CRYOPRESERVATION OF HUMAN ADULT HEPATOCYTES FOR USE IN DRUG-METABOLISM AND TOXICITY STUDIES

1. Human hepatocytes were cryopreserved for up to 14 days at -80-degre es-C and the cryoprotection offered by different media investigated in terms of post-thaw cell viability and function. 2. Optimal cryoprotec tion was offered by a solution containing dimethylsulphoxide, propylen e glycol, acetamide and polyethylene glycol 8000 in Leibowitz L15 medi um. 3. The cytochrome P450 content and activities of the microsomal P4 50 dependent mixed function oxidase system were well maintained at abo ve 70%, of fresh cell values throughout the cryopreservation period. H owever, the activities of the cytosolic enzymes studied, glutathione S -transferase and glutathione reductase, were not well maintained; they declined to < 40% of fresh cell values after storage of cells for 14 days at -80-degrees-C. The membrane environment may protect microsomal enzymes from denaturation by freeze-thaw damage. 4. After cryopreserv ation, viability of human hepatocytes was higher than that of rat hepa tocytes preserved under identical conditions. For human cells maximum post-cryopreservation viability was 67% after 24 h at -80-degrees-C; t his declined to 49% after 14 days storage at - 80-degrees-C. In additi on post-cryopreservation human hepatocytes remained > 70% viable when incubated at 37-degrees-C in suspension compared with only 46% of rat hepatocytes. This indicates that human hepatocytes can withstand freez e-thaw damage better than those from rat. 5. The results of this study define optimal conditions for cryopreserving human hepatocytes. Altho ugh microsomal enzyme activities are retained post-cryopreservation, t he decrease in viability of thawed cells upon incubation at 37-degrees -C suggests that caution should be exercized when using cryropreserved cells to study integrated drug metabolizing pathways in man in vitro.