speed, while the myotube formation (differentiation ratio) decreased in all except for cells cultured on Matrigel coated-surfaces. In contrast, SU9516 supplementation resulted in an increase in both the myotube width and differentiation ratio. Following combined culture with a Matrigel-coated surface and SU9516 supplementation, myotube width was further increased. Additionally, contractile forces produced by the tissue-engineered artificial skeletal muscles was augmented following combined culture. These findings indicate that regulation of the cell-ECM interaction is a promising approach to improve the function of tissue-engineered artificial skeletal muscles.

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Cell culture

C2C12 cells (within 10 passages; ATCC, Manassas, VA, USA) were seeded at the density of 2000 cells/cm2, and cultured in low glucose Dulbecco's modified Eagle's medium (DMEM; Nacalai Tesque, Kyoto, Japan) with 10% fetal bovine serum (FBS; Equitech-Bio, Kerrville, TX, USA), Penicillin-Streptomycin Mixed Solution (Nacalai Tesque), and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; Dojindo, Kumamoto, Japan) at 37°C in a 5% CO2 incubator. To induce myogenic differentiation, growth medium

Effect of ECM proteins on migration, proliferation, differentiation, and fusion of C2C12 cells

To investigate the effects of different ECM proteins on C2C12 cells the C2C12 myoblasts were cultured on the fibronectin, laminin, collagen I, collagen IV, and Matrigel coated culture surface. Time-lapse analysis showed that the C2C12 cells on each ECM coated surface migrated with significantly higher speed than that of cells on the control tissue culture surface (Fig. 1A). Next, to examine the effect of ECM protein on the cell viability of C2C12 cells, we cultured the cells in differentiation

Discussion

In this study, we demonstrated that the physical force generation of artificial skeletal muscle tissues could be improved using SU9516 and Matrigel, which contribute to the creation of a suitable myogenic environment in vitro. For drug screening and testing, the use of established cell lines, such as mouse C2C12 cells provide reproducible and repeatable systems, which resulted in the reduction of animal studies. Furthermore, drug screening using artificial skeletal muscle tissue would

Acknowledgments

This work was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion Science, Japan (Nos. 18K14063). We thank Kyoto University Radioisotope Research Center for use of facilities.

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