C2C12 Differentiation and Orientation Approach Towards Actuation Improvement for Biohybrid Robots

The weak maturity and poor alignment of myotube significantly limit the contractility of C2C12 mouse cell line, which is an important source of engineered skeletal muscle tissue powering biohybrid robots. Currently, substrate material, biochemical factor, topological clue and so on are proved to improve the maturing status and oriented growth of myoblasts, and co-stimulation methods stand a good chance of synergetic enhancement. Therefore, this paper invests C2C12 myogenic differentiation under triple-stimulation: soft substrate, coating and micro-groove structure. The impacts of these three factors are evaluated by the viability, orientation rate and fusion rate of C2C12 myotube, while the viability, obtained by CCK-8 test, is the basic assessment of cell proliferation; the orientation rate and the fusion rate indicates the potential for controllable contractility and the maturity of myotube, and both are analyzed and calculated from fluorescence image processing. After 7 days differentiational culture of C2C12 cell, with a GelMA substrate coated with 10 μg/cm2 fibronectin, the highest viability reaches 78.1%, and the wider (150 μm) and the shallower (25 μm) micro-groove scaffold provide more advantageous support for C2C12 differentiation and orientation. The results of this study are a referable protocol for unidirectional differentiation of C2C12 cell line based biohybrid actuator.