Bile Canaliculi-like Structures Research Articles

Construction of extended and functional bile canaliculi using long-term sandwich-cultured cryopreserved human hepatocytes and the application of hepatocytes for predicting the biliary excretion of pharmaceutical and food-related compounds.

The biliary excretion of pharmaceutical and food-related compounds is an important factor for assessing pharmacokinetics and toxicities in humans, and a highly predictive in vitro method for human biliary excretion is required. We have developed a simple in vitro culture method for generating extended and functional bile canaliculi using cryopreserved human hepatocytes. We evaluated the uptake of compounds by hepatocytes and bile canaliculi, and the biliary excretion index (BEI) was calculated. After 21 days of culture, the presence of extended and functional bile canaliculi was confirmed by the uptake of two fluorescent substrates. Positive BEIs were observed for taurocholic acid-d4, rosuvastatin, pitavastatin, pravastatin, valsartan, olmesartan, and topotecan (reported biliary-excreted compounds in humans), but no difference in BEI was observed for salicylic acid (a nonbiliary-excreted compound). Furthermore, 8 of 21 food-related compounds with specific structures and reported biliary transporter involvement exhibited positive BEIs. The developed in vitro system was characterized by functional bile canaliculus-like structures, and it could be applied to the prediction of the biliary excretion of pharmaceutical and food-related compounds.

HepG2-NIAS cells, a new subline of HepG2 cells that can enhance not only CYP3A4 activity but also expression of drug transporters and form bile canaliculus-like networks by the oxygenation culture via a collagen vitrigel membrane.

We reported the enhanced liver-specific function and structure of HepG2 cells by the oxygenation culture via a collagen vitrigel membrane (CVM). The cells were conditioned in our laboratory for a long period, so their characteristics may change from the original HepG2 cells registered in RIKEN cell bank (RCB) with the number of 1648 (HepG2-RCB1648 cells). We named the conditioned HepG2-RCB1648 cells in our laboratory as HepG2-NIAS cells. Here, we clarified the features of HepG2 cells with three different culture histories by analyzing their morphology and viability, CYP3A4 activity, the potential to form bile canaliculus-like structures, and the expression of drug transporters. On plastic, HepG2-NIAS cells grew as a monolayer without the formation of large aggregates involving dead cells that were observed in HepG2-RCB1648 cells and HepG2-RCB1886 cells. In the oxygenation culture via a CVM, the CYP3A4 activity of HepG2-NIAS cells increased to almost half level in direct comparison to that of differentiated HepaRG cells cultured on a collagen-coated plate; however, that of HepG2-RCB1648 cells and HepG2-RCB1886 cells was almost not detected. HepG2-NIAS cells formed bile canaliculus-like networks in which fluorescein was accumulated after the exposure of fluorescein diacetate, although HepG2-RCB1648 cells and HepG2-RCB1886 cells did not possess the potential. Also, immunohistological observations revealed that HepG2-NIAS cells remarkably enhanced the expression of drug transporters, NTCP, OATP1B1, OATP1B3, BSEP, MDR1, MRP2, and BCRP. These results suggest that HepG2-NIAS cells are a new subline of HepG2 cells useful for drug development studies. HepG2-NIAS cells were registered in RCB with the number of 4679.

Optimization of Canalicular ABC Transporter Function in HuH-7 Cells by Modification of Culture Conditions.

