Fabrication of scaffold-free contractile skeletal muscle tissue using magnetite-incorporated myogenic C2C12 cells

We have fabricated a functional skeletal muscle tissue using magnetite-incorporated myogenic cell line C2C12 and a magnetic field. Magnetite-incorporated C2C12 cells were patterned linearly on a monolayer of fibroblast NIH3T3 cells, using a magnetic field concentrator. After induction of differentiation, the C2C12 cells fused and formed multi-nucleated myotubes. The 3T3 layer became detached in a sheet-like manner after cultivation in differentiation medium for 5-8 days. When two separate collagen films were placed on a culture dish as tendon structures, a cylindrical construct was formed. Histological observation of the fabricated cylindrical tissue revealed the presence of multinucleate cells within it. Immunofluorescence staining of the construct showed the presence of sarcomere structures within the construct. Western blot analysis showed that muscle proteins were expressed in the construct. When the construct was stimulated with electric pulses, it exhibited active tension of approximately 1 μN. These results demonstrate that functional skeletal muscle tissue was formed through magnetic force-based tissue engineering. This is the first report of fabrication of skeletal muscle tissue with active tension-generating capability using magnetic force-based tissue engineering. The scaffold-free skeletal muscle tissue engineering technique presented in this study will be useful for regenerative medicine, drug screening or use as a bio-actuator.