Isochoric supercooling improves preservation of hepatic spheroid viability and structure compared to 4°C cold storage

Cell spheroids and organoids possess three-dimensional cell-cell interactions that more closely recapitulate in vivo tissue architecture than compared to conventional monolayer culture. Pluripotent stem cell-derived organoids demonstrate highly differentiated organ-specific functions and are valuable in applications that range from research and development to clinical therapeutics. Current lack of effective preservation methods is a major bottleneck preventing the broad distribution of spheroids/organoids and restricts their widespread use. Isochoric supercooling is a technology that enables stable, sub-zero, non-freezing preservation of biological material. The isochoric feature significantly reduces the probability of ice nucleation upon impact and vibrational perturbation, an important consideration for the transport and delivery of precious biologic cargo. In this report, we investigated the preservation of self-assembled HepG2 hepatic cell line spheroids in isochoric -6°C supercooling conditions compared to 4°C cold storage. We found that isochoric -6°C supercooling was superior to 4°C cold storage in preserving high viability of hepatic spheroids in University of Wisconsin solution for 3 days. To extend preservation duration to 5 days, it was necessary to use HypoThermosol-FRS as the preservation solution and to implement a gradual cooling and warming protocol. Importantly, isochoric -6°C supercooling for 5 days with gradual cooling/warming preserved both high cell viability and intact spheroid structure, outcomes not achieved with 4°C storage or fast cooling/warming. Our findings indicate that isochoric supercooling may be a critical technology for preserving cellular spheroids and organoids within the timeframe and logistical requirements for facilitating their distribution and utilization.