Human hepatoma cell lines are useful for evaluation of drug-induced hepatotoxicity, hepatic drug disposition, and drug-drug interactions. However, their applicability is compromised by aberrant expression of hepatobiliary transporters. This study was designed to evaluate whether extracellular matrix (Matrigel) overlay and dexamethasone (DEX) treatment would support cellular maturation of long-term HuH-7 hepatoma cell cultures and improve the expression, localization, and activity of canalicular ATP-binding cassette (ABC) transporters, multidrug resistance protein 1 (MDR1/P-glycoprotein/ABCB1), multidrug resistance-associated protein 2 (MRP2/ABCC2), and bile salt export pump (BSEP/ABCB11). Matrigel overlay promoted the maturation of HuH-7 cells toward cuboidal, hepatocyte-like cells displaying bile canaliculi-like structures visualized by staining for filamentous actin (F-actin), colocalization of MRP2 with F-actin, and by accumulation of the MRP2 substrate 5(6)-carboxy-2',7'-dichlorofluorescein (CDF) within the tubular canaliculi. The cellular phenotype was rather homogenous in the Matrigel-overlaid cultures, whereas the standard HuH-7 cultures contained both hepatocyte-like cells and flat epithelium-like cells. Only Matrigel-overlaid HuH-7 cells expressed MDR1 at the canaliculi and excreted the MDR1 probe substrate digoxin into biliary compartments. DEX treatment resulted in more elongated and branched canaliculi and restored canalicular expression and function of BSEP. These findings suggest that hepatocyte polarity, elongated canalicular structures, and proper localization and function of canalicular ABC transporters can be recovered, at least in part, in human hepatoma HuH-7 cells by applying the modified culture conditions. SIGNIFICANCE STATEMENT: We report the first demonstration that proper localization and function of canalicular ABC transporters can be recovered in human hepatoma HuH-7 cells by modification of cell culture conditions. Matrigel overlay and dexamethasone supplementation increased the proportion of hepatocyte-like cells, strongly augmented the canalicular structures between the cells, and restored the localization and function of key canalicular ABC transporters. These results will facilitate the development of reproducible, economical, and easily achievable liver cell models for drug development.

Protein expression and function of organic anion transporters in short-term and long-term cultures of Huh7 human hepatoma cells

Human-derived hepatic cell lines are a valuable alternative to primary hepatocytes for drug metabolism, transport and toxicity studies. However, their relevance for investigations of drug-drug and drug-organic anion (e.g., bile acid, steroid hormone) interactions at the transporter level remains to be established. The aim of the present study was to determine the suitability of the Huh7 cell line for transporter-dependent experiments. Huh7 cells were cultured for 1 to 4 weeks and subsequently were analyzed for protein expression, localization and activity of solute carrier (SLC) and ATP-binding cassette (ABC) transporters involved in organic anion transport using liquid chromatography-tandem mass spectroscopy, immunocytochemistry, and model substrates [3H]taurocholate (TCA), [3H]dehydroepiandrosterone sulfate (DHEAS) and 5(6)-carboxy-2′,7′-dichlorofluorescein (CDF) diacetate. The extended 4-week culture resulted in a phenotype resembling primary hepatocytes and differentiated HepaRG cells: cuboidal hepatocyte-like cells with elongated bile canaliculi-like structures were surrounded by epithelium-like cells. Protein expression of OSTα, OSTβ and OATP1B3 increased over time. Moreover, the uptake of the SLC probe substrate DHEAS was higher in 4-week than in 1-week Huh7 cultures. NTCP, OATP1B1, BSEP and MRP3 were barely or not detectable in Huh7 cells. OATP2B1, MRP2 and MRP4 protein expression remained at similar levels over the four weeks of culture. The activity of MRP2 and the formation of bile canaliculi-like structures were confirmed by accumulation of CDF in the intercellular compartments. Results indicate that along with morphological maturation, transporters responsible for alternative bile acid secretion pathways are expressed and active in long-term cultures of Huh7 cells, suggesting that differentiated Huh7 cells may be suitable for studying the function and regulation of these organic anion transporters.

Evaluation of cellular adhesion and organization in different microporous polymeric scaffolds.

