Use of hepatocytes in primary culture for biochemical studies on liver functions

Summary Rat parenchymal hepatocytes isolated with collagenase were cultured as monolayers in Williams medium E supplemented with calf serum. Freshly isolated cells showed very low activities of various liver functions, and they had to be cultured for 6-24 h to allow recovery of these functions. Insulin and dexamethasone greatly increased cell viability in primary culture. After culture for 24 h, these cells showed various liver functions as seen in vivo and responded well to various added hormones and amino acids. The concentrations of amino acids in the medium regulated synthesis of serum proteins and insulin stimulated lipogenesis, which in turn regulated synthesis of lipoproteins. Insulin also stimulated glycogen synthesis and the stimulation was parallel with the number of insulin receptors. Glucagon stimulated glycogenolysis and its stimulation involved the function of the cytoskeleton. Glucagon and dexamethasone induced various enzymes of amino acid catabolism, such as tryptophan oxygenase, tyrosine aminotransferase and serine dehydratase. These inductions were inhibited by insulin or catecholamine. The effect of catecholamine was due to its -adrenergic action. The -action of isoproterenol was low in freshly isolated cells, but increased during culture of the cells. Acquirement of hormonal responses during neonatal development can be studied in this culture system. Mature hepatocytes in culture are usually quiescent, but when insulin and epidermal growth factor were added, DNA synthesis by the cells increased markedly and they showed density-dependent growth. In this culture system, serum could be omitted for 2 days when the dishes were coated with fibronectin without appreciable change of functions, but serum was needed for longer culture of the cells. A factor that increased cell survival was found in serum and in pituitary gland.These results show that hepatocytes in primary culture are a simple and useful system for studies of liver functions in vitro and related works were also reviewed.     

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.     

Cell density-dependent regulation of albumin synthesis and DNA synthesis in rat hepatocytes by hepatocyte growth factor.

Hepatocyte growth factor (HGF), a potent mitogen for mature hepatocytes, has been considered to act as a hepatotropic factor for liver regeneration. We examined the effect of HGF on albumin synthesis and DNA synthesis of adult rat hepatocytes cultured at various cell densities. HGF stimulated albumin synthesis of hepatocytes by 40-60% when they were cultured at higher cell densities such that there was tight cell-cell contact. But at lower cell densities HGF failed to stimulate albumin synthesis. In contrast, the stimulatory effect of HGF on DNA synthesis of hepatocytes was more potent at lower than at higher cell densities: HGF did not stimulate DNA synthesis of hepatocytes cultured at confluent cell density. Thus, HGF seems to stimulate both albumin synthesis and DNA synthesis of hepatocytes, in a reciprocal relationship depending on cell density. When the effects of various cytokines were examined, epidermal growth factor, transforming growth factor-alpha, and acidic fibroblast growth factor also stimulated albumin synthesis by 20-30%. However, transforming growth factor-beta 1, basic fibroblast growth factor, and interleukin-1 beta had no effect on albumin synthesis, while interleukin-6 inhibited it by 42%. Thus HGF was the most potent in stimulating albumin synthesis in these cytokines. Since HGF is markedly increased in the liver or plasma following various liver insults, HGF may be involved in liver regeneration through the potential to stimulate both cell growth and liver-specific functions such as albumin synthesis in a cell density-dependent manner.     

Direct analysis of growth factor requirements for isolated human fetal hepatocytes

