Production of Contractile Muscle Tissue Constructs Based on Cell Sheet Tissue Engineering

Statement of Purpose: Complex structural organization in the human body is a key factor to produce the appropriate tissue functionality. In mature skeletal muscle, the muscle fibers are highly oriented and the bundle structure is essential to produce its mechanical functions. Therefore, a technique for organizing the microstructure of native muscle tissue is required to produce a biomimetic engineered tissue. Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-grafted surfaces allow the production of a tissue-like cell monolayer, a "cell sheet", and our group have developed this tissue engineering technology for application in regenerative medicine. In this study, a micropatterned thermoresponsive cell culture substrate was prepared to control orientation of myoblasts and harvested as a cell sheet. Based on this anisotropic tissue unit, we have engineered 3D muscle tissues with wellorganized microstructures. Methods: The original procedures for the preparation of the micropatterned surfaces have been reported previously [1]. In this study, a slightly modified procedure was used to prepare the surface. Briefly, polyacrylamide (PAAm) was grafted spatio-selectively on a PIPAAm-grafted substrate through photo-induced polymerization process, resulting in two kinds of stripe patterns composed of PAAm-coPIPAAm and PIPAAm regions (50 m / 50 m stripes). Human skeletal muscle myoblasts were seeded onto the patterned surface. After reached confluence, the cells were harvested as a single continuous cell sheet by lowering culture temperature to 20 °C. To manipulate the harvested cell sheets, a gelatin gel-coated plunger was used. After the plunger was placed on a cell sheet, the cell sheet was layered onto another one. Moreover, the layered cell sheet construct was transferred onto a fibrin gel using the plunger. The construct was cultured for 3 weeks in 2% horse serum containing medium to induce differentiation into myotubes. To investigate the functionality of the tissue construct, the construct was stimulated electrically using electrical pulse generator. Results: Myoblasts seeded on the surface were aligned on the patterned surface and harvested as a cell sheet composed of aligned myoblasts by lowering culture temperature (Figure 1a). Using the gelatin gel-coated plunger, a cell sheet attached to the plunger was able to be transferred onto another cell sheets while maintaining the aligned orientation. As a result, multiple cell sheets were layered as designed three-dimensionally [2]. In this study, to mimic the anisotropic structure of the native skeletal muscle, cell sheets were layered with the same direction. Next, to make the construct mature, the myotubes were cultured on a normal culture dish in the long term. However, most myotubes spontaneously detached from the surface due to the contractile force of mature myotubes. To overcome this problem, cell sheets were transferred onto a fibrin gel in the same manner with the cell sheet layering using a gelatin gel-coated plunger. As shown in Figure 1b, aligned myotubes remained on the gel at 3 weeks after the transfer. Immunostaining images showed that the myotubes formed sarcomere structure which is essential for muscle contraction. In fact, the tissue construct showed contraction by electrical pulse stimulation. Importantly, since the myotubes were aligned even on the fibrin gel, the contraction was regulated directionally by organizing the aligned structure. Furthermore, the response of this tissue construct against drugs was able to be observed. For example, by adding some kinds of calcium ion channel blocker, muscle contraction was suppressed even under electrical pulse stimulation. Moreover, the response of the muscle tissue changed depending on drug concentration. These results indicates that this muscle tissue construct is potentially useful as an in-vitro tissue model for drug discovery.