Chlordecone is a potent in vitro inhibitor of oligomycin-insensitive mg2+ -ATPase of rat bile canaliculi–enriched fraction

The oligomycin-insensitive Mg²⁺ -ATPase (OIMATPase) of rat bile canaliculi–enriched fraction (BCEF) was inhibited by chlordecone (CD) in vitro (IC-50 = 25 μM). Kinetic analysis indicated noncompetitive inhibition. Inhibition of OIMATPase by filipin but not by atractyloside verified plasma membrane origin of activity. The cholestatic agents α-naphthyl isothiocyanate (ANIT) and taurolithocholate (TLC) decreased OIMATPase activity at in vitro concentrations of 33 and 162 μM, while taurocholate (a choleretic bile salt), ethynylestradiol, and manganese did not. Cholestatic drugs with primary intracellular sites of action (colchicine and phalloidin) were ineffective OIMATPase inhibitors in this concentration range. Inhibition of OIMATPase by N-ethylmaleimide (NEM) and dicyclo-hexylcarbodiimide (DCCD) indicated some H⁺ -ATPase activity in BCEF. In vitro sensitivity of OIMATPase of BCEF to CD, ANIT, and TLC suggested the bile canaliculus as a subcellular-level target for their cholestatic actions.     

Formation of bile canaliculi in long-term primary cultures of adult rat hepatocytes on permeable membrane: an ultrastructural study

Adult rat hepatocytes were cultured for 15 days on type I collagen-coated permeable membranes in a hormonally defined Waxman's modified medium supplemented with very low concentrations of insulin, glucagon and dexamethasone. Phase contrast examination showed that 15-day-old cultures still formed a regular monolayer of polygonal cells. In similarly aged cultures, intracellular glycogen was abundant and evenly distributed, while steatosis remained very limited. Scanning and transmission electron microscopy showed that well developed bile canaliculi could be observed on the lateral side of the hepatocyte membrane after 4 days of incubation and persisted for 2 weeks. These canalicular structures probably originated from coalescence of membrane invaginations observed in 1-day-old cultures. Transmission electron microscopy showed that the ultrastructure of the cells was very close to that of normal rat hepatocytes in the intact liver. These results suggest that rat hepatocytes cultured under these experimental conditions are able to develop and maintain tissue-specific cytochemical and morphological properties for at least 15 days.     

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.     

Spontaneous formation of intercellular bile canaliculi and hybrid biliary-pancreatic canaliculi in co-culture of hepatocytes and exocrine pancreas cells from carp

When cultured together in a primary serum-free hormone-free system, hepatocytes and exocrine pancreas cells from the carp, Cyprinus carpio, spontaneously establish unique morphological structures that do not occur in vivo. These structures include intercellular bile canaliculi between neighbouring hepatocytes and hybrid canaliculi between hepatocytes and pancreas cells. In vivo, carp hepatocytes form only unicellular bile canaliculi; hybrid canaliculi between hepatocytes and exocrine pancreas cells do not exist at all in nature. This study shows that, in an artificial environment, cells are able spontaneously to establish novel morphological structures that are absent in the animal from which the cells have been obtained. Received: 3 January 1996 / Accepted: 17 March 1997     

Primary cultures of rat hepatocytes in hollow fiber chambers

Summary Hepatocyte culture may represent an alternative to the use of animals to study drug detoxification by the liver. An ideal in vitro system should closely mimic the in vivo environment by providing continuous media perfusion and oxygenation, and should facilitate sampling of cells and culture media. To meet these criteria, a hollow fiber bioreactor seeded with isolated rat hepatocytes was developed and tested by measuring the formation of three products of the oxidative metabolism of diazepam and the glucuronidation of phenolsulfonphthalein (PSP). To compare the performance of conventional monolayer culture to that of the bioreactor system, diazepam metabolism was studied for 45 days in both systems. The oxygen dependency of diazepam metabolism was evaluated by perfusing the bioreactor in an oxygen-rich atmosphere (30%). Total diazepam metabolism was twofold higher in the O₂-rich perfused hollow fiber cultures than in the cultures perfused under normal conditions, reflecting an increase in temazepam and oxazepam production. Diazepam detoxification activity was significantly enhanced by oxygen (P≤0.001) over the life of the perfused cultures. PSP metabolism was similar in all three culture systems. By Day 10, diazepam metabolism in the oxygenated bioreactor system was 44% of the in vivo activity of rat hepatocytes. This activity dropped to 30% by Day 25 of culture. These results justify the use of perfused culture systems for in vitro detoxification studies as an alternative to animal use and emphasize the capacity of a culture device perfused under O₂-enriched conditions to maintain long-term P450 activity of rat hepatocytes.     