Summary Hepatocytes were isolated from human fetal liver in order to analyze the direct effects of growth factors and hormones on human hepatocyte proliferation and function. Mechanical fragmentation and then dissociation of fetal liver tissue with a collagenase/dispase mixture resulted in high yield and viability of hepatocytes. Hepatocytes were selected in arginine-free, ornithine-supplemented medium and defined by morphology, albumin production and ornithine uptake into cellular protein. A screen of over twenty growth factors, hormones, mitogenic agents and crude organ and cell extracts for effect on the stimulation of hepatocyte growth revealed that EGF, insulin, dexamethasone, and factors concentrated in bovine neural extract and hepatoma cell-conditioned medium supported attachment, maintenance and growth of hepatocytes on a collagen-coated substratum. The population of cells selected and defined as differentiated hepatocytes had a proliferative potential of about 4 cumulative population doublings. EGF and insulin synergistically stimulated DNA synthesis in the absence of other hormones and growth factors. Although neural extracts enhanced hepatocyte number, no effect on DNA synthesis of neural extracts or purified heparin-binding growth factors from neural extracts could be demonstrated in the absence or presence of defined hormones, hepatoma-conditioned medium or serum. Hepatoma cell-conditioned medium had the largest impact on both hepatocyte cell number and DNA synthesis under all conditions. Dialyzed serum protein (1 mg/ml) at 10 times higher protein concentration had a similar effect to hepatoma cell-conditioned medium (100 μg/ml). The results suggest that hepatoma cell conditioned medium may be a concentrated and less complicated source than serum for purification and characterization of additional normal hepatocyte growth factors. This work was supported by NIH grant DK35310. Editor’s statement Many investigators have struggled with the special problems associated with culture of differentiated hepatocytes. In this paper attention is given to the specific growth factor requirements for fetal human hepatocytes. The observation that factors from hepatoma conditioned medium or neural extracts enhanced the growth of the cells may indicate that additional growth factors are to be identified that are important in the survival and proliferation of hepatocytes, and may also indicate that the malignant transformation of these cells may involve the production of autocrine growth stimulators.     

Density-dependent growth control of adult rat hepatocytes in primary culture

Adult rat hepatocytes in primary culture, which show various liver functions, did not show any mitosis at confluent cell density, although they entered the S phase and remained in the G2 phase, judging by cytofluorometry, when insulin and epidermal growth factor (EGF) were added to 2-day cultures (Tomita, Y., Nakamura, T., & Ichihara, A. (1981) Exp. Cell Res. 135, 363-371). However, when the cell density was decreased by half or one third, the number of nuclei and cell number increased to 1.5-2.0 times that after culture for 35 h with insulin and EGF. Moreover, at these lower densities, DNA synthesis started much earlier, although at the usual high density DNA synthesis with these two hormones did not start until the hepatocytes had been cultured for over 40 h. These results suggest that proliferation of mature rat hepatocytes is regulated by the cell density. First, cells in G0 enter the G1 phase density-dependently; then cells in the G1 phase seem to be stimulated to enter the S phase by insulin and EGF, and a low cell density may permit cells after DNA synthesis to enter the M phase. DNA synthesis of rat hepatocyte cultures at low cell density was strongly inhibited by co-culture with a dense culture. Therefore, the density-dependent mechanism of hepatocyte proliferation seems to involve regulation by a soluble inhibitor(s) secreted by the hepatocytes into the culture medium.     

Cultivation of adult rat hepatocytes on 3T3 cells: expression of various liver differentiated functions

Adult rat hepatocytes were maintained in culture for at least 1 month without losing the expression of their differentiated functions; they were cultured on lethally treated 3T3 fibroblasts inoculated at 35,000 cells/cm2 with medium containing 10-25 micrograms/ml hydrocortisone. Hepatocytes showed their typical morphology; they formed bile canaliculi, microvilli, and intercellular junctions with desmosomes and nexus; some formed structures that may resemble the perisinusoidal space of Disse. In addition, they showed DNA synthesis and expressed some liver-specific functions. They synthesized albumin and other proteins, which were exported to the culture medium. Like parenchymal liver cells in vivo, de novo fatty acid synthesis and esterification took place, and more than 80% of the lipids synthesized by the hepatocytes were secreted into the medium as triglycerides; they also showed cytochrome-P450 activity that was inducible with phenobarbital, suggesting that the hepatocytes have the capacity to metabolize drugs. These culture conditions allow the study of various hepatocyte differentiated functions, and they may provide the means to analyze the effect on liver of hormones, viruses and hepatotoxic chemicals and drugs; they may also indicate conditions adequate for serial growth of hepatocytes.     

