Abstract
In the present study we evaluated viability and detoxifying enzyme capacity of cryopreserved hepatocytes from various species, including man, immobilized in calcium alginate gels. Ethoxyresorufin O-deethylase, phenacetin deethylase, pentoxyresorufin O-dealkylase, tolbutamide hydroxylase, S-mephenytoin hydroxylase, dextromethorphan demethylase, and nifedipine oxidation corresponding to the major cytochromes P450 (CYP) involved in xenobiotic metabolism as well as whole glutathione S-transferase (GST) activity were measured using specific substrates and after exposure or not to prototypical inducers. After deep-freeze storage, viability of immobilized hepatocytes was only slightly reduced and most CYP-related monooxygenase activities were well preserved, being expressed at levels close to those measured in unfrozen hepatocyte monolayers. By contrast, total GST activity was decreased by around 50%. However, as did CYP1A- and 3A-related enzymes, rat GST remained capable of responding to prototypical inducers. The fold increases were comparable in unfrozen and frozen immobilized hepatocytes and in unfrozen hepatocyte monolayers. The duration of storage, even when exceeding one year, did not affect viability and functions. In conclusion, after cryopreservation, alginate-entrapped hepatocytes remain highly viable and capable of expressing most detoxifying enzymes at levels close to those expressed in corresponding unfrozen hepatocyte monolayers and of responding to prototypical inducers.
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Adams RM, Wang M, Crane AM, Brown B, Darlington GJ, Ledley FB. Effective cryopreservation and long-term storage of primary human hepatocytes with revovery of viability, differentiation and replicative potential. Cell Tranplant. 1995;4:579-86.
Borel Rinkes I, Toner M, Sheeha SJ, Tompkins RG, Yarmush M. Long-term functional recovery of hepatocytes after cryopreservation in a three-dimensional culture configuration. Cell Transplant. 1992;1:281-92.
ChesnëC, Guillouzo A. Cryopreservation of isolated rat hepatocytes: a critical evaluation of freezing and thawing conditions. Cryobiology. 1988;25:323-30.
ChesnëC, Guyomard C, Fautrel A, et al.Viability and function in primary culture of adult hepatocytes from various animal species and human beings after cryopreservation. Hepatology. 1993;18:406-14.
Coundouris JA, Grant MH, Simpson JG, Hawksworth GM. Drug metabolism and viability studies in cryopreserved rat hepatocytes. Cryobiology. 1990;27:288-300.
Coundouris JA, Grant MH, Engeset J, Pë trie JC, Hawksworth GM. Cryopreservation of human adult hepatocytes for use in drug metabolism and toxicity studies. Xenobiotica. 1993;23:1399-409.
De Sousa G, Langouet S, Nicolas F, Placidi M, Rahmani R, Guillouzo A. Induction of cytochrome P-450 1A and glutathione S-transferase transcripts in primary cultures of hepatocytes from dog, monkey and man after cryopreservation. Cell Biol Toxicol. 1996;12:351-8.
Diener B, Abdel-Latif H, Arand M, Oesch F. Xenobiotic metabolizing enzyme activities and viability are well preserved in EDTA-isolated rat liver parenchymal cells after cryopreservation. Toxicol Appl Pharmacol. 1995;130:149-53.
Dixit V, Darvasi R, Arthur M, Lewin K, Gitnick G. Cryopre-served microencapsulated hepatocytes: transplantation studies in Gunn rats. Transplantation. 1993;55:616-22.
Dou M, De Sousa G, Lacarelle B, et al. Thawed human hepatocytes in primary culture. Cryobiology. 1992;29:454-69.
Fautrel A, Joly B, Guyomard C, Guillouzo A. Long-term maintenance of drug-metabolizing enzyme activities in rat hepatocytes after cryopreservation. Toxicol Appl Pharmacol. 1997;147:110-14.
Frë mond B, Malandain C, Guyomard C, ChesnëC, Guillouzo A, Campion JP. Correction of bilirubin conjugation in the Gunn rat using hepatocytes immobilized in alginate gel beads as an extracorporeal bioartificial liver. Cell Transplant. 1993;2:453-60.
Guguen-Guillouzo C, Guillouzo A. Methods for preparation of adult and fetal hepatocytes. In: Guillouzo A, Guguen-Guillouzo C eds, Isolated and cultured hepatocytes. Paris: Les Editions INSERM and Libbey Eurotext; 1986:1-12.
Guillouzo A. Liver cell models in in vitro toxicology. Environ Health Perspect. 1998;106:511-32.
