D-Glucose uptake in fish hepatocytes: mediated by transporter in rainbow trout (Oncorhynchus mykiss), but only by diffusion in prespawning lamprey (Lampetra fluviatilis) and in RTH-149 cell line

The transport of D-glucose into rainbow trout (Oncorhynchus mykiss) and river lamprey (Lampetra fluviatilis) hepatocytes, as well as into rainbow trout hepatoblastoma cell line RTH-149 was studied using tracer methods. The half-time for D-glucose equilibration was 15 s for rainbow trout. The half-times for the non-metabolizable D-glucose analog, 3-O-methyl-D-glucose equilibration were 8 s, 37 s and 38 s for rainbow trout, lamprey and RTH-149 cells, respectively. The 3-O-methyl-D-glucose was taken up by rainbow trout hepatocytes by facilitated diffusion in addition to simple diffusion. The uptake showed saturation kinetics with the K(m) of 37 mM and V(max) of 62 mmol kg(-1) cells min(-1). The uptake was sensitive to phloretin and cytochalasin B, but not affected by ouabain. The 3-O-methyl-D-glucose uptake by lamprey hepatocytes and RTH-149 cells showed no indication of saturation up to 160 mM, and was not affected by phloretin, cytochalasin B or ouabain, which suggests the mode of transport to be by passive diffusion. However, immunocytochemical stainings revealed the existence of mammalian type GLUT1 and GLUT2 transporters in all cells studied. The lack of a functioning carrier-mediated glucose uptake in lamprey hepatocytes might be due to its physiological state (prespawning starvation). The minor 3-O-methyl-D-glucose uptake into RTH-149 cells compared to freshly isolated rainbow trout hepatocytes might reflect low metabolic activity of the cell lines. Under the conditions applied the RTH-149 cell line is no suitable in vitro model for glucose transport in fish cells.     

Differences in metallothionein gene expression in primary cultures of rainbow trout hepatocytes and the RTH-149 cell line

Primary cultures of rainbow trout, Salmo gairdneri, hepatocytes were used to study the expression of metallothionein (MT) genes in response to steroid hormone treatment. The expression pattern was compared to that of an immortal cell line (RTH-149). MT mRNA accumulated in both cell cultures after exposure to zinc while 17 beta-oestradiol had no effect in either system. Treatment with cortisol and corticosterone resulted in a 2-fold increase of metallothionein mRNA levels in the primary cultures but had no effect in the RTH-149 cell culture. Primary cultures that were exposed to zinc or cortisol showed a high temporal correlation (r = 0.974) between MT mRNA and MT protein levels. The basal level expression was 3-4-fold higher in primary cultures than in RTH-149 cells. The present study demonstrates the inducibility of rainbow trout MT genes in response to glucocorticoids. It further indicates that primary cultures are to be preferred to immortal cell lines when investigating the inducibility of MT mRNA.     

Use of trout serum to prepare primary attached monolayer cultures of hepatocytes from rainbow trout (Salmo gairdneri)

Summary The influence of trout serum on the attachment and spreading of isolated trout hepatocytes maintained in primary culture at different temperatures was evaluated. Hepatocytes were obtained from young rainbow trout (Salmo gairdneri) by collagenase dissociation and maintained in modified Leibowitz L15 medium at 10° or 27° C for 24 h in plastic dishes previously coated with type I bovine collagen. In the absence of serum, fewer than 10% of hepatocytes attached and none of them spread on the collagen substrate. Trout serum at concentrations as low as 1.25% in the medium resulted in a pronounced concentration-dependent increase in hepatocyte attachment, as determined by direct counts by phase contrast microscopy, or by percentage of lactate dehydrogenase activity attached to the dishes after washing away unattached cells. Attachment rates were greater at the lower temperature (10° C). Trout serum also substantially increased the proportion of attached hepatocytes that spread as monolayers on the collagen substrate, especially at 10° C. By comparison, fetal bovine serum had little influence on the attachment or spreading of trout hepatocytes. These studies demonstrate a simple inexpensive method for preparing attached monolayer trout hepatocyte cultures. This procedure may be useful in toxicologic or functional studies in which fish hepatocyte attachment is an operational requirement.     

