Cultures with cryopreserved hepatocytes: applicability for studies of enzyme induction

The use of hepatocyte cultures is well established for the study of drug-dr ug interactions. However, the major hindrance for the use of human hepatocy te cultures is that human hepatocytes are only occasionally available. This problem could be overcome by cryopreservation. Although cryopreserved hepa tocytes have been recommended for short term applications in suspension, st udies on induction of enzyme activity, requiring a more prolonged maintenan ce of cryopreserved hepatocytes in culture, represent a new field of resear ch. In the present study, we established a technique that allows preparatio n of rat hepatocyte co-cultures, using cryopreserved hepatocytes. After inc ubation with phenobarbital (0.75 mM; 72 h) induction factors for the isoenz yme-dependent regio and stereoselective testosterone hydroxylations were 1. 6, 2.2, 1.0, 2.1, 5.6, 2.4, 3.6, 4.5 and 0.9 for 2 alpha-, 2 beta-, 6 alpha , 6 beta-, 7 alpha, 15 beta-, 16 alpha- and 16 beta-hydroxytestosterone and 4-androsten-3,17 dione. Regarding induction factors of less than 2-fold, a s questionable these induction factors were similar to those of cultures wi th freshly isolated hepatocytes and the induction pattern of the individual hydroxylation products was similar to the in vivo situation. In addition 3 -methylcholanthrene (5 mu M; 72 h) induced exclusively the formation of 7 a lpha-hydroxytestosterone (6.6-fold) in cultures with cryopreserved hepatocy tes. This specificity also correlates to that obtained in rats. Although th ese induction factors were clearly satisfactory in cryopreserved cultures, the absolute activities of the main testosterone hydroxylation products wer e reduced when compared to fresh cultures. For instance. 6 beta-hydroxytest osterone, the main metabolite in solvent controls was reduced to 79%, 7 alp ha-hydroxytestosterone, the main metabolite after induction with 3-MC, was reduced to 66% and 16 beta-hydroxytestosterone, the main metabolite after i nduction with PB. was reduced to 52%. Similarly, EROD activity after induct ion with 3-methylcholanthrene in cryopreserved cultures was reduced to 62%, compared with that in fresh cultures. Although further optimization and va lidation is required, the data show that cytochrome P450 activities can cle arly be induced in co-cultures of cryopreserved hepatocytes, in a fashion w hich for the investigated inducers, is similar to that in cultures from fre shly isolated hepatocytes and similar to the in vivo situation. (C) 2000 El sevier Science Ireland Ltd. All rights reserved.