Ultrastructural characteristics of intercellular contacts and bile canaliculi in neonatal rat hepatocytes in primary culture

The ultrastructure of the cellular contacts and bile canaliculi was examined in cultured neonatal (day 5) rat hepatocytes to elucidate the development of cellular polarity. A new scanning electron microscopic technique for cultured hepatocytes allowed a view of cell-cell attachment and the entire cell surface, including the underside on plastic dishes. At 3 h after plating, neonatal hepatocytes were shown to be round, with loss of the preferential localization of cell organelles. After 6 h of culture, the cells had become oblong; they were aggregated in groups of several cells and the cellular contacts were not as rigid or as straight as those in adult hepatocytes. Transmission electron microscopy showed the biliary functional polarity to be like that in vivo. On the undersurfaces of adjacent neonatal heptocytes a hemicanalicular structure lined with microvilli was found, which probably corresponds to the ultrastructure of bile canaliculi in vivo. However, no canaliculi or orifices of bile channels were found in adult hepatocytes. These results suggest that in neonatal rat hepatocyts the formation of tight rigid cellular contacts was suppressed. Modulation of cell membranes appeared on the undersurfaces of neonatal hepatocytes in early culture stages. The difference in the development of cellular polality could be caused by the proliferating activity of neonatal hepatocytes.     

Effects of medium and substratum conditions on the rates of DNA synthesis in primary cultures of bile ductular epithelial cells

Summary Select medium and substratum conditions were investigated for their effects on semiconservative DNA synthesis in essentially pure primary cultures of bile ductular epithelial cells that were initially isolated from cholestatic rat livers at 6 to 10 wk after bile duct ligation. DNA synthesis in these cultured cells was serum-dependent, being at its highest level when the concentration of fetal bovine serum present in the medium was maintained at 10%. This serum-dependent DNA synthesis was completely inhibited when 10 mM hydroxyurea was also included in the medium, and bile ductular cells cultured in the continued presence of 1.0% fetal bovine serum showed only marginal DNA synthesis during 8 to 10 d of primary culture when compared with no-serum controls. Maximum rates of serum-dependent DNA synthesis were obtained when the bile ductular cells were cultured for 7 to 14 d on tissue culture plastic coated with obtained when the bile ductular cells were cultured for 7 to 14 d on tissue culture plastic coated with either fibronectin from bovine plasma or type I rat-tail collagen. Cells cultured on plastic coated with basement membrane Matrigel exhibited the lowest levels of DNA synthesis, whereas those on plastic alone had intermediate amounts. Furthermore, the addition of epidermal growth factor (50 ng·ml⁻¹·d⁻¹) to medium supplemented with 1.0% fetal bovine serum greatly enhanced the rate of DNA synthesis in bile ductular cells after 6 d in primary culture on type I collagen-coated plastic over that measured in solvent control cultures. These findings indicate that our bile ductular epithelial cell culture model is potentially useful in the study of biliary cell growth regulation and carcinogenesis. This investigation was supported by USPHS grant RO1 CA 39225 to A. E. Sirica by the National Cancer Institute, Department of Health and Human Services, Bethesda, MD. During the period of this study, G. A. Mathis was a recipient of a Fellowship from the Fund for Academic Career Development of the State of Zurich, Switzerland.     

Isolated hepatocyte couplets and bile duct units - Novel preparations for the in vitro study of bile secretory function

Studies of the mechanisms of bile formation have been facilitated by the development of in-vitro models that enable the transport functions of hepatocytes and bile duct epithelial cells to be analyzed in isolation. Isolated hepatocyte couplets represent a primary bile secretory unit that retain secretory polarity. Isolated bile duct units are intact polarized segments of interlobular bile duct segments. Both preparations can be maintained in short term culture, respond appropriately to choleretic agonists and facilitate a variety of functional applications.     