Comparison of trout hepatocyte culture on different substrates

Summary Comparisons were made of attachment and viability of rainbow trout (Salmo gairdneri) hepatocytes in short-term (2 days), primary culture on plastic, collagen-coated or extracellular matrix (ECM) coated dishes. Hepatocyte isolation routinely yielded cells with good viability (96%). Cells plated on ECM attached with high efficiency (93%) in contrast to cells cultured on plastic or collagen (∼20%). The cells plated on ECM flattened out and formed monolayers, while the cells on plastic and collagen rounded up and formed multi-cell aggregates in suspension. Viability of cells in all substrates remained high over the 2 day culture period. ECM is the first substrate to support trout-hepatocyte attachment in primary culture. Differentiated liver function was maintained in cells cultured on ECM as evidence by the induction of tyrosine aminotransferase by hydrocortisone (200%). This work was supported in part by research grant R809599010 from the U. S. Environmental Protection Agency. Editor's Statement This paper reports improved methods for culture of trout liver-derived cells that make in vitro investigations of fish metabolism, carcinogenesis and chemical toxicity more feasible than previously applied techniques. Recent interest in fish as models for study and indicators of effects of envionmental and food-related toxins make this work timely, poarticularly since many of the compounds of interest are primarily metabolized by hepatocytes or act on liver as a major target. David W. Barnes     

Maintenance of rat hepatocytes under inflammation by coculture with human orbital fat-derived stem cells

Preservation of hepatocyte functions in vitro will undoubtedly help the management of acute liver failure. The coculture system may be able to prevent functional decline of hepatocytes. It has already been shown that hepatocytes, when cocultured with bone marrow mesenchymal stem cells, could undergo long-term culture in vitro without loss of functions. In this study, human orbital fat-derived stem cells were isolated and cocultured with rat hepatocytes. When treated with serum from an acute liver failure patient, rat hepatocyte monoculture showed reduction of cell viability and loss of liverspecific functions. However, rat hepatocytes in the coculture system were still able to secret albumin and synthesize urea. IL-6 was significantly elevated in the coculture of rat hepatocyte with orbital fat-derived stem cells, and it might be the key immunoregulator which protects rat hepatocytes against inflammation. Our data confirmed that orbital fat-derived stem cells, or other adipose tissue-derived stem cells, are an ideal candidate to support rat hepatocyte functions in vitro.     

Isolation and characterization of biliary epithelial cells from rainbow trout liver

Summary Lectin binding and density gradient centrifugation were explored for isolating epithelial cells from trout liver. Hepatocytes exhibited preferential attachment to coverslips coated withPhaseolus vulgaris erythroagglutinin. Biliary epithelial cells attached with glycine max agglutinin; however, significant attachment of cellular debris limited the use of glycine max agglutinin. Percoll-density gradient centrifugation separated liver cells into two distinct populations with biliary cells and hepatocytes banding at densities of 1.04 and 1.09, respectively. A discontinuous gradient composed of 13% Ficoll (wt/wt) separated biliary cells from hepatocytes. The recovery of highly enriched biliary epithelial cells from trout liver using Ficoll gradients yielded approximately 8 million cells (0.1 ml packed cells) from 10 g liver. Western blot analysis demonstrated that the cytokeratin profile for extracts from biliary epithelial cell-enriched populations differ significantly from those seen with whole liver extracts or with extracts from hepatocyte-enriched populations. Ficoll-gradient purified biliary cells and hepatocytes attached to culture plates coated with trout skin extract and carried out linear incorporation of leucine into protein and thymidine into DNA for 24 h. A mixture of growth hormones (insulin, epidermal growth factor, and dexamethasone) stimulated thymidine incorporation into DNA; however, long-term culture of dividing biliary epithelial cells was not achieved. Chemical analysis of neutral and acidic glycolipids indicated that hepatocytes and biliary cells have similar glycolipid profiles with an exception in the region of GM3 mobility, which is attributable to differences in the ceramide moiety. These studies provide a starting point for further characterization of unique cell types of the trout liver that may be important in their response to toxic and carcinogenic agents.     