The lack of prediction accuracy during drug development and screening risks complications during human trials, such as drug-induced liver injury (DILI), and has led to a demand for robust, human cell-based, in vitro assays for drug discovery. Microporous polymer-based scaffolds offer an alternative to the gold standard flat tissue culture plastic (2D TCPS) and other 3D cell culture platforms as the porous material entraps cells, making it advantageous for automated liquid handlers and high-throughput screening (HTS). In this study, we optimized the surface treatment, pore size, and choice of scaffold material with respect to cellular adhesion, tissue organization, and expression of complex physiologically relevant (CPR) outcomes such as the presence of bile canaliculi-like structures. Poly-l-lysine and fibronectin (FN) coatings have been shown to encourage cell attachment to the underlying substrate. Treatment of the scaffold surface with NaOH followed with a coating of FN improved cell attachment and penetration into pores. Of the two pore sizes we investigated (A: 104 ± 4 μm; B: 175 ± 6 μm), the larger pore size better promoted cell penetration while limiting tissue growth from reaching the hypoxia threshold. Finally, polystyrene (PS) proved to be conducive to cell growth, penetration into the scaffold, and yielded CPR outcomes while being a cost-effective choice for HTS applications. These observations provide a foundation for optimizing microporous polymer-based scaffolds suitable for drug discovery. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:505-514, 2018.

Decellularized caprine liver extracellular matrix as a 2D substrate coating and 3D hydrogel platform for vascularized liver tissue engineering

Development of a vascularized liver tissue construct is a need of an hour to circumvent the current demand of liver transplantation in health care sector. An appropriate matrix must support liver cell viability, functionality, and development of microvasculature. With this perspective, here, we report the use of decellularized caprine liver extracellular matrix (CLECM) derived hydrogel for tissue engineering applications. First, CLECM was used as a substrate coating material for 2D hepatocyte culture. HepG2 cells cultured on CLECM-coated surface showed higher albumin, urea, glycogen, and GAGs synthesis in comparison with collagen-coated surface (taken as control for the study). Thereafter, the cells were encapsulated in CLECM hydrogels for 3D culture. In CLECM hydrogels, HepG2 cells showed highly differentiated and polarized phenotype with the appearance of bile canaliculi-like structures and enhanced expression of mature hepatocyte markers. We further showed that CLECM hydrogels also supported the development of microvasculature in vitro, thus making it a suitable candidate for development of a prevascularized liver tissue construct. In conclusion, we proved the superiority of CLECM over collagen for 2D/3D human hepatocyte and endothelial cell culture. CLECM could serve as an efficient biomaterial platform in the development of a liver tissue construct for application in tissue engineering.

Functional Maturation of Induced Pluripotent Stem Cell Hepatocytes in Extracellular Matrix—A Comparative Analysis of Bioartificial Liver Microenvironments

: Induced pluripotent stem cells (iPSCs) are new diagnostic and potentially therapeutic tools to model disease and assess the toxicity of pharmaceutical medications. A common limitation of cell lineages derived from iPSCs is a blunted phenotype compared with fully developed, endogenous cells. We examined the influence of novel three-dimensional bioartificial microenvironments on function and maturation of hepatocyte-like cells differentiated from iPSCs and grown within an acellular, liver-derived extracellular matrix (ECM) scaffold. In parallel, we also compared a bioplotted poly-l-lactic acid (PLLA) scaffold that allows for cell growth in three dimensions and formation of cell-cell contacts but is infused with type I collagen (PLLA-collagen scaffold) alone as a "deconstructed" control scaffold with narrowed biological diversity. iPSC-derived hepatocytes cultured within both scaffolds remained viable, became polarized, and formed bile canaliculi-like structures; however, cells grown within ECM scaffolds had significantly higher P450 (CYP2C9, CYP3A4, CYP1A2) mRNA levels and metabolic enzyme activity compared with iPSC hepatocytes grown in either bioplotted PLLA collagen or Matrigel sandwich control culture. Additionally, the rate of albumin synthesis approached the level of primary cryopreserved hepatocytes with lower transcription of fetal-specific genes, α-fetoprotein and CYP3A7, compared with either PLLA-collagen scaffolds or sandwich culture. These studies show that two acellular, three-dimensional culture systems increase the function of iPSC-derived hepatocytes. However, scaffolds derived from ECM alone induced further hepatocyte maturation compared with bioplotted PLLA-collagen scaffolds. This effect is likely mediated by the complex composition of ECM scaffolds in contrast to bioplotted scaffolds, suggesting their utility for in vitro hepatocyte assays or drug discovery. Through the use of novel technology to develop three-dimensional (3D) scaffolds, the present study demonstrated that hepatocyte-like cells derived via induced pluripotent stem cell (iPSC) technology mature on 3D extracellular matrix scaffolds as a result of 3D matrix structure and scaffold biology. The result is an improved hepatic phenotype with increased synthetic and catalytic potency, an improvement on the blunted phenotype of iPSC-derived hepatocytes, a critical limitation of iPSC technology. These findings provide insight into the influence of 3D microenvironments on the viability, proliferation, and function of iPSC hepatocytes to yield a more mature population of cells for cell toxicity studies and disease modeling.

