In-vitro drug testing on precision-cut liver slices (PCLS) provides the most realistic biochemical metabolic profile of in-situ conditions for predicting drug toxicity and metabolism during early stage drug development. An ample supply of cryopreserved human liver slices obtained from viable donor livers and hepatic resects of split liver transplant surgeries could be used for in-vitro drug testing and replace the need of experimental animals. However, imperfect preservation techniques that degrade viability and reduce drug testing incubation periods have limited the potential of PCLS. Prior research efforts to cryopreserve PCLS have encountered fundamental problems of ice formation at extra- and intracellular sites and toxic levels of cryoprotectants which have resulted in impaired xenobiotic metabolism and biotransformation capacities. The working hypothesis that this feasibility study seeks to address is that ice-free cryopreservation of precision-cut rat liver slices employing molecular ice control compounds will enhance xenobiotic metabolism and hepatic viability for extended incubation periods compared with existing vitrification and conventional cryopreservation technologies. It is hypothesized that vitreous cryopreservation of PCLS using synthetic molecular iceblockers will retain xenobiotic metabolic functions for longer incubation periods. The xenobiotic metabolism functions and cellular integrity of cryopreserved PCLS will be assessed using various viability and histological assays. Following successful completion of the aims of this Phase I SBIR feasibility proposal, scale up issues will be addressed in Phase II SBIR submission to fully develop an effective storage protocol for banking of human and large animal liver slices. It is anticipated that the PCLS banking will invariably reduce and in many case replace the need of experimental animals required for drug testing. This technology development will provide multiple benefits to the US health care system by expediting drug screening process and reducing drug development costs.
The specific aims of Phase I of this proposal are the in- vitro evaluation of natural and synthetic molecular ice control compounds with selected cryoprotectants proven to produce ice-free cryopreservation and maintain tissue function and viability. First, the selected formulations will be tested in vitro for cytotoxicity. Second, formulations with the greatest biocompatibility will be evaluated for ice formation in PCLS during preservation. Rewarmed PCLS will be evaluated for metabolic function over extended incubation periods. The molecular ice control formulations which provide the highest tissue viability and least interstitial ice formation will be tested in larger animal and human PCLS tissues in a sub-sequent Phase II SBIR proposal.
