0000000000076934
AUTHOR
Bernd Diener
Malignantly transformed non-parenchymal liver epithelial cells and transformed oval cells suppress the homotypical gap junctional intercellular communication of co-cultured rat liver parenchymal cells.
Isolated rat liver parenchymal cells (PC) were co-cultured with a non-parenchymal rat liver epithelial cell line (NEC) or with an oval cell line. The homotypical gap junctional intercellular communication (GJIC) between the liver PC was measured after microinjection of Lucifer Yellow by dye transfer. The rat liver PC were dye coupled between 87% and 100% for at least 1 week in both co-cultures, in contrast to PC In monoculture between which no dye coupling was left after 1 week. When liver PC were co-cultured with a transformed and tumorigenic NEC or with a transformed and tumorigenic oval cell line the homotypical GJIC between the liver PC was drastically decreased with culture time, and t…
Cell Systems for Use in Studies on the Relationship Between Foreign Compound Metabolism and Toxicity
: Since the metabolism of most foreign compounds is predominantly controlled by hepatic in metabolism, isolated hepatocytes in most cases quite well predict the pattern of the overall metabolism of a given compound. Methods have been developed for cryopreserving isolated hepatocytes from man and other species with satisfactory maintenance of foreign compound metabolizing enzyme activities. The installation of a bank of cryopreserved hepatocytes from different species is possible and may be used for rational species extrapolation. It is necessary for some toxicological investigations to have hepatocytes which retain their differentiated status in culture for a sufficient time period. This mi…
Cryopreserved primary hepatocytes as a constantly available in vitro model for the evaluation of human and animal drug metabolism and enzyme induction.
The use of primary hepatocytes is now well established for both studies of drug metabolism and enzyme induction. Cryopreservation of primary hepatocytes decreases the need for fresh liver tissue. This is especially important for research with human hepatocytes because availability of human liver tissue is limited. In this review, we summarize our research on optimization and validation of cryopreservation techniques. The critical elements for successful cryopreservation of hepatocytes are (1) the freezing protocol, (2) the concentration of the cryoprotectant [10% dimethyl-sulfoxide (DMSO)], (3) slow addition and removal of DMSO, (4) carbogen equilibration during isolation of hepatocytes and…
The gap junctional intercellular communication is no prerequisite for the stabilization of xenobiotic metabolizing enzyme activities in primary rat liver parenchymal cells in vitro.
In primary monocultures of adult rat liver parenchymal cells (PC), the activities of the xenobiotic metabolizing enzymes microsomal epoxide hydrolase (mEHb), soluble epoxide hydrolase (sEH), glutathione S-transferases (GST), and phenolsulfotransferase (ST) were reduced after 7 d to values below 33% of the initial activities. Furthermore, the gap junctional intercellular communication (GJIC), measured after microinjection by dye transfer, decreased from 90% on Day 1 to undetectable values after 5 d in monoculture. Co-culture of PC with nonparenchymal rat liver epithelial cells (NEC) increased (98% on Day 1) and stabilized (82% on Day 7) the homotypic GJIC of PC. Additionally, most of the mea…
Cultures with cryopreserved hepatocytes: applicability for studies of enzyme induction
The use of hepatocyte cultures is well established for the study of drug-drug interactions. However, the major hindrance for the use of human hepatocyte cultures is that human hepatocytes are only occasionally available. This problem could be overcome by cryopreservation. Although cryopreserved hepatocytes have been recommended for short term applications in suspension, studies on induction of enzyme activity, requiring a more prolonged maintenance of cryopreserved hepatocytes in culture, represent a new field of research. In the present study, we established a technique that allows preparation of rat hepatocyte co-cultures, using cryopreserved hepatocytes. After incubation with phenobarbit…
Viability, attachment efficiency, and xenobiotic metabolizing enzyme activities are well maintained in EDTA isolated rat liver parenchymal cells after hypothermic preservation for up to 3 days in University of Wisconsin solution
Rat liver parenchymal cells were isolated by EDTA perfusion and were subsequently purified by Percoll centrifugation. The freshly isolated liver cells had a mean viability of 95% as judged by trypan blue exclusion. Isolated liver parenchymal cells were then stored at 0°C for up to 1 wk in University of Wisconsin solution (UW). During this hypothermic preservation, the viability was only slightly reduced to 92% after 1 d and to 85% after 3 d at 0°C. Thereafter, the viability decreased rapidly. After cold storage for up to 3 d, it was possible to use the parenchymal liver cells either in short-term suspension or in cell culture. The attachment efficiency in cell culture was the same for fresh…
Direct Analysis of Phase I Metabolites, Phenol Sulfates, Glucuronides and Glutathione Conjugates of Benzo[a]pyrene in Freshly Isolated, Hypothermically Stored and Cryopreserved Hepatocytes
Abstract The complex biotransformation of benzo[a]pyrene (BaP), the prototype of the class of carcinogenic polycyclic aromatic hydrocarbons, can be used as a tool to characterize the capacity of in vitro systems for the biotransformation of xenobiotics. In order to account for the ability of liver parenchymal cells to metabolize BaP, a method was developed for the isolation, separation and quantitation of its phase I metabolites, e.g. tetrahydrotetraols, trans-dihydrodiols, quinones and phenols, as well as its phase II metabolites, e.g. sulfates, glucuronides and glutathione conjugates, employing a combination of extractive and chromatographic steps. Upon incubation of BaP with freshly isol…
Gap junctional intercellular communication of cultured rat liver parenchymal cells is stabilized by epithelial cells and their isolated plasma membranes
The gap junctional intercellular communication (GJIC) determined by measuring dye coupling with Lucifer yellow, decreased within 3 d from 66% to 28% in monocultures of rat liver parenchymal cells. Coculturing of the parenchymal cells with a nonparenchymal epithelial cell line from rat liver resulted in increased and stabilized intercellular communication (83% after 3 d). The presence of isolated plasma membrane vesicles of the nonparenchymal epithelial cells also stabilized the intercellular communication between the liver parenchymal cells (70% after 3 d). When liver parenchymal cells were cocultured with a rat liver fibroblast cell line the gap junctional communication between the parench…
Xenobiotic metabolizing enzyme activities and viability are well preserved in EDTA-isolated rat liver parenchymal cells after cryopreservation
Rat liver parenchymal cells (PC) were isolated by EDTA perfusion and were purified by a subsequent Percoll centrifugation. The isolated PC had a viability of 95%, as judged by trypan blue exclusion. Freshly isolated PC were cryopreserved with an optimized protocol in a computer-controlled freezer. After thawing, the PC still retained a viability of 89%. The activities of representative xenobiotic metabolizing enzymes were compared between freshly isolated and cryopreserved PC after thawing. The cytochrome P450 content and the cytochrome P450 2C11 isoenzyme activity, determined by hydroxylation of testosterone in intact cells, were not affected by the cryopreservation. The following phase II…
Characterization of cryopreserved rat liver parenchymal cells by metabolism of diagnostic substrates and activities of related enzymes
The metabolism of testosterone and benzo(a)pyrene (BaP) which is mediated by diverse enzymes was determined in cryopreserved rat liver parenchymal cells and compared with that found in freshly isolated cells. In addition, the activities of single xenobiotic-metabolizing enzymes were measured by using specific substrates. The cytochrome P450 (P450)-mediated total metabolic conversion of testosterone was reduced to 55% in cryopreserved cells. The metabolite profile, i.e. the formation of single metabolites compared with total metabolic conversion, was however unchanged when compared with freshly isolated cells. A concomitant reduction in the activities of the involved P450 isoenzymes can ther…