Thermo-responsive polymer aided spheroid culture in cryogel based platform for high throughput drug screening.

In high throughput cell culture, the paradigm is now shifting from 2D to 3D systems. However, in 3D cell culture systems, it is important that the cells form spheroids with robust cell-cell interactions. We fabricated poly(N-isopropylacrylamide-co-gelatin) cryogel scaffolds for cell culture and inserted them into open-ended 96-well plates that formed a drainage and leakage protected, easy to handle high throughput platform. This platform was used to screen for the optimal concentration of poly(N-isopropylacrylamide) (pNIPAAm) as an external aid to the formation of cellular spheroids. It was found that hepatic cells (Hep G2) seeded in the presence of 0.03% pNIPAAm formed better hepatic spheroids in terms of morphology (as assessed by microscopic analysis and formation of bile canaliculi-like structures) and functionality by day 5 of culture. An increase of 44.22%, 15.75%, 36.44%, 32.05% and 27.02% was observed in glucose consumption (1.925 mM per day per 10(4) cells), albumin synthesis (164.18 ng per day per 10(4) cells), CYP1A1 (304.92 pg per min per 10(4) cells), CYP2A6 (441.23 nM per min per 10(4) cells) and phase II metabolic activity (386.18 nM per min(-1) per 10(4) cells), respectively, upon using 0.03% pNIPAAm, as compared to the 3D control. The platform was tested with other cells such as breast and lung cancer cells and found to be compatible. The cell spheroids were subjected to drug toxicity screening in cryogel based open-ended platforms. It was observed that the spheroids were more resistant to anticancer drugs, as compared to 2D and 3D controls, with approximately 11%-67% increase in the IC50 values of tamoxifen and paclitaxel. The platform also showed dose dependent and reproducible responses to drugs.

Polarized location of SLC and ABC drug transporters in monolayer-cultured human hepatocytes

Human hepatocytes cultured in a monolayer configuration represent a well-established in vitro model in liver toxicology, notably used in drug transporter studies. Polarized status of drug transporters, i.e., their coordinated location at sinusoidal or canalicular membranes, remains however incompletely documented in these cultured hepatocytes. The present study was therefore designed to analyze transporter expression and location in such cells. Most of drug transporters were first shown to be present at notable mRNA levels in monolayer-cultured human hepatocytes. Cultured human hepatocytes, which morphologically exhibited bile canaliculi-like structures, were next demonstrated, through immunofluorescence staining, to express the influx transporters organic anion transporting polypeptide (OATP) 1B1, OATP2B1 and organic cation transporter (OCT) 1 and the efflux transporter multidrug resistance-associated protein (MRP) 3 at their sinusoidal pole. In addition, the efflux transporters P-glycoprotein and MRP2 were detected at the canalicular pole of monolayer-cultured human hepatocytes. Moreover, canalicular secretion of reference substrates for the efflux transporters bile salt export pump, MRP2 and P-glycoprotein as well as sinusoidal drug transporter activities were observed. This polarized and functional expression of drug transporters in monolayer-cultured human hepatocytes highlights the interest of using this human in vitro cell model in xenobiotic transport studies.