Isolated trout liver cells: Establishing short-term primary cultures exhibiting cell-to-cell interactions

Summary Composition and interactions of cell types in rainbow trout (Oncorhynchus mykiss) liver digested with collagenase and cultured in serum-free media were investigated. Suspensions obtained after digesting trout liver with collagenase contained all the cell types present in the liver, including liver parenchymal cells (hepatocytes), biliary epithelial cells, sinusoidal endothelium, fat-storing cells of Ito, and macrophages. A major cell pellet, mainly hepatocytes but containing significant numbers of biliary epithelial cells, was obtained by centrifuging the cell suspension at 120×g for 1 min. Cells present in this pellet quantitatively attached to culture plates coated with a trout skin extract and remain attached for 4 to 6 d with good retention of intracellular enzymes and DNA. When in culture, significant changes in and among the cells were observed. Initial preparations were rounded, single cells. Within several hours, however, cellular interactions leading to aggregation became evident and aggregates increased in size for 2 to 3 d. Scanning electron microscopy (EM) showed frequent shaftlike projections from margins of the aggregates. Transmission EM indicated that these projections represent biliary ductules forming in vitro. Adjacent hepatocytes also showed plasma membrane specializations forming junctional complexes and canaliculi characteristics of normal trout liver. After 5 to 6 d in culture, significant numbers of the cell aggregates dislodged from the plate. Analysis showed the dislodged cells were viable but vacuolated. The reestablishment in vitro of morphologic relationships resembling in situ tissue components suggest these culture preparations may have significant utility in cooperative metabolic studies of cell interactions in trout liver. Supported by grant CA45131 from the National Cancer Institute, Bethesda, MD.     

Serum-free cryopreservation of porcine hepatocytes

The use of porcine hepatocytes in xenotransplantation, bioartificial liver support or pharmacological approaches demands serum-free cryopreservation protocols yielding high quality, viable, functional hepatocytes. Here, primary porcine hepatocytes were frozen without serum in liquid nitrogen by the use of a computer-assisted freezing device. After thawing, more than 90% of the initial hepatocytes were lost, in part because of damage to genomic DNA. When cryoprotectants were used, the loss was lowered to 70% of the initial cell number; 90% of the remaining cells excluded trypan blue indicating a high degree of viability. Cells were seeded serum-free onto collagen-coated plastic dishes to determine proliferation and retainment of specific functions representing prominent features of hepatocytes in vivo. Whereas no cells adhered to the substratum effectively in conventional culture medium, the addition of conditioned medium derived from hepatic non-parenchymal cells improved attachment. Cells proliferated, retained hepatocyte-specific functions, such as urea production and cytochrome P450 activity, and expressed liver-specific genes to levels observed in non-cryopreserved hepatocytes. Thus, serum-free cryopreserved primary porcine hepatocytes may serve as a valid source of cells for downstream applications. The cells seem to function adequately when an appropriate environment is chosen for recovery after cryopreservation, an ultimate demand for the clinical application of human hepatocytes.     