Changes in some chromatin and cytoplasmic enzymes of perinatal rat hepatocytes during culture

Summary Hepatocytes prepared from rats at various perinatal stages were cultured in selective medium that does not allow fibroblastic cell growth. Cell population remained homogeneous during the culture. Hepatocytes undergo divisions for a period, which varies according to the stage of development of the rat. Light and electron microscope observations showed the presence of numerous cytoplasmic organelles; moreover, hydrocortisone-induced structures similar to bile canaliculi. Chromatin protein kinase decreased rapidly during culture except in samples prepared from 17-day fetuses in which it remained unchanged for 2 days and decreased to a lesser extent afterwards. Chromatin nonhistone proteins were incubated with (γ-³²P) ATP and the phosphorylation pattern analyzed on polyacrylamide gels. Many radioactive peaks were observed in chromatin proteins from 17-day fetuses; they were much lower in proteins from 19-day fetuses. The phosphorylation pattern was analyzed in hepatocytes after 2 days of culture. Many radioactive peaks were observed with proteins from heapatocytes taken from 17-day fetuses; no radioactivity was observed in proteins from 19-day fetuses. This is in contrast with the absence of radioactive peaks in chromatin proteins from adult rat hepatocytes. In cytoplasm, aldolase and pyruvate kinase specific activities varied according to the age of the rat. They strongly decreased during culture except in hepatocytes from 15-and 17-day fetuses, in which they remained stable for at least 5 days. The stability of chromatin and cytoplasmic enzymes in hepatocytes from 17-day fetuses could result from their ability to be regulated by hormones that are secreted at this stage of development.     

Rapid and enhanced repolarization in sandwich-cultured hepatocytes on an oxygen-permeable membrane

In this study, we established rat primary hepatocyte sandwich cultures on oxygen-permeable membranes and investigated the change in their repolarization. Functional bile canaliculi in sandwich-cultured hepatocytes on oxygen-permeable polydimethylsiloxane (PDMS) membranes were re-established more quickly than those in a conventional sandwich culture on polystyrene (PS). This enhanced biliary excretory activity was also observed in hepatocytes on another oxygen-permeable membrane plate but not on a PDMS surface whose oxygen permeability is blocked. An apical efflux transporter protein, Mrp2, was more rapidly distributed in hepatocytes cultured on PDMS membranes than in hepatocytes cultured on conventional PS plates. Moreover, the area of distribution of the Mrp2 in polarized hepatocytes cultured on PDMS membranes was more widespread than that for the hepatocytes grown on sandwich-cultured PS plates. The observation of ultrastructure in transmission electron microscopy clearly confirmed the presence of bile canalicular lumens possessing microvilli and tight junctions. Additionally, we demonstrated that the 7-ethoxyresorufin-O-deethylation activity of hepatocytes on PDMS membranes was also improved as compared to those on a PS surface. Therefore, sandwich-cultured hepatocytes on oxygen-permeable substrates can provide a simple tool for predicting the hepatic metabolism and toxicity of xenobiotics in vivo with short span and low cost in the course of drug discovery and evaluation.     

Effect of hydrocortisone and nicotinamide on gamma glutamyltransferase in primary cultures of rat hepatocytes

Summary Isolated rat hepatocytes cultured on collagen coated plates exhibit a gradual fetal phenotypic change during time in culture. The fetal liver marker gamma glutamyltransferase (GGT) was used to follow this change. Inasmuch as a significant overgrowth of nonparenchymal liver derived cells is seen frequently in primary cultures of hepatocytes, a technique was utilized that corrects for the presence of nonparenchymal cells. In media supplemented with either hydrocortisone (10⁻⁵ M) or nicotinamide (25 mM) the original epithelial morphology of hepatocytes was preserved for a longer period of time than in unsupplemented media. Hepatocytes in unsupplemented media exhibited an increase in GGT specific activity over time. Hydrocortisone (10⁻⁵ M) induced an increase in GGT activity compared to controls. Nicotinamide (25 mM) inhibited the increase in GGT activity compared to the unsupplemented hepatocytes. Our results indicate that GGT is regulated by hydrocortisone and nicotinamide. This study was supported by NIH Grant CA30241-01.     