Signalling pathways involved in the process of mesenchymal stem cells differentiating into hepatocytes

End‐stage liver disease can be the termination of acute or chronic liver diseases, with manifestations of liver failure; transplantation is currently an effective treatment for these. However, transplantation is severely limited due to the serious lack of donors, expense, graft rejection and requirement of long‐term immunosuppression. Mesenchymal stem cells (MSCs) have attracted considerable attention as therapeutic tools as they can be obtained with relative ease and expanded in culture, along with features of self‐renewal and multidirectional differentiation. Many scientific groups have sought to use MSCs differentiating into functional hepatocytes to be used in cell transplantation with liver tissue engineering to repair diseased organs. In most of the literature, hepatocyte differentiation refers to use of various additional growth factors and cytokines, such as hepatocyte growth factor (HGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), oncostatin M (OSM) and more, and most are involved in signalling pathway regulation and cell–cell/cell–matrix interactions. Signalling pathways have been shown to play critical roles in embryonic development, tumourigenesis, tumour progression, apoptosis and cell‐fate determination. However, mechanisms of MSCs differentiating into hepatocytes, particularly signalling pathways involved, have not as yet been completely illustrated. In this review, we have focused on progress of signalling pathways associated with mesenchymal stem cells differentiating into hepatocytes along with the stepwise differentiation procedure.     

Modulation of functional activities in cultured rat hepatocytes

Rat hepatocytes isolated by enzymatic dissociation of the liver must attach in order to survive for more than a few hours. In conventional culture conditions, they rapidly lose their highly differentiated functions, e.g. adult isozymic forms, enzyme response to specific hormones and cytochrome P-450-dependent monooxygenase activities. Incompletely differentiated cells such as perinatal and regenerating hepatocytes, can transiently exhibit a more differentiated state. Therefore, regulation of hepatic functions, particularly enzyme activities cannot be studied for more than a few days. Hepatocyte survival rate and maintenance of specific functions are dependent on nutrient composition of the medium as well as the substrate. Complex matrices, particularly that derived from the connective liver biomatrix, appear to have an important favorable effect. However, regardless of culture conditions specific functions cannot be quantitatively maintained for more than several days. Recent observations strongly suggest that such a problem may be overcome by mimicking in vivo specific cell-cell interactions. Thus when co-cultured with a liver epithelial cell line, probably derived from biliary ductular cells, adult hepatocytes remain able to synthesize high levels of albumin and to conjugate drugs. In these conditions, the cells secrete an abundant heterogeneous extracellular material. The co-cultures can be maintained in a serum-free medium and specific liver functions can be altered experimentally. Such a model could be appropriate for studying long-term induction and modulation of liver enzyme activities under defined experimental conditions.     

Control of DNA synthesis and ornithine decarboxylase activity by hormones and amino acids in primary cultures of adult rat hepatocytes

The conditions for stimulation of ornithine decarboxylase (ODC) and DNA synthesis in primary monolayer cultures of non-growing, highly differentiated hepatocytes from adult rats were compared. The syntheses of ODC and DNA were not stimulated by hormones on the 1st day of culture, but they were induced markedly by insulin (10⁻⁸ M) and epidermal growth factor (EGF, 0.1 μg/ml) in cells cultured for 40 h. The effects of insulin and EGF were synergistic, and the ODC activity as well as the DNA synthesis in the presence of these hormones was comparable to that of cultured hepatocytes from partially hepatectomized liver. Other factors had different effects on the two processes. Dexamethasone induced ODC slightly, but it inhibited DNA synthesis strongly. Putrescine inhibited ODC activity, but it had no effect on DNA synthesis. Asparagine and glutamine induced ODC activity, but they inhibited DNA synthesis; their inhibitory effects on DNA synthesis were specific to primary cultured liver cells and were not seen in an established rat liver cell line or in mouse L cells. These results show that although there is some correlation between ODC induction and DNA synthesis, the former is not essential for cell growth. There was no indication of cell division under conditions where maximal ODC induction and DNA synthesis were observed. Cytofluorometry of cells treated with insulin and EGF showed that the DNA content increased from 2 N to 4 N, and to 8 N in some cells. Therefore, under the present culture conditions, mature liver cells could enter G2 phase through S phase, but could not enter M phase.     