Trout hepatocyte culture: Isolation and primary culture

Summary Rainbow trout (Salmo gairdneri) hepatocytes were isolated using a two-step perfusion through the portal vein. A typical perfusion yielded 2.92×10⁶ liver cells with a mean viability of 96.3%. Hepatocytes comprised 93.4% of the total cell isolate. Survival of hepatocytes in suspension culture was dependent on fetal bovine serum concentration and temperature of incubation. Serum concentrations of 5, 10, and 20% produced the highest survival during primary culture. Hepatocyte survival was in inverse proportion to the incubation temperature. Trout hepatocyte DNA synthesis and mitosis decreased during the culture period. Cytochromep ₄₅₀ activity decreased rapidly during the first 2 d of culture and then remained low but measurable during the remaining 8 d of culture. Culture temperature also influenced thep ₄₅₀ activity with lower temperatures producing greater activity. Morphologic changes occurred in the cells during culture. Isolated hepatocytes self-aggregated, forming strands and clumps that increased in size with time in culture. Junctional complexes between cells were evident within the aggregates. Nuclear atypia, increases in size and number of autophagic vacuoles, and the appearance of bundles of intermediate filaments also were observed with increased time in culture. This work was supported in part by an American Cancer Society Grant (Ohio Division, Inc.) and an NIH Biomedical Research Support Grant 5507RR05700010.     

Integrin-like substrate adhesion in RTG-2 cells, a fibroblastic cell line derived from rainbow trout

Fluorometric cell attachment assays together with competitive inhibitors of adhesion were used to probe for the presence of integrins, a diverse family of heterodimeric cell-surface glycoproteins involved in cell-cell and cell-extracellular matrix adhesion, in the fibroblastic rainbow trout cell line, RTG-2. The adhesive properties of this cell line were evaluated. RTG-2 cells adhered poorly to TC plastic in the absence of serum but as little as 2.5% fetal bovine serum allowed over 75% of the cells to attach after 5 h. Surfaces coated with the extracellular matrix proteins collagen I, collagen IV, fibrin, fibrinogen, or fibronectin were able to support attachment of RTG-2 cells. Adhesion of RTG-2 cells to fibronectin varied linearly with fibronectin coating densities in the range 0 to 65 ng/mm(2). Oligopeptides containing the sequence Arg-Gly-Asp (RGD) caused dose-dependent inhibition of adhesion to microtiter plates coated with fibrin, fibrinogen, and fibronectin, whereas attachment to collagen I and collagen IV was less severely affected. In all cases, peptides containing Arg-Gly-Glu (RGE) or Asp-Gly-Arg (DGR) sequences caused no reduction of cell attachment. Since many integrins mediate adhesion by binding to RGD sequences in their target ligands, these results suggest the presence of integrin-like adhesion molecules on the surface of RTG-2 cells.     

Effects of genetically modified human skin fibroblasts,stably overexpressing hepatocyte growth factor,on hepatic functions of cocultured C3A cells

Diseases leading to terminal hepatic failure are among the most common causes of death worldwide. Transplant of the whole organ is the only effective method to cure liver failure. Unfortunately, this treatment option is not available universally due to the serious shortage of donors. Thus, alternative methods have been developed that are aimed at prolonging the life of patients, including hepatic cells transplantation and bridging therapy based on hybrid bioartificial liver devices. Parenchymal liver cells are highly differentiated and perform many complex functions, such as detoxification and protein synthesis. Unfortunately, isolated hepatocytes display a rapid decline in viability and liver‐specific functions. A number of methods have been developed to maintain hepatocytes in their highly differentiated state in vitro, amongst them the most promising being 3D growth scaffolds and decellularized tissues or coculture with other cell types required for the heterotypic cell‐cell interactions. Here we present a novel approach to the hepatic cells culture based on the feeder layer cells genetically modified using lentiviral vector to stably produce additional amounts of hepatocyte growth factor and show the positive influence of these coculture conditions on the preservation of the hepatic functions of the liver parenchymal cells' model—C3A cells.     

