Long-term cryopreservation and revival of tissue engineered skeletal muscle.

Tissue engineered skeletal muscle plays an important role not only in the field of regenerative medicine, but also in emerging areas such as soft robotics, organ-on-a-chip disease models, and drug testing. However, further expansion of the applications of tissue engineered skeletal muscle models requires a suitable method for their long-term storage and shipment. Cryopreservation has long been the standard for cell storage, but the freezing of 3D tissues is accompanied by many complications due to heat and mass transfer limitations. In this study, we used a tissue engineered skeletal muscle bioactuator as a model to characterize the effects of freezing on muscle viability, gene expression, myotube structure, and force generation. We optimized the protocol for cryopreservation by comparing outcomes when tissue was frozen undifferentiated and differentiated. Our optimized protocol, in which skeletal muscle was frozen undifferentiated, not only maintained cell viability, but led to a 3-fold increase in force production as compared to unfrozen muscle. Furthermore, we enhanced muscle lifetime through inhibition of cysteine proteases. The reported timeline for skeletal muscle tissue fabrication, freezing, revival, and long-term culture not only promotes a more streamlined fabrication process, but enables multi-site collaborative research efforts through the shipment of pre-formed skeletal muscle constructs.