This study was directed to use the genetically developed isoprenaline-sensitive (S), isoprenaline-resistant (R) and spontaneous hypertensive rats (SHR) as standard diseased animal models for in vitro liver function evaluation of drug biotransformation. Hepatic hexobarbital hydroxylase and glutathione transferase (GST) were evaluated by using hexobarbital and l-chloro-2,4-dinitrobenzene (CDNB) as substrates, at concentrations of 0.21 mmol/l and 1 mmol/l, respectively. The assay was conducted by using isolated hepatocytes in suspension and hepatocytes in a bioreactor configuration. The data demonstrate that there are certain cellular pharmacokinetic differénces in hexobarbital hydroxylase and GST activities in hepatocytes obtained from Wistar, SHR, R and S strains which can be better demonstrated, when using the model of perfused and immobilized hepatocytes.
This study deals with the application of the previously developed immobilized and perfused isolated hepatocytes as a cellular system for the study of representative phase 1 and phase II of biotransformation reactions. To illustrate phase I reactions, aminopyrine (0.17-4.25 mmol/1) and hexobarbital (0.2 mmol/1) were selected. For phase II reactions, glutathione transferase activity was evaluated by using l-chloro-2,4-dinitrobenzene (CDNB) as a substrate (0.125-2.0 mmol/1). Formaldehyde, that was formed from aminopyrine, increased steadily in the perfusion medium with time. The perfused hepatocytes eliminated hexobarbital at a much higher rate than the hepatocytes in suspension. At several time points the amount of CDNB-glutathione conjugate formed per one million hepatocytes in the bioreactor was almost twice the amount formed by the hepatocytes in suspension. The present data illustrate the successful application of the hepatocyte bioreactor in phase I and phase II of xenobiotic metabolism and indicate that the cells were metabolically more active than the cells in suspension.
In the present study, a method has been employed for hepatocyte immobilization in agarose threads which allows for cell perfusion. The rat hepatocytes are isolated from the liver. A 1.8 % low-gelling agarose solution is prepared in warm Krebs-Henseleit solution. The agarose solution is mixed 1:1 with the hepatocytes and the cells are immobilized in agarose threads by extruding the agarose-cell mixture through cooled Chemfluor teflon (TFE) tubing. Light and electron microscopy studies indicated the integrity of the hepatocytes in the gel matrix. This system allows for liver cell perfusion and viability studies to be carried out non-invasively on the cells and provides data that are comparable to those obtained with a perfused isolated liver. Immobilized hepatocytes are an in vitro system worthy of further evaluation which may be useful in the studies of liver cell metabolism and the response of the liver to foreign chemicals.
The present study was designed to investigate the ameliorative effect of cyclosporine A (CsA) pretreatment on an anoxia/reoxygenation injury model by using immobilized perfused hepatocytes. Rats received an i.p. injection of two successive doses of CsA (5 mg/kg/day). Twenty-four hours later hepatocytes were isolated from CsA-treated and control rats. After hepatocyte isolation, immobilization, perfusion, induction of anoxia/reoxygenation, the structural and functional integrity of the hepatocytes was followed in a perfusion medium by measuring the leakage of lactate dehydrogenase (LD) and the time course of urea biosynthesis. CsA pretreatment reduced the initial rate of urea synthesis during normoxia but reduced the drop in the relative percentage rate of urea synthesis during the period of anoxia. LD leakage was increased threefold by anoxia and sevenfold by reoxygenation in cells of untreated animals. After CsA pretreatment in vivo, hepatocytes showed no increase in LD leakage into the medium. These findings demonstrate that the perfused immobilized hepatocytes can be used as a cellular model to assess the effects of liver insults such as anoxia/reoxygenation injury and that CsA modulates the injury. The mechanisms of CsA beneficial effects at the experimental level remain to be elucidated.
An overview of the concept of cellular immobilization and perfusion as a small laboratory bioreactor model is presented. The cellular systems currently used may be described as static. This is due to conditions of hypoxia and waste product build-up that affect cell physiology. Cellular immobilization and perfusion is, therefore, expected to maintain the cells for very long periods of time under approximately physiological conditions. A number of applications of immobilized perfused hepatocytes and other cellular systems such as adipocytes and Sertoli cells are described in addition to various other cell lines. Moreover, it is suggested that the bioreactor may have potential use as a bioartificial organ.