Extended expression of differentiated function in primary cultures of adult liver parenchymal cells maintained on nitrocellulose filters : I. Induction of phosphoenolpyruvate carboxykinase and tyrosine aminotransferase

A new support system has been developed which provides long-term maintenance of non-dividing adult rat liver parenchymal cells in monolayer cultures. The hepatocytes, attached to Millipore (MP) filters, are maintained as free-floating cultures which express differentiated liver cell functions for up to 13 days. After 8 days of culture on MP filters, the hepatocytes are still capable of inducing tyrosine aminotransferase 3- to 4-fold and phosphoenolpyruvate carboxykinase 10-to 15-fold. The advantage of using floating MP filters to support the hepatocytes over the more conventional culture supports such as glass or plastic dishes are: (1) the functional lifespan of cultured hepatocytes is doubled, permitting experiments requiring 4–8 days to complete; (2) it permits rapid and easy transfer of cells from one set of culture conditions to another; (3) sections can be cut from one filter permitting multiple samples from a single culture; (4) the filters containing the cells can be processed without losing the orientation of cell surfaces, an important consideration when employing techniques such as autoradiography and/or electron microscopy; and (5) this culture technique can readily be adapted for co-cultivation experiments in order to directly examine biological and biochemical effects of secreted products of one cell type on another.     

Stimulative effect of non-parenchymal liver cells on ability of tyrosine aminotransferase induction in hepatocytes

Hepatocytes and non-parenchymal liver cells were isolated from adult rat liver and co-cultured for 48 hours as a monolayer on polystyrene culture dishes. The ability of tyrosine aminotransferase (TAT) induction in hepatocytes was examined in the presence of dexamethasone and dibutyryl cAMP. Non-parenchymal cells greatly enhance the ability of TAT induction of hepatocytes. A soluble factor with molecular weight of more than 10,000 is responsible for this enhancement, because conditioned medium prepared from non-parenchymal cells is also stimulatory. Non-parenchymal cells restored the ability in hepatocytes damaged with the addition of D-galactosamine. Conditioned medium prepared from non-parenchymal cells treated with D-galactosamine had higher activity of enhancement than the medium from normal cells. The soluble factor might be released in response to some signal of injury. Hepatocytes and non-parenchymal cells were immobilized within Ca-alginate, and although immobilized hepatocytes rapidly lost the ability to induce TAT, hepatocytes co-immobilized with non-parenchymal cells maintained the ability during 4 days of culture. These results indicated that non-parenchymal liver cells, as well as hepatocytes, could be used to construct a bioartificial liver support system.     

Isolation and culture of hepatocytes from human liver of immediate autopsy

Summary Human livers were removed at immediate autopsy (IA) from brain death patients within 1 h after cessation of cardiac function. Viable hepatocytes were isolated successfully from these IA livers by perfusion of an intack lobe with collagenase or by digestion of a small tissue wedge with collagenase-dispase. The yields of hepatocytes ranged from 1 to 3 × 10⁶ cells/g liver in the five cases studied. Approximately 70 to 90% of the cells excluded trypan blue dye. In the isolated hepatocytes, 632 pmol/mg protein of cytochromep ₄₅₀ and 536. pmol/mg protein cytochromeb ₅ were measured. The cells attached to the dishes in 4 h and produced monolayer cultures with a high success rate. The cells maintained in primary cultures for several days and developed ultrastructural features characteristic of human hepatocytes in vivo. The cultured hepatocytes can hydroxylate benzo[a]pyrene, conjugate the metabolites, and have a benzo[a]pyrene hydroxylase activity of 48.7 pmol/mg DNA per h, which is comparable to that of rat hepatocytes. The liver cells repaired DNA damage caused by exposures to aminofluorene and acetylaminofluorene in culture. This work was supported by EPA Grants R-809835-01-1, R-809599010 and DOE Contract DE-A505-83ER60158. Cobtribution no. 1762 from the Cellular Pathobiology Laboratory, University of Maryland School of Medicine.     