Hepatotoxicity studies with primary cultures of rat liver cells

Summary A method for preparing primary monolayer cultures of postnatal rat hepatocytes has been developed in our laboratory. Growing cultures in arginine-deficient medium inhibits fibroblast overgrowth, and relatively pure cultures of parenchymal hepatocytes are obtained. This cell culture system has been used to study the cytotoxicity of two hepatotoxic agents, tetracycline and norethindrone. Caffeine was evaluated as an agent thought to be relatively nontoxic to liver. Cytotoxicity was evaluated by phase-contrast microscopy of cellular morphology and by measurement of leakage of intracellular enzymes [arginosuccinate lyase (ASAL), lactate dehydrogenase (LDH), glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), and acid phosphatase (AP)] into the culture medium. Hepatic cultures were treated with each of the agents in concentrations ranging from 5×10⁻⁶ to 1×10⁻³ m and for durations from 1 to 24 hr. ASAL was found to be the most sensitive in predicting early cell injury and AP the least sensitive; the other three enzymes tested were intermittent in value and equally sensitive in evaluating cytotoxicity. Treatment of the cultures with tetracycline (5×10⁻⁴ m) for 6 hr resulted in ASAL leakage that was 400% of control values; and norethindrone (5×10⁻⁴ m) for 6 hr caused a 250% increase relative to controls. The hepatotoxic agents demonstrated a dose- and timedependence of cytotoxicity in the cultures. In contrast, caffeine was relatively nontoxic to the cultures. Part of this investigation was presented orally at the 17th Annual Meeting of the Society of Toxicology, San Francisco, March 13, 1978.     

Cytochrome P450 activities in pure and co-cultured rat hepatocytes. Effects of model inducers

Summary The stability and inducibility of several P450 activities (namely, P450 1A1, 2A1, 2B1/2, 2C11, and 3A1) were studied in rat hepatocytes co-cultured with the MS epithelial cell line derived from monkey kidney. The results revealed that these monooxygenase activities were systematically higher in co-cultures than in conventional hepatocyte cultures. Pure cultures showed a rapid loss of monooxygenase activities, which were undetectable after 5 days. In contrast, all isozymes assayed were measurable in co-cultured hepatocytes on Day 7 (about 15 to 40% of the initial activities of Day 0 of culture). The beneficial effects of the co-culture system seemed to be more selective for certain cytochrome P450 isoforms, with P450 1A1 and 3A1 being the best stabilized isozymes after 1 wk. A clear response to inducers was observed in co-cultures, each isozyme showing a different induction pattern. 3-Methylcholanthrene produced a strong increase in P450 1A1 (7-ethoxyresorufin O-deethylase) activity and a low increase in P450 2A1 (testosterone 7α-hydroxylation), whereas no changes were observed in the other activities. Phenobarbital treatment resulted in increases in P450 2B1/2 (7-pentoxyresorufin O-depentylase and 16α- and 16β-hydroxylation of testosterone) activities, while minor effects were observed on P450 3A1 (testosterone 6β-hydroxylation) activity. Dexamethasone markedly increased P450 3A1 (testosterone 6β- and 15β-hydroxylation) activity and, to a lesser extent, P450 2B1/2 (16β-hydroxylation).     