Importance of cell aggregation for expression of liver functions and regeneration demonstrated with primary cultured hepatocytes

Adult rat hepatocytes aggregated to form floating multicellular spheroids when cultured in Primaria dishes, which have a positively charged surface, in serum-free Williams' medium E (WE) supplemented with insulin and epidermal growth factor (EGF). These hormones were essential for maintenance of the spheroids, whereas the size of the spheroids depended on the inoculum cell density. The spheroids retained in vivo levels of expressions of albumin and glucokinase and synthesized scarcely any DNA even in the presence of insulin and EGF. On transfer to type I collagen-coated dishes, the spheroids gradually disaggregated and the cells formed monolayers, in which the expressions of albumin and glucokinase were suppressed and DNA synthesis and hexokinase activity were increased. DNA synthesis of hepatocytes in monolayer culture was maximal 24 hr after transfer of the spheroids, ～80% of the hepatocyte nuclei were labelled with bromodeoxyuridine during culture for 48 hr, and the mitotic index was ～70% after 60 hr. These results suggest that, in spheroids, hepatocytes remained in the G₀ phase, but that when they formed monolayers, they progressed to the G₁ phase and proceeded through the cell cycle in the presence of insulin and EGF. This work shows that the cell cycle of hepatocytes in culture can be manipulated by providing conditions for quiescence as spheroids or growth as monolayers and that the shape of hepatocytes is important for regulating their growth and liver-specific functions. © 1993 Wiley-Liss, Inc.     

Reciprocal modulation of growth and liver functions of mature rat hepatocytes in primary culture by an extract of hepatic plasma membranes

A cell-surface modulator with the ability to mimic the reciprocal effects of cell density on cell growth and liver-specific functions of mature rat hepatocytes in primary culture (Nakamura, T., Yoshimoto, K., Nakayama, Y., Tomita, Y., and Ichihara, A. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 7229-7233) was solubilized with 4% octyl glucoside and 4 M guanidine HCl from plasma membranes purified from adult rat liver. The membrane-free modulator showed activities for inhibition of DNA synthesis stimulated by insulin with epidermal growth factor and stimulation of tyrosine aminotransferase induction by dexamethasone. The apparent Mr of the factor was estimated as 670,000 by gel filtration on a Sephacryl S-400 column equilibrated with 0.5% octyl glucoside and 4 M guanidine HCl. The modulator was heat-labile and sensitive to trypsin. These results suggest that the reciprocal regulations of cell growth and hepatocyte-specific functions are mediated via cell to cell contact by a cell-surface modulator(s) that is an integral membrane protein.     

Propagation and functional characterization of serum-free cultured porcine hepatocytes for downstream applications