Cell detachment mediated by hyaluronic acid

Exogenous hyaluronic acid (HA) added to a confluent monolayer of 3T3 BALB cells facilitates cell detachment which can be enhanced by gently pipetting. When HA is added to a cell culture with the cell inoculum, the cells are able to grow and form a confluent monolayer, but the cellular density is lower than in the control cultures, in a concentration-dependent way. This difference seems due to the ease of detachment promoted by HA on the cells near confluency. In fact only near confluency is the amount of the detached cells greater in the culture plates containing HA than in controls. Culture dishes containing substrate-attached material (SAM) left behind by the confluent 3T3 BALB cells have been prepared by removing the cells with different detaching agents. The SAM-containing dishes have been incubated in the presence of HA for 24 h and, after washing, were used for cell cultures. The cells grown on such HA-treated dishes show a very low density at confluency and in some cases are prevented from forming a confluent monolayer. When the SAM-containing dishes are treated with Streptomyces hyaluronidase, the effect of HA is abolished and the cells are able to grow normally. Among the chondroitin sulphates, only chondroitin sulphate C shows the same effects as HA, whereas A and B are ineffective.     

Primary cultures and the levels of cytochromeP450 in hepatocytes from mouse,rat, hamster,and rabbit liver

Summary Hepatocyte primary cultures (HPC) derived from rat, mouse, hamster, and rabbit liver were characterized for a variety of parameters. The conditions that maximized recovery, attachment, and survival varied between species. Hepatocytes from all four species were capable of attaching in serum-free Williams’ medium E (WME), but optimal attachment as monolayer cultures was achieved for mouse and hamster HPC in medium receiving 1% calf serum supplementation. Hamster hepatocytes required additional cations, whereas rabbit and rat hepatocytes displayed maximal attachment in medium supplemented with 10% calf serum. Survival of mouse and rabbit hepatocytes after 24 h in serum supplemented media was in the order of 90%. Rat and hamster hepatocyte 24 h survival was approximately 70 and 60%, respectively, and was not significantly affected by serum supplementation. Hepatocytes from each species varied in their content of cytochromeP450 at the time of isolation and in the rate of reduction during culture. Mouse and rat hepatocytes demonstrated the most rapid decline in content during the initial 24 h in culture, whereas concentrations in rabbit hepatocytes were virtually unchanged. The rate of decline inP450 concentrations in hamster hepatocytes was intermediate between those displayed by rat and rabbit hepatocytes. These studies have delineated conditions useful for the culture of hepatocytes from four species and have documented the status of an important parameter of their functional capability. This study was supported by EPA contract 68-01-6179. C. J. Maslansky was a recipient of a Monsanto Fund Fellowship in Toxicology.     

Oxidative stress response induced in rat primary hepatocyte monolayers by mechanical removal of adherent cells

We show that mechanical separation of adherent rat primary hepatocytes after the monolayer-forming stage causes the induction of the oxidative stress genes HO-1 (haem oxygenase) and MnSOD (manganese superoxide dismutase). The procedures for enzymatically breaking up liver tissue structure and isolating hepatocytes do not cause HO-1 and MnSOD activation. Only after a 3-h incubation, during which hepatocytes form a monolayer on culture dishes, does the hydrodynamic shearing away of necrotic cells sticking to the monolayer surface activate these two genes. Analysis of this injury-response pathway shows that oxidative stress and mitochondrial dysfunction play a role, as activation can be repressed by antioxidants and by respiratory inhibitors. Recovery of the cells takes a further 24-h incubation during which HO-1 and MnSOD expression returns to basal levels.     

