Elsevier

Toxicology in Vitro

Volume 16, Issue 1, February 2002, Pages 89-99
Toxicology in Vitro

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

https://doi.org/10.1016/S0887-2333(01)00099-6Get rights and content

Abstract

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.

Introduction

Primary cultures of hepatocytes are widely used in pharmacological, toxicological and metabolic studies (Berry et al., 1991), and form the mainstay of cell transplantation (Mito and Sawa, 1997) or bioartificial organ support systems (Rozga et al., 1993) in clinical medicine. In both applications their usefulness is limited by a loss of important liver-specific functions, in particular phase I cytochromes P450 (CYP) (LeClyse, Bullock, & Parkinson, 1996a, LeClyse, Bullock, Parkinson, & Hochman, 1996b). Early loss of CYP after hepatocyte isolation has recently been suggested to be due to activation of nitric oxide synthesis, with a peak being observed 4–8 h post-isolation, undetectable after 24 h, by the reactive oxygen species generated during the conventional collagenase-based isolation procedure (Duval, Sieg, & Billings, 1995, Wang, Gao, Fukumoto, Tademoto, Sato, & Hirai, 1998, Rodriguez-Ariza, & Paine, 1999, Tirmenstein, Nicholls-Grzemski, Schmittgen, Zakrajsek, & Fariss, 2000). Overproduction of nitric oxide has indeed been shown to be directly related to CYP inactivation (Vink, Osawa, Darbyshire, Collins, Eshenaur, & Nims, 1993, Stadler, Trockfield, Scmilix, Brill, Seiwert, Greim, & Doemer, 1994, Lopez-Garcia, 1998, Lopez-Garcia, & Sanz-Gonzalez, 2000). The effect of isolation on other important liver metabolic functions, such as phase II UDP-glucuronosyl transferase (UGT) and glutathione transferase (GST) has, to our knowledge, not been reported.

The first aim of the present study was to assess how hepatocytes cope with the oxidative stress generated during perfusion. For this, we evaluated the antioxidant status, i.e. the cellular lipid peroxidation rate, by measuring the concentration of 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 hepatocytes immediately after isolation and after attachment in culture, and compared to that of whole livers. Other major liver metabolic functions such as microsomal CYP- and UGT-dependent activities were also assessed in hepatocytes after isolation and attachment and compared to “ex vivo” microsomal liver data.

Several attempts have been directed at increasing the longevity and differentiated functions of individual and aggregated hepatocytes in primary cultures. Two popular configurations of stable culture matrix systems are the sandwich configuration of collagen–collagen or collagen–Matrigel that elicits a planar cell morphology and two-dimensional multicellular arrays reminiscent of the liver plate, and the single layer configuration on Matrigel where cells form multicellular three-dimensional aggregates (LeClyse, Bullock, & Parkinson, 1996a, LeClyse, Bullock, Parkinson, & Hochman, 1996b, Moghe, Coger, Toner, & Yarmush, 1997, Powers, Rodriguez, & Griffith, 1997, Semler, Ranucci, & Moghe, 2000). These culture configurations have been described as increasing hepatocyte longevity in culture compared to cultures on single layers of collagen (LeClyse, Bullock, & Parkinson, 1996a, LeClyse, Bullock, Parkinson, & Hochman, 1996b, Qiao, & Farrell, 1999), suggesting that hepatocytes in the former culture configuration are less subjected to oxidative stress-induced cell damage than the latter, as has been described for rat hepatocytes co-cultured with rat epithelial cells compared to pure cultures (Mertens et al., 1993).

In terms of expression of various CYP isoenzymes, reports are conflicting. Some authors reported re-expression of several CYP in rat and human hepatocytes cultured on Matrigel (Silva et al., 1998) and in rat hepatocyte cultures in a collagen–collagen sandwich configuration (Dunn et al., 1991) vs on single collagen layer, and others reported equivalent decreases in all culture systems of rat (Kocarek et al., 1993) and pig (Hosagrahara et al., 2000) hepatocytes. These discrepancies are most probably due to differences in media formulations, which, in addition to matrix, influence CYP maintenance, as previously reported (LeCluyse et al., 1999). The effect of culture configuration on UGT and GST activities has, to our knowledge, not been described.

The second aim of the present study was thus to evaluate cell viability and morphology, antioxidant status and microsomal CYP- and UGT-dependent activities in different rat hepatocyte culture configurations in the presence of the same medium formulation: directly on plastic culture dishes, on collagen-coated or Matrigel-coated dishes and as collagen–collagen or collagen–Matrigel sandwich cultures.

Section snippets

Materials

All culture media, sera, glutaMAX media supplement, and non-essential amino acids (NEAA) were obtained from Gibco BRL (Grand Island, NY, USA). Insulin, transferrin, selenium, linoleic acid and bovine serum albumin (BSA) supplement (ITS+) and Matrigel® were purchased from Collaborative Biomedical Research (Bedford, MA, USA). Collagenase (CLS 2) was from Worthington Biochemical Corporation (Lakewood, NJ, USA). Collagen, type I (Vitrogen®) was obtained from CelTrix (Santa Clara, CA, USA). Petri

Antioxidant status

The overall antioxidant status in hepatocyte homogenates immediately after isolation was not significantly different from that of liver homogenates (Table 1). After attachment to collagen, 4 h after seeding, lipid peroxidation, as assessed by the concentration of TBARS, as well as GSH content, GST, GSSG-R and GSH-Px activities, were not significantly different compared to liver homogenates and homogenates from freshly isolated rat hepatocytes. However, catalase activity was significantly

Acknowledgements

This study was financially supported in part by Hoffmann-LaRoche (Basel, Switzerland), and Aventis (Sophia Antipolis, France). The authors wish to thank Catherine Guyon, Alexandre Bonet and Summer Jolley for their technical assistance.

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