Guillouzo A, ChesnëC. Xenobiotic metabolism in epithelial cell cultures. In: Shaw AJ, ed. Cell culture models of epithelial tissues: a practical approach. Oxford: University Press; 1996:67-85.
Guillouzo A, Guyomard C, Fautrel A, ChesnëC. Storage of isolated hepatocytes. In: Berry M, ed. The hepatocyte review. Amsterdam: Kluwer; 2000 [In press].
Guyomard C, Rialland L, Fremond B, ChesnëC, Guillouzo A. Influence of alginate gel entrapment and cryopreservation on survival and xenobiotic metabolism capacity of rat hepatocytes. Toxicol Appl Pharmacol. 1996;141:349-58.
Innes GK, Fuller BJ, Hobbs KEF. Functional testing of hepatocytes following their recovery from cryopreservation. Cryobiology. 1988;25:23-30.
Koebe HG, Dunn JC, Tonner M, et al. A new approach to the cryopreservation of hepatocytes in a sandwich culture configuration. Cryobiology. 1990;27:576-84.
Koebe HG, Dahnhardt C, Muller-Hocker J, Wagner H, Schildberg F. Cryopreservation of porcine hepatocytes. Cryobiology. 1996;33:127-41
Labarca C, Paigen K. A simple, rapid and sensitive DNA assay procedure. Anal Biol. 1980;102:344-52.
Lawrence JN, Benford DJ. Development of an optimal method for the cryopreservation of hepatocytes and their subsequent monolayer culture. Toxicol in Vitro. 1991;5:39-50
Loretz LJ, Li AP, Flye MW, Wilson AGE. Optimization of cryopreservation procedures for rat and human hepatocytes. Xenobiotica. 1989;19:489-98.
Madan A, Dehaan R, Mudra D, Carroll K, Lecluyse E, Parkinson A. Effect of cryopreservation on cytochrome P-450 enzyme induction in cultured rat hepatocytes. Drug Metab Dispos. 1999;27:327-35.
Miners JO, Smith KJ, Robson RA, McManus ME, Veronese ME, Birkett DJ. Tolbutamide hydroxylation by human liver microsomes. Biochem Pharmacol. 1988;37:1137-42.
Powis G, Santone KS, Melder DC, Thomas L, Moore DJ, Wilke TJ. Cryopreservation of rat and dog hepatocytes for studies of xenobiotic metabolism and activation. Drug Metab Dispos. 1987;15:826-32.
Rozga J, Morsiani E, LePage E et al. Isolated hepatocytes in a bioartificial liver: a single group view and experience. Biotech Bioeng. 1994;43:645-53.
Sonderfan AJ, Aleotto MP, Dutton DR McMillen SK, Parkinson A. Regulation of testosterone hydroxylation by rat liver microsomal cytochrome P450. Arch Biochem Biophys. 1987;255:27-41.
Strom SC, Dorko K, Thompson MT, Pisarov LA, Nussler AK. Large scale isolation and culture of human hepatocytes. In: Franco D, Boudjema K, Varet B, eds. Ilots de Langerhans et hë patocytes. Paris: Les Editions INSERM; 1998:196-205.
Swales NJ, Luong C, Cadwell J. Cryopreservation of rat and mouse hepatocytes. Comparative viability stuides. Drug Metab Dispos. 1996;24:1218-23.
Swales NJ, Utesch D. Metabolic activity of fresh and cryopreserved dog hepatocyte suspensions. Xenobiotica. 1998;28: 937-48.
Utesch D, Diener B, Molitor E, Oesch F, Platt KL. Characterization of cryopreserved rat liver parenchymal cells by metabolism of diagnostic substrates and activities of related enzymes. Biochem Pharmacol. 1992;44:309-15.
Watts P, Grant MH. Cryopreservation of rat hepatocyte mono-layer cultures. Hum Exp Toxicol. 1996;15:30-7.
Zaleski J, Richburg J, Kauffman FC. Preservation of the rate and profile of xenobiotic metabolism in rat hepatocytes stored in liquid nitrogen. Biochem Pharmacol. 1993;46: 111-16.
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Rialland, L., Guyomard, C., Scotte, M. et al. Viability and drug metabolism capacity of alginate-entrapped hepatocytes after cryopreservation. Cell Biol Toxicol 16, 105–116 (2000). https://doi.org/10.1023/A:1007690009927
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DOI: https://doi.org/10.1023/A:1007690009927