Differentiation of liver progenitor cell line to functional organotypic cultures in 3D nanofibrillar cellulose and hyaluronan-gelatin hydrogels

Physiologically relevant hepatic cell culture models must be based on three-dimensional (3D) culture of human cells. However, liver cells are generally cultured in two-dimensional (2D) format that deviates from the normal in vivo morphology. We generated 3D culture environment for HepaRG liver progenitor cells using wood-derived nanofibrillar cellulose (NFC) and hyaluronan-gelatin (HG) hydrogels. Culture of undifferentiated HepaRG cells in NFC and HG hydrogels induced formation of 3D multicellular spheroids with apicobasal polarity and functional bile canaliculi-like structures, structural hallmarks of the liver tissue. Furthermore, hepatobiliary drug transporters, MRP2 and MDR1, were localized on the canalicular membranes of the spheroids and vectorial transport of fluorescent probes towards the biliary compartment was demonstrated. Cell culture in 3D hydrogel supported the mRNA expression of hepatocyte markers (albumin and CYP3A4), and metabolic activity of CYP3A4 in the HepaRG cell cultures. On the contrary, the 3D hydrogel cultures with pre-differentiated HepaRG cells showed decreasing expression of albumin and CYP3A4 transcripts as well as CYP3A4 activity. It is concluded that NFC and HG hydrogels expedite the hepatic differentiation of HepaRG liver progenitor cells better than the standard 2D culture environment. This was shown as improved cell morphology, expression and localization of hepatic markers, metabolic activity and vectorial transport. The NFC and HG hydrogels are promising materials for hepatic cell culture and tissue engineering.

A long-term three dimensional liver co-culture system for improved prediction of clinically relevant drug-induced hepatotoxicity

Drug-induced liver injury (DILI) is the major cause for liver failure and post-marketing drug withdrawals. Due to species-specific differences in hepatocellular function, animal experiments to assess potential liabilities of drug candidates can predict hepatotoxicity in humans only to a certain extent. In addition to animal experimentation, primary hepatocytes from rat or human are widely used for pre-clinical safety assessment. However, as many toxic responses in vivo are mediated by a complex interplay among different cell types and often require chronic drug exposures, the predictive performance of hepatocytes is very limited. Here, we established and characterized human and rat in vitro three-dimensional (3D) liver co-culture systems containing primary parenchymal and non-parenchymal hepatic cells. Our data demonstrate that cells cultured on a 3D scaffold have a preserved composition of hepatocytes, stellate, Kupffer and endothelial cells and maintain liver function for up to 3months, as measured by the production of albumin, fibrinogen, transferrin and urea. Additionally, 3D liver co-cultures maintain cytochrome P450 inducibility, form bile canaliculi-like structures and respond to inflammatory stimuli. Upon incubation with selected hepatotoxicants including drugs which have been shown to induce idiosyncratic toxicity, we demonstrated that this model better detected in vivo drug-induced toxicity, including species-specific drug effects, when compared to monolayer hepatocyte cultures.In conclusion, our results underline the importance of more complex and long lasting in vitro cell culture models that contain all liver cell types and allow repeated drug-treatments for detection of in vivo-relevant adverse drug effects.