Crude liver membrane fractions as substrate preserve liver-specific functions in long-term,serum-free rat hepatocyte cultures

Summary Over time, rat hepatocytes cultured on collagen lose the capacity to express liver-specific functions. The influence on this degradation process of an alternative substratum—crude membrane fractions prepared from the liver of the same rat strain—was investigated. Freshly isolated rat hepatocytes were cultured in serum-free Williams E medium supplemented with aprotinin, selenium, dexamethasone, and insulin in flasks coated with a mixture of rat liver crude membrane fractions:collagen type I (100:1). The cells adhered firmly, exhibiting minimal spreading and remaining grouped in columns or in cell islands, and retained their liver-specific functions for more than 1 wk. Hepatocytes secreted substantially higher amounts of albumin than cells cultured on collagen-coated dishes, and on Days 1 and 9 in culture the total P-450 content was 72 and 40%, respectively, of that of freshly isolated cells. On Day 6, the 7-ethoxyresorufin-O-de-ethylase and the aldrin epoxidase activities were still more than 50% that of freshly isolated hepatocytes. Exposure to phenobarbital on Days 3 to 6 increased the total cytochrome P-450 content twofold; exposure to 3-methylcholanthrene increased the activity of the corresponding cytochrome P-450 isoforms to 20 times that observed in untreated cultures and 6 times that observed in freshly isolated cells. Thus, given the ease with which they are prepared, the use of crude membrane fractions combined with culture medium supplemented with aprotinin and selenium can facilitate the preparation of reproducible cultures suitable for long-term in vitro pharmacotoxicologic studies using rat hepatocytes.     

Quantitative comparison of rat hepatocyte functions in two improved culture systems with or without rat liver epithelial cell line

We quantitatively evaluated two recently-developed novel techniques for hepatocyte cultivation in a dish level; that is, spheroid culture and membrane-supported collagen (CN) gel sandwich culture, in terms of cellular maintenance, albumin secretion and 7-ethoxycoumarin (7EC) metabolism to 7-hydroxycoumarin (7HC) as a marker for cytochrome P450 IA1 activity in the presence and absence of rat liver epithelial cell line (RLEC) during one month of culture, together with conventional coculture with RLEC in CN-coated dishes as a control. RLEC prevented spheroid loss caused by its detachment from the culture dishes often occurring in pure culture. CN-gel sandwich by itself improved remarkably hepatocyte maintenance when compared with CN-gel free systems, thereby resulting in enhancement of overall functional expressions as compared with CN-gel free systems. RLEC in CN-gel sandwhich, however, reduced cellular sustainment probably due to its suppression of hepatocyte growth. Although there were no significant differences in albumin secretion per cell among the five cultures examined, CN-gel sandwich expressed markedly higher 7EC metabolizing activity per cell, where RLEC presence had a preferable influence. Consequently, membrane-supported CN-gel sandwich was the most superior technique for hepatocyte cultivation from the standpont of both cellular maintenance and its functional expressions per cell.     

Selective and organotypic culture of intrahepatic bile duct cells from adult pig liver

Summary Secondary culture of nontransformed bile duct epithelium has been difficult to achieve. STO feeder cell-dependent secondary cultures of adult pig bile duct cells were established from primary cultures of adult pig liver cells. Adult pig hepatocytes exhibited limited or no replication and were lost from the secondary culture at Passage 3 or 4. In contrast, adult pig bile duct cells replicated and were carried for 4–8 passages in secondary culture. A simple method to produce nearly pure pig intrahepatic bile duct cultures was first to freeze a relatively crude liver cell preparation. Upon subsequent thawing, all hepatocytes and most macrophages were lysed. Bile duct cells composed 95% of the surviving cells after the freeze/thaw, and they grew out rapidly. The bile duct cells grew on top of the STO feeder cells as closely knit epithelial, colonial outgrowths. Histocytochemical and biochemical analyses demonstrated high levels of gamma-glutamyltranspeptidase activity and low levels of P450 activity in the bile duct cultures. The bile duct cells spontaneously adopted a multicellular ductal morphology after 7–10 d in static culture which was similar to that found in in vivo pig liver. Transmission electron microscopic examination revealed complex junctions and desmosomes typical of epithelium, and lumenally projecting cilia typical of in vivo intrahepatic bile ductules. This simple method for the coculture of pig intrahepatic bile duct cells which adopt in vivo-like structure may facilitate biological studies of this important, but difficult to culture, cell type.