Vitrification and rapid rewarming of precision-cut liver slices for pharmacological and biomedical research

High-throughput in vitro pharmacological toxicity testing is essential for drug discovery. Precision-cut liver slices (PCLS) provide a robust system for screening that is representative of the complex 3D structure of the whole liver. However, PCLS are not available as off-the-shelf products. Cryopreservation could solve this bottleneck by effectively preserving PCLS indefinitely until their time of use. With cryopreservation, slices could be shipped to laboratories without access to fresh tissue or used in planned experiments independent of surgical schedules. Here, we explore an "ice-free" cryopreservation approach called vitrification and focus on culturing and assessing PCLS for 3 days post-vitrification and rewarming, given that most acute drug toxicity tests are conducted over 24 h. Rat liver slices were diffusively loaded with a cryoprotective agent (CPA) cocktail consisting of ethylene glycol and sucrose. The CPA-loaded PCLS were placed on a polymer cryomesh, vitrified in liquid nitrogen, and rapidly rewarmed in CPA. The vitrified and rewarmed PCLS were subsequently cultured in serum-free media for 3 days. The cryopreserved PCLS maintained high viability, morphology, function, enzymatic activity, and drug toxicity response. Results show that the vitrified PCLS perform comparably to untreated controls and significantly outperform conventionally cryopreserved PCLS in all assessments (p < 0.05).