Changes in Expression of Connexin 32, Bile Canaliculus-Like Structures, and Localization of Alkaline Phosphatase in Primary Cultures of Fetal Rat Hepatocytes

We devised an experimental design in primary cultures of fetal rat hepatocytes for studying hepatocyte differentiation over a short period. In the present study, hepatocytes were first cultured for 3 days in dexamethasone-supplemented medium and then for an additional 3 days in dexamethasone- or epidermal growth factor-supplemented medium. In hepatocytes cultured continuously in dexamethasone-supplemented medium, the expression of connexin 32 increased and bile canaliculus-like structures and localization of alkaline phosphatase in the plasma membrane around bile canaliculus-like structures were maintained. Few cells incorporated bromodeoxyuridine. On the other hand, in most of the hepatocytes cultured in epidermal growth factor-supplemented medium, the expression of connexin 32 was minimally recognized, bile canaliculus-like structures were shortened or eliminated, and alkaline phosphatase was localized as numerous fine spots throughout the cytoplasm. More than 20% of all hepatocytes incorporated bromodeoxyuridine.The present study suggests that in hepatocytes, there is a close relationship among connexin 32 expression, the maintenance of bile canaliculus-like structures, and the localization of alkaline phosphatase to the plasma membrane around the bile canaliculus-like structures, and this indicates that the present experimental model is useful for studying hepatocyte differentiation over a short period.

AKAP350 Is involved in the development of apical “Canalicular” structures in hepatic cells HepG2

Hepatocytes are epithelial cells whose apical poles constitute the bile canaliculi. The establishment and maintenance of canalicular poles is a finely regulated process that dictates the efficiency of primary bile secretion. Protein kinase A (PKA) modulates this process at different levels. AKAP350 is an A-kinase anchoring protein that scaffolds protein complexes involved in modulating the dynamic structures of the Golgi apparatus and microtubule cytoskeleton, facilitating microtubule nucleation at this organelle. In this study, we evaluated whether AKAP350 is involved in the development of bile canaliculi-like structures in hepatocyte derived HepG2 cells. We found that AKAP350 recruits PKA to the centrosomes and Golgi apparatus in HepG2 cells. De-localization of AKAP350 from these organelles led to reduced apical cell polarization. A decrease in AKAP350 expression inhibited the formation of canalicular structures and impaired F-actin organization at canalicular poles. Furthermore, loss of AKAP350 expression led to diminished polarized expression of the p-glycoprotein (MDR1/ABCB1) at the apical "canalicular" membrane. AKAP350 knock down effects on canalicular structures formation and actin organization could be mimicked by inhibition of Golgi microtubule nucleation by depletion of CLIP associated proteins (CLASPs). Our data reveal that AKAP350 participates in mechanisms which determine the development of canalicular structures as well as accurate canalicular expression of distinct proteins and actin organization, and provide evidence on the involvement of Golgi microtubule nucleation in hepatocyte apical polarization.

Serum-free collagen sandwich cultures of adult rat hepatocytes maintain liver-like properties long term: A valuable model for in vitro toxicity and drug–drug interaction studies

Cultures of primary hepatocytes from various species, including human, are used in several applications during pre-clinical drug development. Their use is however limited by cell survival and conservation of liver-specific functions in vitro. The differentiation status of hepatocytes in culture strongly depends on medium formulation and the extracellular matrix environment. We incubated primary rat hepatocytes for 10 days on collagen monolayer and in collagen sandwich cultures with or without serum. Restoration of polygonal cell shape and formation of functional bile canaliculi-like structures was stable only in serum-free sandwich cultures. Variations in general cell viability, as judged by the cellular ATP content, LDH release or apoptosis, were less pronounced between alternative cultures. The intracellular glutathione content was preserved close to in vivo levels especially in serum-free sandwich cultures. Basal activities of cytochrome P450 enzymes (P450) varied strongly between cultures. There was a minor effect on CYP1A but CYP2B activity was only detectable in the serum-free sandwich culture after 3 days and beyond. CYP2C activity was slightly elevated in both sandwich cultures, whereas CYP3A showed increased levels in both serum-free cultures. Inducibility of these P450s was fully maintained over time in serum-free collagen sandwich only. Gene expression was largely constant over time in serum-free sandwich cultures that was closest to liver. This liver-like property was supported by protein profiling results. Taken together, the serum-free collagen sandwich culture of primary rat hepatocytes maintained liver-like features over 10 days and is therefore a suitable model for long-term toxicity and drug–drug interaction studies.

