Development of Supercooling Preservation Method for Adherently Cultured Endothelial Cells and Its Application to Microphysiological Systems

Microphysiological systems (MPS) that recapitulate human organ functions have gained attention as alternatives to animal experiments in drug discovery, regenerative medicine, and toxicity assessments. However, preserving MPS with adherent cells remains a significant challenge. In this study, we developed a supercooling preservation method that enables the low-temperature storage of human-derived adherent cells without freezing. Using human hepatic sinusoidal endothelial cells (TMNK-1), we optimized the preservation conditions by assessing the temperature, cooling and rewarming rates, and preservation solutions. Under optimized conditions (preservation at −4 °C, −0.028 °C/min cooling, and +1.0 °C/min rewarming), high cell viability and preserved morphology were maintained for up to 7 days. When these conditions were applied to both two- and three-dimensional MPS containing TMNK-1 or HepG2 cells, post-preservation viability remained high, and no cell death or cytoskeletal disruption was observed. This supercooling preservation method has the potential to serve as a practical strategy for the temporary storage of MPS.