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 degrees C and the cryoprotection offered by different media investigated in terms of post-thaw cell viability and function. 2. Optimal cryoprotection was offered by a solution containing dimethylsulphoxide, propylene glycol, acetamide and polyethylene glycol 8000 in Leibowitz L15 medium. 3. The cytochrome P450 content and activities of the microsomal P450 dependent mixed function oxidase system were well maintained at above 70% of fresh cell values throughout the cryopreservation period. However, 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 cryopreservation, viability of human hepatocytes was higher than that of rat hepatocytes preserved under identical conditions. For human cells maximum post-cryopreservation viability was 67% after 24 h at -80 degrees C; this declined to 49% after 14 days storage at -80 degrees C. In addition 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 freeze-thaw damage better than those from rat. 5. The results of this study define optimal conditions for cryopreserving human hepatocytes. Although microsomal enzyme activities are retained post-cryopreservation, the decrease in viability of thawed cells upon incubation at 37 degrees C suggests that caution should be exercized when using cryopreserved cells to study integrated drug metabolizing pathways in man in vitro.