Extended primary culture of human hepatocytes in a collagen gel sandwich system

Summary To develop a strategy for extended primary culture of human hepatocytes, we placed human hepatocytes between two layers of collagen gel, called a “collagen gel sandwich.” Maintenance of hepatocellular functions in this system was compared with that of identical hepatocyte preparations cultured on dry-collagen coated dishes or co-cultured with rat liver epithelial cells. Human hepatocytes in a collagen gel sandwich (five separate cultures) survived for more than 4 wk, with the longest period of culture being 78 d. They maintained polygonal morphology with bile canaliculuslike structures and high levels of albumin secretion throughout the period of culture. In contrast, hepatocytes on dry-collagen became feature-less, and albumin secretion could not be detected after 14 d of culture. This loss of albumin secretion was partially recovered by overlaying one layer of collagen gel. Ethoxyresorufin O-deethylase activity, associated with cytochrome P450 1A2, was detected basally up to 29 d in collagen gel sandwich culture. These activities were induced four- to eightfold after induction with dibenz(a,h)anthracene. Cocultures also maintained basal activity up to 29 d. However, their inducibility was lower than that of hepatocytes in collagen gel sandwich. No ethoxyresorufin O-deethylase activity was detected in hepatocytes cultured on dry-collagen at 7 d. Thus, the collagen gel sandwich system preserves differentiated morphology and functions of human hepatocytes in primary culture for a prolonged period of time. This system is a promising model for studying human hepatocellular function, including protein synthesis and drug metabolism in vitro.     

Uptake and transport of glucagon by rat liver: evidence for transcytotic pathway

Summary The binding of¹²⁵I-glucagon to the cell surface and the pathway of intracellular transport of this hormone by rat hepatocytes in vivo were studied by light and EM autoradiography. Radiolabeled glucagon injected into the blood stream was taken up predominantly by the hepatocytes. Negligible radioactivity was found to be associated with other cell types such as endothelial or Kupffer cells. Our results indicate that at early time points after injection glucagon has been preferentially interacting with the sinusoidal domain of the hepatocytes and found to be associated with coated pits and uncoated vesicles corresponding to endosomes. At 15–20 min time intervals glucagon grains were found within hepatocyte interior. Later, at 30 min after injection glucagon grains accumulate in the Golgi-lysosomal region of hepatocyte often in close proximity to the opening of the bile canaliculi. Accordingly a portion of internalized¹²⁵I-glucagon was found to be released into the bile thereby indicating that a transcytotic pathway may be involved in this peptide's clearance process.     

Liver Ischemia and Ischemia–Reperfusion Induces and Trafficks the Multi-specific Metal Transporter Atp7b to Bile Duct Canaliculi: Possible Preferential Transport of Iron into Bile

Both Atp7b (Wilson disease gene) and Atp7a (Menkes disease gene) have been reported to be trafficked by copper. Atp7b is trafficked to the bile duct canaliculi and Atp7a to the plasma membrane. Whether or not liver ischemia or ischemia–reperfusion modulates Atp7b expression and trafficking has not been reported. In this study, we report for the first time that the multi-specific metal transporter Atp7b is significantly induced and trafficked by both liver ischemia alone and liver ischemia–reperfusion, as judged by immunohistochemistry and Western blot analyses. Although hepatocytes also stained for Atp7b, localized intense staining of Atp7b was found on bile duct canaliculi. Inductive coupled plasma-mass spectrometry analysis of bile copper, iron, zinc, and manganese found a corresponding significant increase in biliary iron. In our attempt to determine if the increased biliary iron transport observed may be a result of altered bile flow, lysosomal trafficking, or glutathione biliary transport, we measured bile flow, bile acid phosphatase activity, and glutathione content. No significant difference was found in bile flow, bile acid phosphatase activity, and glutathione, between control livers and livers subjected to ischemia–reperfusion. Thus, we conclude that liver ischemia and ischemia–reperfusion induction and trafficking Atp7b to the bile duct canaliculi may contribute to preferential iron transport into bile.     

Demonstration of 5′-nucleotidase activity in unfixed cryostat sections of rat liver using a combined light- and electron-microscope procedure

