Metabolic activity of fresh and cryopreserved dog hepatocyte suspensions

1. Dog hepatocytes were cryopreserved at 6 x 10(6) 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/10(6) cells in freshly isolated whole hepatocytes and 463 +/- 24 pmol/min/10(6) CP whole hepatocytes, but they were equivalent in fresh and CP hepatocyte homogenates supplemented with 250 mu M NADPH. 5. Phase 2 enzymes were functional in freshly thawed CP hepatocytes but they required exogenous addition of cofactors (20 mu M UDPGA and 1.7 mu M 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 metabolism since enzyme functions in CP hepatocytes were stabilized. Cofactors in freshly thawed CP hepatocytes should be measured and controlled for optimal use.