Characterization of a primary hepatocyte culture system for toxicological studies

An hepatocyte culture system was developed for potential use in toxicological studies in vitro. Rat hepatocytes were isolated by two-step collagenase perfusion and cultured on Vitrogen-coated Permanox™ dishes in a modified Chee's medium containing 1 μM dexamethasone and 1% dimethylsulfoxide. Th...

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Veröffentlicht in:In vitro cellular & developmental biology. Animal 1996-04, Vol.32 (4), p.211-220
Hauptverfasser: Zurlo, J. (Johns Hopkins Center for Alternatives to Animal Testing, Baltimore, MD.), Arterburn, L.M
Format: Artikel
Sprache:eng
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RAT
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Zusammenfassung:An hepatocyte culture system was developed for potential use in toxicological studies in vitro. Rat hepatocytes were isolated by two-step collagenase perfusion and cultured on Vitrogen-coated Permanox™ dishes in a modified Chee's medium containing 1 μM dexamethasone and 1% dimethylsulfoxide. The cells remained highly viable for at least 10 d as determined by lactate dehydrogenase release and total protein levels. Albumin secretion into the medium, as a measure of differentiated function, was maintained at elevated levels over the course of 10 d in culture. A number of CYP activities were determined by the analysis of testosterone metabolism in freeze-thawed cells, diazepam metabolism in live cells, and specific assays for CYP 1A1/2, 2B1/2, 2E1, and 3A. Results of these assays indicated that a wide range of CYP isozymes were maintained, some activities were enhanced under the conditions of culture and some activities were inducible. Activities of the phase II enzymes, glutathione S-transferase and UDP-glucuronosyltransferase, and glutathione levels were also maintained in the cultured hepatocytes for at least 6 d. These results strongly support the use of this hepatocyte culture system for in vitro toxicological studies.
ISSN:1071-2690
1543-706X
DOI:10.1007/BF02722948