Hepatocyte transplantation is considered an alternative to whole organ transplantation. However, the availability of human cadaveric livers for the isolation of transplantation-quality hepatocytes is increasingly restricted. Xenogeneic porcine hepatocytes may therefore serve as an alternate cell ressource. The propagation of hepatocytes is often necessary to yield a sufficient cell number for downstream applications in xenotransplantation and in, for example, bioartificial liver support or pharmacological and toxicological studies. Our goal has been to propagate primary porcine hepatocytes in vitro and to determine the functional maintenance of the propagated cells. Porcine hepatocytes were cultured under serum-free conditions in the presence of hepatocyte growth factor and epidermal growth factor and passaged several times. The viability, proliferation and maintenance of liver-specific functions were determined as culture proceeded. Total cell number increased by 12-fold during four sequential passages, although the proliferative capacity was higher in primary cells and early passages as compared with late passages. Xenobiotics metabolism and urea synthesis gradually decreased with ongoing culture but could be restored by treatment with appropriate stimuli such, as β-naphthoflavone and cAMP. The expression of hepatocyte-specific genes was generally lower at the beginning than at later time-points of culture of individual passages. Porcine hepatocytes can thus be propagated in vitro. The partial loss of hepatocyte function may be restored in vitro by appropriate stimuli. This may also be achieved in a recipient liver after hepatocyte transplantation provided that the proper physiological environment for the maintenance of the differentiated hepatocyte phenotype is present. This study was supported by grants to B. Christ from the German Ministry of Education and Research (01 ZZ 0109 and NBL3-NG4) as well as by grants from the Federal State of Saxonia-Anhalt through the Wilhelm-Roux-Program at the Medical Faculty of the Martin-Luther-University of Halle-Wittenberg to B. Christ (09/07 and 04/03).     

Growth stimulation of primary rat hepatocytes by 2,3,7,8-tetrachlorodibenzo-p-dioxin

The modulation of liver growth control by the tumor promoter, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), was investigated in primary hepatocytes of adult rats. Under defined conditions in serum-free cultures, the interaction of TCDD with growth-related hormones was studied. TCDD-treatment of the cultured hepatocytes for two days caused a transient stimulation of both DNA synthesis and mitotic activity. This effect was maximal at the very low nontoxic concentration of 10^–12 M TCDD, i.e., two orders of magnitude below the optinzal concentrations for induction of drug metabolizing enzymes. Growth stimuladon by TCDD was dependent on the presence of growth-related hormones; in primary rat hepatocytes, TCDD acted synergistically with insulin and epidermal growth factor (EGF) and antagonized the growth inhibition by dexamethasone. Under culture conditions allowing high rates of DNA synthesis, e.g., at low concentrations of dexamethasone, in the presence of EGF plus alphal-adrenergic agonists or rat serum, no significant effect of TCDD on cellular growth was observed. Furthermore, TCDD failed to stimulate DNA synthesis in a rat hepatoma cell line, H4IIE, which is less sensitive to growth controlling factors than normal hepatocytes. Therefore, the results suggest that the growth modulation of primary rat hepatocytes by TCDD is the most sensitive parameter of the agent thus far observed. This effect may involve both a release from the growth inhibition caused, for instance, by glucocorticoids, as well as a direct growth-stimulating effect, synergistic to the one induced by insulin.Abbreviations Ah aryl hydrocarbon - EGF epidermal growth factor - EROD 7-ethoxyresorufin-0-deethylase - ³HdT [³H]thymidine - TCB 3,4,3,4-tetrachlorobiphenyl - TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin     

Hormonal inducibility of liver-specific enzymes in cultured rat embryos

In monolayer cultures, hepatocyte-specific enzymes are inducible by hormones as soon as hepatocytes differentiate from the embryonic foregut (15-somite stage). Though offering an excellent opportunity for quantitative studies, several features of a normal cell environment are lost in such a model system. To determine the inducibility of such tissue-specific enzymes in intact organisms, rat embryos were cultured in vitro for 48 h and exposed to the hormonal factors that had been found effective in monolayer culture, viz. dexamethasone, triiodothyronine and dibutyryl cyclic AMP. Normal development of the embryos during culture in vitro was assessed by general criteria reflecting growth, morphogenesis and cytodifferentiation. Development of external features, organogenesis, the distribution of cell divisions and the appearance of tissue-specific proteins such as alpha-fetoprotein and glutamate dehydrogenase served as parameters. Despite undisturbed development of the embryos as judged by these criteria, irrespective of whether the culture was started at day 10 or at day 11 of gestation (just before, respectively after the appearance of the liver primordium), induction of hepatocyte-specific enzymes like carbamoylphosphate synthetase by hormones could not be demonstrated immunohistochemically. However, induction of this enzyme by hormones could be demonstrated in monolayers of hepatocytes isolated from such embryos after 48 h of culture, providing yet another demonstration of the adequate culture conditions. In addition, an adequate uptake of hormones by the embryo during culture could be shown with radio-actively labeled dexamethasone and triiodothyronine and with a radioreceptor assay for cyclic AMP. Therefore, the presence of factors in young embryos that inhibit tissue-specific enzyme synthesis has to be postulated.     

