Metabolic activity of fresh and cryopreserved dog hepatocyte suspensions

Abstract

1. Dog hepatocytes were cryopreserved at 6 × 10⁶ viable cells/ml in a suspension buffer containing 10% DMSO and were stored in liquid nitrogen. 2. The exclusion of trypan blue dye was 96± 2 and 85± 9% in fresh and cryopreserved (CP) hepatocytes, respectively. Albumin synthesis was unaffected by freezing. 3. Ethoxycoumarin and ethoxyresorufin O-deethylase activities were equivalent in fresh and CP hepatocytes. 4. The profile of testosterone metabolism was unaffected by freezing. Total hydroxylase activities were 815± 33 pmol/min/10⁶ CP cells in freshly isolated whole hepatocytes and 463± 24 pmol/min/10⁶ CP whole hepatocytes, but they were equivalent in fresh and CP hepatocyte homogenates supplemented with 250 μM NADPH. 5. Phase 2 enzymes were functional in freshly thawed CP hepatocytes but they required exogenous addition of cofactors (20 μM UDPGA and 1.7 μM PAPS). 6. When placed in suspension for longer times, fresh and CP cell viabilities were 88± 6 and 64± 2% after 4 h. ECOD and EROD activities were equivalent in fresh and CP hepatocyte suspensions, over 4 h. Testosterone hydroxylase activities were well maintained in fresh cell suspensions but they declined to 63± 6% of the initial activity after 4 h in CP hepatocytes. 7. These results indicate that CP dog hepatocytes are a suitable in vitro system for xenobiotic metabolisms in ce enzyme functions in CP hepatocytes were stabilized. Cofactors in freshly thawed CP hepatocytes should be measured and controlled for optimal use.