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 three-dimensional 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 also led to a three-fold increase in force production 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 also enables multisite collaborative research efforts through the shipment of preformed skeletal muscle constructs. The ability to freeze, revive, and prolong the lifetime of tissue-engineered skeletal muscle without incurring any loss of function represents a significant advancement in the field of tissue engineering. Cryopreservation enables the efficient fabrication, storage, and shipment of these tissues. This in turn facilitates multidisciplinary collaboration between research groups, enabling advances in skeletal muscle regenerative medicine, organ-on-a-chip models of disease, drug testing, and soft robotics. Furthermore, the observation that freezing undifferentiated skeletal muscle enhances functional performance may motivate future studies developing stronger and more clinically relevant engineered muscle.