Currently, the fabrication of scaffolds for engineered skeletal muscle tissues is unable to reach the millimeter size. The main drawbacks are the poor nutrient diffusion, lack of an internal structure to align the precursor cells, and poor mechanical and electric properties. Herein, we present a combination of gelatin-carboxymethyl cellulose materials polymerised by a cryogelation process that allowed us to reach scaffold fabrication up to millimeter size and solve the main problems related to the large size muscle tissue constructs. (1) By incorporating carbon nanotubes (CNT), we can improve the electrical properties of the scaffold, thereby enhancing tissue maturation when applying an electric pulse stimulus (EPS). (2) We have fabricated an anisotropic internal three-dimensional microarchitecture with good pore distribution and highly aligned morphology to enhance the cell alignment, cell fusion and myotube formation. With this set up, we were able to generate a fully functional skeletal muscle tissue using a combination of EPS and our doped-biocomposite scaffold and obtain a mature tissue on the millimeter scale. We also characterized the pore distribution, swelling, stiffness and conductivity of the scaffold. Moreover, we proved that the cells were viable and could fuse in three-dimensional (3D) functional myotubes throughout the scaffold. In conclusion, we fabricated a biocompatible and customizable scaffold for 3D cell culture suitable for a wide range of applications such as organ-on-a-chip, drug screening, transplantation and disease modelling.
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