Cryopreservation of liver tissue slices for pharmacological applications

There is a significant need in pharmacology for better in-vitro drug testing and discovery models given the poor rates of success for translation of drugs from pre-clinical to clinical trials. Liver tissue slices have the potential to serve as simple and versatile models representing aspects of the whole organ architecture, phenotype, and function, allowing for understanding non-hepatocyte cell interactions in drug metabolism and helping mitigate inaccurate drug clearance rates that hepatocyte-only based models suffer from. However, at present, they are not easily cultured, nor are they “off the shelf” available for testing. One way to address scaling and availability of liver slices is cryopreservation. Cryopreservation can not only be applied to both animal and human tissue, enabling ideal testing, but also allowing for preservation of pooled effects across diverse population groups. In this project we successfully cryopreserved rat liver tissue slices, 250 μm in thickness and 5mm in diameter, prepared using a vibratome after coring. The slices were diffusively loaded with 7.7M Ethylene Glycol and Sucrose based cryoprotective agent in a stepwise manner. The loaded tissue was then vitrified and rewarmed using an ETFE cryomesh. The tissues were then cultured along with controls for a week in 96 well plates with serum-free, chemically defined media in low volumes (100-250 μl). The viability and function of the tissues were evaluated using a variety of assays including live/dead staining, LDH release, urea production and oxygen consumption. Results show that the cryopreserved slices maintained viability and functionality throughout the culture period, with measures comparable to controls. This work demonstrates the feasibility of cryopreservation and banking of liver tissue slices, which could potentially enable a dramatic shift in the effectiveness of pharmacological drug discovery and testing. The simplicity of the system may be used in the future for large animal and human tissues.