Long-Term Cryopreservation and Revival of Tissue-Engineered Skeletal Muscle

Abstract

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 threefold 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.

Biohybrid Design Gets Personal: New Materials for Patient‐Specific Therapy

“…Advancing biomanufacturing technologies must be coupled with concurrent advances in protocols for long‐term storage of biofabricated materials, to enable off‐site manufacture and shipping of biohybrid machines and systems. We have done some preliminary work in this field by proving that engineered skeletal muscle tissue‐integrated hydrogels can be frozen, stored for months, and revived to completely recover contractile function without a loss in cell viability . Interestingly, this process even introduces microscale pores into the natural hydrogel extracellular matrix of the tissue, improving myotube alignment and generating larger forces in response to external stimulation.…”

“…We have done some preliminary work in this field by proving that engineered skeletal muscle tissue-integrated hydrogels can be frozen, stored for months, and revived to completely recover contractile function without a loss in cell viability. [70] Interestingly, this process even Adv. Mater.…”

Modeling muscle

“…To address this need, we have developed a laboratory-based curriculum to teach biohybrid design to undergraduates and have validated that this technique teaches biofabrication and tissue engineering principles more clearly than a traditional format (14). Transferring our protocol to other scientists and laboratories has also generated new knowledge of the dynamics of neuromuscular junctions (15), muscle lifetime and proteolytic degradation (16), long-term muscle cryopreservation and revival (17), and muscle force enhancement through tissue scalability (18).…”

Bio-hybrid soft robots with self-stimulating skeletons

“…Later, the same bio-robotic device was lightcontrolled remotely by optogenetically-modified skeletal muscle cells which contract upon blue light stimulation [40] . In addition, this device demonstrated self-healing [50] , adaptability [40] , integration of motor neurons for advanced stimulation [51] , long-time preservation [52,53] , scalability [54] or their integration with micro-electrodes [55] . The integration of neuronal and skeletal muscle tissue in one single biobot has been of great interest, as it resembles the structure of native muscle to obtain improved controllability of the bio-robotic systems [24,56] .…”