Growth stimulation and apoptosis induced in cultures of neonatal rat liver cells by repeated exposures to epidermal growth factor/urogastrone with or without associated pancreatic hormones

Summary In untreated primary cultures of neonatal rat liver kept in high-calcium (1.8 mmol/l), foetal bovine serum (10%v/v)-containing minimal essential medium (FBSMEM), the absolute numbers of hepatocytes did not change between day 4 and day 9 because ongoing cell loss was counterbalanced by proliferation of a discrete sub-population of the cells. By contrast, the number of stromal cells increased linearly with time. Growth of hepatocytes and stromal cells was differently affected by the daily addition, between day 4 and day 8 of culture, of fresh medium to which peptide mitogen(s) in concentrations ranging from 10^-14 to 10^-8 mol/l had been added. Epidermal growth factor/urogastrone (EGF/URO) with or without equimolar mixtures of glucagon and insulin, induced first hyperplasia of hepatocytes and stromal cells and then apopotosis (degeneration and death) of the progeny of the stimulated cells. By contrast, equimolar mixtures of glucagon and insulin caused a progressive increase in the number of hepatocytes and stromal cells unbalanced by any increase in cell death. At subphysiological concentrations glucagon, in synergism with EGF/URO and/or some other unknown heat-stable component of serum, acted as a trophic factor for hepatocytes. By contrast, insulin alone did not enhance growth of hepatocytes, but rather blocked the mitogenic effects of EGF/URO. The three hormones exerted neither mitogenic nor apoptotic effects when administered in a low calcium (0.01 mmol/l) FBS-MEM medium.These results reveal that EGF/URO may control the size of cell populations in neonatal liver by calcium-dependent mechanisms that make it unlikely to be a promoter of hepatocyte tumours. They also show that glucagon acts as a positive trophic regulator for hepatocytes.     

A long-term culture of human hepatocytes which show a high growth potential and express their differentiated phenotypes

The present study succeeded for the first time in cultivating for more than 2 months human normal hepatocytes which showed a high growth potential and expressed their differentiated phenotypes. Constituents of culture medium were critical for this culture, and the medium optimized for their growth contained fresh human serum, fetal bovine serum, Swiss 3T3-cell conditioned medium, L-ascorbic acid 2-phosphate, epidermal growth factor, nicotinamide, and dimethyl sulfoxide. Hepatocytes steadily replicated and formed colonies which continued to increase in size up to around 35 days. The number of hepatocytes in the most replicative colonies increased 17-fold during 31 days. Cells in colonies expressed normal differentiated hepatocytic phenotypes for as long as 35 days. These hepatocytes retained normal liver functions at least for 70 days such as to secrete albumin, and to metabolize lidocaine and D-galactose.     

