Cryopreservation Alters Tissue Structure and Improves Differentiation of Engineered Skeletal Muscle

Impact statementThis study examines the effects of cryopreservation on engineered skeletal muscle tissue structure and uncovers the mechanisms by which these changes impact tissue development and function. These insights help inform the engineering of stronger muscle tissues and highlight the need to investigate cryopreservation-mediated changes to extracellular matrix as we develop suitable cryopreservation protocols for other tissues and organs. Tissue-engineered skeletal muscle can play an important role in regenerative medicine, disease modeling, drug testing, as well as the actuation of biohybrid machines. As the applications of engineered muscle tissues expand, there exists a growing need to cryopreserve and store these tissues without impairing function. In a previous study, we developed a cryopreservation protocol in which engineered skeletal muscle tissues are frozen before myogenic differentiation. In that study, we found that this cryopreservation process led to a three-fold increase in the force generation of the differentiated muscle. Here, we perform further testing to determine the mechanisms by which cryopreservation enhances engineered skeletal muscle function. We found that cryopreservation alters the microstructure of the tissue by increasing pore size and decreasing elastic modulus of the extracellular matrix (ECM), which leads to increased expression of genes related to cell migration, cell-matrix adhesion, ECM secretion, and protease activity. Specifically, cryopreservation leads to the upregulation of many ECM proteins, including laminin, fibronectin, and several types of collagens, as well as integrins and matrix metalloproteinases. These changes to ECM structure and composition were associated with enhanced myogenic differentiation, as evidenced by the upregulation of late-stage myogenic markers and increased force generation. These results highlight the need to understand the effects of cryopreservation on the ECM of other tissues as we strive to advance tissue and organ cryopreservation protocols for regenerative medicine.