Summary In the present study a technique was developed to demonstrate 5′-nucleotidase activity in unfixed cryostat sections of rat liver at the light- and electron-microscope level using a semipermeable membrane. In order to retain the ultrastructure of the unfixed material as much as possible, incubations were also performed at 4°C rather than at 37°C. The optimized incubation medium contained 300 mm Tris-maleate buffer, pH 7.2, 5 mm adenosine monophosphate as substrate, 30 mm cerium chloride as capturing agent for liberated phosphate, 10 mm magnesium chloride as activator and 1.5% agar. At the light-microscope level, similar localizations of 5′-nucleotidase activity were obtained when incubations were performed at 37°C and 4°C. Enzyme activity was present mainly at bile canalicular membranes and at sinusoidal membranes of hepatocytes; total activity was higher in pericentral than in periportal areas. Cytophotometric analyses revealed that specific formation of final reaction product (FRP) (test minus control reaction) at 37°C followed a hyperbolic curve with time. A linear relationship was found between specific amounts of FRP and section thickness up to 8μm. 5′-Nucleotidase activity was about three-fold higher after incubation for 30 min at 37°C than at 4°C. At the electron-microscope level, it was demonstrated that the ultrastructure of rat liver was rather well-preserved after incubating unfixed cryostat sections attached to a semipermeable membrane and electron-dense FRP was found at bile canalicular and sinusoidal plasma membrane of hepatocytes. The most distinct changes in ultrastructure after incubation at 37°C, in comparison with that at 4°C, were the appearance of multi-lamellar structures at bile canaliculi at 37°C. We conclude that the present method is valid for the demonstration of 5′-nucleotidase activity in unfixed cryostat sections of rat liver at both the light- and electron-microscope levels and that hypothermic incubations improve ultrastructural morphology substantially.     

Plasma membranes of the rat liver. Isolation and enzymatic characterization of a fraction rich in bile canaliculi

A method is described for the rapid isolation of a plasma membrane fraction containing a high concentration of intact bile canaliculi from the rat liver. Isolated bile canaliculi retain most of the ultrastructural features exhibited in the intact liver cell. The final fraction contains 5''-nucleotidase activity at approximately the same concentration as that in previous preparations of plasma membranes. In the presence of 0.01 M Mg⁺⁺, 5''-nucleotidase exhibits a double pH optimum at pH values of 7.5 and 9.5. The activities of glucose-6-phosphatase and alkaline phosphatase are present in low amounts. Cytochrome P-450 is not detectable. Na⁺-K⁺-activation of ATPase is observed to the extent of 20–36% in about half of the assays. The availability of a method for preparation of intact bile canaliculi should prove useful for studying the biochemical events associated with the transport of bile constituents into canaliculi.     

Synthesis and secretion of lipoproteins by human hepatocytes in culture

Summary Confluent monolayers of normal human hepatocytes obtained by collagenase perfusion of liver pragments were incubated in a serum-free medium. Intracellular apolipoproteins apo AI, apo C, apo B, and apo E were detected between Day 1 and Day 6 of the culture by immunoenzymatic staining using polyclonal antibodies directed against these apoproteins and monoclonal antibodies directed against both forms of apo B (B100 and B48). Translation of mRNA isolated from these hepatocytes in an acellular system revealed that apo AI and apo E were synthesized as the precusor forms of mature plasma apo AI and apo E. Three lipoprotein fractions corresponding to the density of very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL) were isolated from the medium at Day 5 of culture and examined by electron microscopy after negative staining. VLDL and LDL particles are similar in size and shape to plasma lipoproteins; spherical HDL are larger than normal plasma particles isolated at the same density. Their protein represented 44, 19.5, and 36.5% respectively, of the total lipoprotein protein. The secretion rate of VLDL protein corresponded to that measured in primary cultures of rat hepatocytes. After incorporation of [³H]glycerol, more than 92% of the [³H]triglyceride secreted into the medium was recovered in the VLDL fraction. These results demonstrate that primary cultures of normal human hepatocytes are able to synthesize and secrete lipoproteins and thus could be a useful model to study lipoprotein metabolism in human liver.     

Colony isolation and secondary culture of fetal porcine hepatocytes on STO feeder cells

Summary The secondary culture of non-transformed parenchymal hepatocytes has not been possible. STO feeder cell-dependent secondary cultures of fetal pig hepatocytes were established by colony isolation from primary cultures of 26-d fetal livers. The liver cells had the typical polygonal morphology of parenchymal hepatocytes. They also spontaneously differentiated to form small biliary canaliculi between individual cells or progressed further to large multicellular duct-like structures or cells undergoing gross lipid accumulation and secretion. The secondary hepatocyte cultures expressed alpha-fetoprotein (AFP), albumin, and β-fibrinogen mRNA, and conditioned medium from the cells contained elevated levels of transferrin and albumin. STO feeder cell co-culture may be useful for the sustainable culture of hepatocytes from other species.