Insulin and heregulin-beta1 upregulate guanylyl cyclase C expression in rat hepatocytes: reversal by phosphodiesterase-3 inhibition

Guanylyl cyclase C (GC-C) is the receptor for the hormones guanylin and uroguanylin. Although primarily expressed in the rat intestine, GC-C is also expressed in the liver during neonatal or regenerative growth or during the acute phase response. Little is known about the hepatic regulation of GC-C expression. The influence of various hepatic growth or acute phase regulators on GC-C expression was evaluated by immunoblot analysis of protein from primary rat hepatocytes grown in a serum-free medium. Insulin and heregulin-beta1 strongly stimulated GC-C expression by 24 h of cell culture. Several different hormones and agents suppressed this action, including transforming growth factor beta (TGF-beta), as well as inhibitors of phosphatidylinositol 3-kinase (PI-3-kinase) and phosphodiesterase 3 (PDE-3, an insulin- and PI-3-kinase-dependent enzyme). The compartmental downregulation of cAMP levels by PDE-3 may be a critical step in the hormonal action that culminates in GC-C synthesis.     

Gene transfection of multicellular spheroid of hepatocytes on an artificial substrate

The handling of hepatocytes, a major cell population in the liver, is an important technique in both liver tissue engineering and hepatology. However, these cells are so fragile that it has been impossible to harvest hepatocytes with high viability from tissue culture dishes after a period of culture in vitro. In this study, we employed an artificial substrate for transfection of multilayer hepatocytes and harvested these cells with high viability after transfection. Hepatocytes cultured on an amphiphilic artificial substrate form multilayer aggregates (spheroids) in the presence of growth factors during gene transfection with cation liposomes. Compared to cells cultured on a collagen-coated plate, these spheroids are easily harvested with high viability by pipetting in EDTA solution. In addition, these spheroids rapidly spread on collagen after transfer from the artificial substrate, demonstrating that hepatocytes in the center of the spheroids were viable. Epidermal growth factor (EGF) increased the transfection efficiency into hepatocytes while hepatocyte growth factor (HGF) alone did not increase the efficiency. However, HGF synergestically increased the effect of EGF on transfection. Interestingly, this transfection required the process of spheroid formation because the gene was not transfected once the spheroid formation completed or under conditions where hepatocytes did not form spheroids. This method using spheroidal hepatocytes for in vitro transfection is promising for the development of ex vivo gene therapy.     

Modulation of mitogen-independent hepatocyte proliferation during the perinatal period in the rat

Summary Late gestation fetal rat hepatocytes can proliferate under defined in vitro conditions in the absence of added mitogens. However, this capacity declines with advancing gestational age of the fetus from which the hepatocytes are derived. The present studies were undertaken to investigate this change in fetal hepatocyte growth regulation. Examination of E19 fetal hepatocyte primary cultures using immunocytochemistry for 5-bromo-2′-deoxyuridine (BrdU) incorporation showed that approximately 80% of these cells traverse S-phase of the cell cycle over the first 48 h in culture. Similarly, 65% of E19 hepatocytes maintained in culture under defined mitogen-free conditions for 24 h showed nuclear expression of proliferating cell nuclear antigen (PCNA). These in vitro findings correlated with a high level of immunoreactive PCNA in immunofluorescent analyses of E19 liver. In contrast, E21 (term) liver showed little immunoreactive PCNA. The in vivo finding was recapitulated by in vitro studies showing that E21 hepatocytes had low levels of BrdU incorporation during the first day in culture and were PCNA negative shortly after isolation. However, within 12 h of plating, E21 hepatocytes showed cytoplasmic staining for PCNA. Although maintained under mitogen-free conditions, PCNA expression progressed synchronously to a nucleolar staining pattern at 24 to 48 h in culture followed by intense, diffuse nuclear staining at 60 h which disappeared by 72 h. This apparently synchronous cell cycle progression was confirmed by studies showing peak BrdU incorporation on the third day in culture. Whereas DNA synthesis by both E19 and E21 hepatocytes was potentiated by transforming growth factor α (TGFα), considerable mitogen-independent DNA synthesis was seen in hepatocytes from both gestational ages. These results may indicate that fetal hepatocytes come under the influence of an exogenous, in vivo growth inhibitory factor as term approaches and that this effect is relieved when term fetal hepatocytes are cultured.     

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.