Skeletal Muscle Spheroids as Building Blocks for Engineered Muscle Tissue.

ABSTRACT

Spheroids exhibit enhanced cell-cell interactions that facilitate improved survival and mimic the physiological cellular environment in vivo. Cell spheroids have been successfully used as building blocks for engineered tissues, yet the viability of this approach with skeletal muscle spheroids is poorly understood, particularly when incorporated into three-dimensional (3D) constructs. Bioprinting is a promising strategy to recapitulate the hierarchical organization of native tissue that is fundamental to its function. However, the influence of bioprinting on skeletal muscle cell spheroids and their function are yet to be interrogated. Using C2C12 mouse myoblasts and primary bovine muscle stem cells (MuSCs), we characterized spheroid formation as a function of duration and cell seeding density. We then investigated the potential of skeletal muscle spheroids entrapped in alginate bioink as tissue building blocks for bioprinting myogenic tissue. Both C2C12 and primary bovine MuSCs formed spheroids of similar sizes and remained viable after bioprinting. Spheroids of both cell types fused into larger tissue clusters over time within alginate and exhibited tissue formation comparable to monodisperse cells. Compared to monodisperse cells in alginate gels, C2C12 spheroids exhibited greater MyHC expression after 2 weeks, while cells within bovine MuSC spheroids displayed increased cell spreading. Both monodisperse and MuSC spheroids exhibited increased expression of genes denoting mid- and late-stage myogenic differentiation. Together, these data suggest that skeletal muscle spheroids have the potential for generating myogenic tissue via 3D bioprinting and reveal areas of research that could enhance myogenesis and myogenic differentiation in future studies.