Novel method for fabrication of skeletal muscle construct from the C2C12 myoblast cell line using serum-free medium AIM-V.

We have fabricated muscle tissue from murine myoblast cell line C2C12 by modifying the previously reported method. Fabrication of skeletal muscle tissue has been performed in many ways including the use of a biodegradable scaffold, a collagen gel-embedded culture, or cell sheet tissue engineering, but the extent of tension generation remains low. Recently, a new skeletal muscle tissue engineering technique involving self-dissociation of a cell sheet from a laminin-coated polydimethylsiloxane surface was reported which mostly involved a primary cell culture or co-culture of C2C12 and 10T1/2 cells. In this study, we succeeded in fabricating muscle tissue using C2C12 cells alone by enhancing cell-cell attachment by the use of serum-free medium AIM-V. C2C12 cells were seeded on to a laminin-coated PDMS surface in a 35 mm culture dish with two silk sutures of 5 mm in length each pinned at two places 18 mm apart. Then, cells were allowed to differentiate in AIM-V, and the cells started to dissociate in a sheet-like manner after 5-8 days of differentiation. The cells remained attached to the silk sutures, and tissue having a cylindrical morphology was fabricated. After the cylindrical morphology had been obtained, the medium was changed to DMEM supplemented with 2% horse serum, followed by culture for an additional 5-8 days for maturation. Tissue fabricated using this method was excitable with electric pulse stimulation and the generated active tension was approximately 1.4x greater than that reported previously for a co-culture of C2C12 and 10T1/2 cells. Immuno-fluorescence study revealed the presence of a sarcomere structure within the fabricated tissue, and Western blotting confirmed the expression of muscle specific-proteins. The increased active tension generation compared to that with the previously reported method is probably attributable to the increased proportion of myogenic cells in the tissue. Myooid fabricated from mono-culture of C2C12 will be useful in the muscle study, especially in the area where gene modification is needed.