Cryopreserved Rat Liver Slices: A Critical Evaluation of Cell Viability, Histological Integrity, and Drug-Metabolizing Enzymes

Abstract

The effects of a cryopreservation procedure on the biochemical, morphological and functional integrity of rat liver slices just after thawing and after 24 h culture were evaluated. Freshly prepared slices were incubated in modified University of Wisconsin solution containing 50% fetal calf serum and 10% dimethyl sulfoxide for 20 min on ice prior to a rapid cooling in liquid nitrogen. After 10–40 days, slices were thawed rapidly at 42°C. Total protein content and (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) (MTT) reduction were well preserved at thawing, whereas ATP content was markedly decreased relative to freshly prepared slices (−83%). The major microscopic findings in sections of just-thawed liver slices consisted of hepatocellular dissociation and minimal apoptosis. The qualitative profile of antipyrine (AP) metabolism was well preserved in cryopreserved slices, but the amounts of phase I and phase II AP metabolites produced over a 3-h incubation period were markedly reduced relative to fresh slices (−58 to −71%). When cryopreserved slices were cultured for 24 h after thawing, the viability was markedly reduced, as reflected by the almost complete absence of MTT reduction and the loss of ATP content. Histological examinations showed extensive cellular necrosis. The amount of AP metabolites produced by cryopreserved slices was similar after a 3- or a 24-h culture period, indicating that AP metabolism capacities were lost at 24 h culture. In conclusion, our results suggest that cryopreserved rat liver slices may be a useful model for short-term in vitro determination of drug metabolism pathways. Further work is required to extend their use for toxicological studies.