Alignment of Skeletal Muscle Cells Cultured in Collagen Gel by Mechanical and Electrical Stimulation

For in vitro tissue engineering of skeletal muscle, alignment and fusion of the cultured skeletal muscle cells are required. Although the successful alignment of skeletal muscle cells cultured in collagen gel has been reported using a mechanical force, other means of aligning cultured skeletal muscle cells have not been described. However, skeletal muscle cells cultured in a two-dimensional dish have been reported to align in a uniform direction when electrically stimulated. The purpose of this study is to determine if skeletal muscle cells cultured three-dimensionally in collagen gels can be aligned by an electrical load. By adding direct current to cells of the C2C12 skeletal muscle cell line cultured in collagen gel, it was possible to align C2C12 cells in a similar direction. However, the ratio of alignment was better when mechanical force was used as the means of alignment. Thus for tissue engineering of skeletal muscle cells, electrical stimulation may be useful as a supplementary method. 1. Introduction In order to construct functional skeletal muscle in vitro for use in tissue engineering, it is important to align skeletal muscle cells in a uniform direction. In a three-dimensional (3D) collagen gel culture without any applied stress, skeletal muscle cells do not orient uniformly. Although some research groups have shown the alignment of skeletal muscle cells in collagen gel using mechanical force [1, 2], other means of aligning skeletal muscle cells in 3D culture have not been reported. In monolayer culture, the application of a direct current (DC) electrical field to muscle cells causes myoblast axes to orient perpendicular to the electrical current [3–8]. However, it has not been determined if skeletal muscle cells will align in a 3D collagen gel culture using a similar electrical load. Thus we cultured skeletal muscle cells three-dimensionally under either a mechanical load or an electrical load and compared the rate of alignment. 2. Materials and Methods 2.1. Skeletal Muscle Cells Mature murine myogenic cell line C2C12 cells were purchased from Riken Cell Bank (Tsukuba, Japan). The culture medium was Dulbecco’s modified essential medium (DMEM, Sigma, St. Louis) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). 2.2. Mechanical and Electrical Load We made ring-shaped collagen gels containing C2C12 cells. Cells were suspended in 0.25% Trypsin/EDTA, centrifuged, and embedded in type I collagen gel solution (Cell-Matrix 3D cell culture kit, Nitta Gelatin Inc., Japan) at 3 × 105 cells/mL. The collagen gels were