Cryogenic, but not hypothermic, preservation disrupts the extracellular matrix of cell sheets.

Cell sheet (CS)-based approaches hold significant potential for tissue regeneration, relying on the extracellular matrix (ECM) for success. Like in native tissues, the ECM provides structural and biochemical support for cellular homeostasis and function. Effective preservation strategies that maintain ECM integrity are critical to enhance the therapeutic potential of CS-based approaches. While cryogenic and hypothermic preservation methods offer potential solutions, their impact on CS ECM structure is not fully understood. Therefore, a comprehensive analysis of the ECM of hASCs CS following cryogenic and hypothermic preservation for 3 and 7 days, was conducted. Although proteomic analysis indicated that cryopreservation had no significant effect on the overall composition of the ECM, it induced significant ECM structural alterations, particularly disrupting collagen organization, which was not observed following hypothermic preservation. These structural changes were accompanied by alterations in mechanical properties, including a reduction in elastic modulus. In contrast, hypothermic preservation maintained ECM integrity and mechanical properties similar to the control. The notable ECM structural changes following cryogenic preservation can potentially impact cellular behavior, including adhesion, proliferation, and differentiation, thereby affecting the efficacy of CS therapies in vivo. This suggests that hypothermia may offer a promising alternative to cryopreservation for preserving CS integrity and functionality.