Evaluation of the effect of culture configuration on morphology, survival time, antioxidant status and metabolic capacities of cultured rat hepatocytes

• Published in: Toxicology in vitro Vol. 16; no. 1; pp. 89 - 99
• Main Authors: Richert, L, Binda, D, Hamilton, G, Viollon-Abadie, C, Alexandre, E, Bigot-Lasserre, D, Bars, R, Coassolo, P, LeCluyse, E
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Ltd 01.02.2002; Elsevier Science
• Subjects: Animals; Antioxidant status; Antioxidants - metabolism; Biological and medical sciences; Cell Culture Techniques - methods; Cells, Cultured; Culture matrices; Enzymes - analysis; Enzymes - metabolism; Fundamental and applied biological sciences. Psychology; General aspects. Methods; glutahione reductase; Glutathione - metabolism; Hepatocytes - cytology; Hepatocytes - enzymology; Lipid Peroxidation - physiology; Liver. Bile. Biliary tracts; Male; Medical sciences; Metabolic capacities; Microsomes, Liver - enzymology; Rat hepatocytes after isolation and in culture; Rats; Rats, Sprague-Dawley; Time Factors; Toxicology; Vertebrates: digestive system; PBS; GSH-Px; UGT; DMSO; GSSG-R; GST; TBARS; Antioxidant status; BSA; 3D; DMEM; 2D; PB; NEAA; Culture matrices; Metabolic capacities; FCS; Rat hepatocytes after isolation and in culture; OFR; CYP; HBSS; GSH; Culture medium; Cell culture; Unspecific monooxygenase; Rat; Enzyme; Isozyme; Cytochrome P450; Liver; Rodentia; Detoxification; Antioxidant; Metabolism; In vitro; Vertebrata; Mammalia; Enzymatic activity; Hepatocyte; Animal; Collagen; Morphology; Primary culture; Drug-metabolizing enzyme; Extracellular matrix; Oxidoreductases
• ISSN: 0887-2333; 1879-3177
• DOI: 10.1016/S0887-2333(01)00099-6

We evaluated the antioxidant status, namely cellular lipid peroxidation, by measuring thiobarbituric acid reactive substances (TBARS), cellular reduced glutathione (GSH) content, glutathione reductase (GSSG-R), glutathione transferase (GST), glutathione peroxidase (GSH-Px) and catalase activities in rat liver, hepatocytes immediately after isolation and in two-dimensional (2D) culture (on non-coated or collagen-coated dishes, as collagen–collagen or collagen–Matrigel sandwich cultures) or three-dimensional (3D) culture on Matrigel-coated dishes. Microsomal cytochrome P450 (CYP)- and UDP-glucuronosyl transferase (UGT)- dependent activities were also assessed in rat livers and hepatocyte cultures. The overall antioxidant status of rat hepatocytes immediately after isolation was not significantly different from that of rat livers. During culture, GSH was increased in 2D but not in 3D cultures in accordance with morphological observations; that is that matrix–cell interactions involving GSH, important in 2D, are minimal in 3D cultures. While UGT- and GST-dependent activities were equivalent in cultured hepatocytes and in rat livers, both catalase and GSH-Px activities decreased with time in all culture configurations. Constitutive CYP-dependent activities were drastically decreased in hepatocytes after isolation and attachment and did not recover in any culture configuration tested. Our results highlight that, although 2D sandwich cultures and 3D cultures on Matrigel allow longevity of rat hepatocyte cultures and optimal induction of CYPs, an imbalance in phase I/phase II detoxication processes in cultured rat hepatocytes occurs, whatever the culture configuration.