A Cryopreservation Strategy for Myoblast Storage in Paper-Based Scaffolds for Inter-Laboratory Studies of Skeletal Muscle Health

3D tissue-engineered models are poised to facilitate understanding of skeletal muscle pathophysiology and identify novel therapeutic agents to improve muscle health. Adopting these culture models within the broader biology community is a challenge as many models involve complex methodologies and significant investments of time and resources to optimize manufacturing protocols. To alleviate this barrier, a protocol with commercially available reagents is developed to cryopreserve myoblasts in a 96-well compatible format that allows tissues to be transferred to users without expertise in 2D or 3D skeletal muscle cell culture. This report validates that myoblasts encapsulated in a hydrogel and cryopreserved in paper-based scaffolds maintain cell viability, differentiation, and function via acetylcholine-induced transient calcium responses. Furthermore, successful shipping of myoblasts cryopreserved in paper-based scaffolds to intra-provincial and international collaborators is demonstrated who successfully thaw, culture, and use the 3D muscle tissues. Finally, the application of this method is confirmed for studying muscle endogenous repair by seeding freshly isolated skeletal muscle stem cells to cryopreserved then differentiated and injured tissues, demonstrating expected responses to a known stimulator of muscle stem cell self-renewal, p38 alpha/beta MAPKi. Altogether, the 3D myoblast cryopreservation protocol offers broadened access of a complex skeletal muscle tissue model to the research community. 3D tissue-engineered models facilitate studying skeletal muscle health but are challenging to adopt. This report describes a method to cryopreserve myoblasts encapsulated in a hydrogel and paper-based scaffold to allow the transfer of engineered tissues to users without model expertise. Cryopreserved myoblasts maintain cell viability, differentiation, and function. International collaborators successfully thawed, cultured, and used the engineered tissues. image