Water rotational relaxation time of preservation solutions relates to storage lifespan of cells in isochoric cryopreservation system

Biological materials can be stored in an ice-free liquid state using isochoric cryopreservation (isochoric supercooling and isochoric freezing) to minimize cryoinjury from ice damage. However, the mechanism underlying the relationship between storage lifespan and the physical-chemistry of water in solutions during storage in isochoric cryopreservation system remains unknown. In this work, we evaluated the rotational relaxation time of water molecules of preservative solutions in isochoric cryopreservation by dielectric spectroscopy. The cell viability change during isochoric cryopreservation was also experimentally assessed to clarify the relationship between the water relaxation times and cellular deterioration rate. The findings reveal that although initial high pressure caused by isochoric freezing does sudden damage to cells, and regardless of isochoric supercooling or isochoric freezing, the cellular deterioration rate only depends on the water relaxation times in preservative solution. Notably, when considering the same cryopreservation temperature, the cellular deterioration time under isochoric freezing conditions tends to be longer than that under isochoric supercooling conditions, primarily due to the longer water relaxation time in isochoric freezing compared to isochoric supercooling. This work gives valuable guidance to understand the relationship between the kinetics of water molecules and the storage lifespan of bio-matter during storage inside isochoric cryopreservation system.