Cytotoxicity of Fumonisin B1 in Spheroid and Monolayer Cultures of Rat Hepatocytes

Fumonisin B1 (FB1), the most prevalent member of toxins produced by several species of Fusarium molds, which occur mainly in maize, causes several fatal hepatopathies and nephropathies of animals. The current study was scrutinized to ascertain different cytotoxic and morphological transformations in rat hepatocytes induced by the treatments of diverse concentrations (300, 500, or 1000 μM) of fumonisin B1 in vitro, using both monolayer and spheroid cultures. In each hepatocyte culture, the cytotoxicity of FB1 was augmented in dose- and time-response manners. Morphological transformations among FB1-treated groups integrated accumulation of lipid droplets, cytoplasmic vacuolation in hepatocyte monolayers, and bleb formation in the hepatocyte spheroids. Additionally, electron microscopy revealed the loss of microvilli, mitochondrial swelling, and formation of lamellar membranous whorl in the vacuoles and bile canaliculi-like structures. Appearance of electron dense bodies in the monolayers, and loss of cell-to-cell contact in spheroids were depicted in 1000 μM FB1-treated hepatocytes. These outcomes insinuate different vital events in explaining morphological transformations in the cell membrane and organelles, induced by fumonisins in rat hepatocytes.

Thermoreversible gelation polymer induces the emergence of hepatic stem cells in the partially injured rat liver

Focal injury of the adult liver causes formation of granulomatous tissue and fibrosis. When thermoreversible gelation polymer (TGP) was applied to such defects of the rat liver, complete recovery of hepatic tissues was observed without granulation. We analyzed the mechanism of the regeneration. TGP is a chemically synthesized biocompatible polymer material whose sol-gel transition is reversible by changing the temperature. Cooled TGP was poured into a penetration lesion of the rat liver. Immunohistochemistry and polymerase chain reaction were carried out using tissues and cultured cells isolated from ductular structures. Immunocytochemical and ultrastructural analyses were also conducted. Seven days after TGP treatment, ductular reactions were observed around the wound and ductules elongated to the injured area. Cells in the structures were alpha-fetoprotein (AFP) positive, albumin+, CK19+, c-Kit+, and Thyl+. Hepatocyte-like cells possessing glycogen appeared around the tips of the ductules from day 9. The defect was completely replaced with hepatocytes by day 28. Cells isolated from the ductules expressed Musashi-1, c-Kit, Thyl, AFP, albumin, transferrin, connexin 43, and CK19. When the cultured cells were covered by TGP, they rapidly proliferated to form colonies, whereas without TGP cells gradually died. Morphologically and ultrastructurally the cells were similar to hepatocytes. They expressed not only albumin and transferrin but TAT, CYP2E1, and CCAAT/enhancer binding protein a. Some cells formed bile canaliculus-like structures. In conclusion, TGP may trigger the initiation of hepatic stem cells in biliary ductules, and stem cell activation may occur even in the regeneration of the normal liver.

Effects of cell culture conditions on primary rat hepatocytes—Cell morphology and differential gene expression

We incubated primary rat hepatocytes on collagen monolayer as well as in collagen sandwich cultures with serum-containing or serum-free medium formulations. Morphological monitoring of hepatocytes revealed that hepatocytes cultured on collagen monolayer adopted their polygonal shape and started to create aggregates earlier than sandwich-cultured cells. Bile canaliculi-like structures were observed in every cell culture system but were more prominent in serum-free cultures. Hepatocytes in collagen-sandwich configuration and serum-free medium were the most viable after 72 h of culture, still displaying polygonal shape, clear cytoplasm and stable canaliculi-like structures. Differential gene expression patterns were determined for each cell culture condition using quantitative TaqMan ® Low Density Arrays (LDA). Gene expression analysis revealed distinct profiles in monolayer versus sandwich cultures and in particular in serum-free versus serum-containing culture medium. The hepatocytes cultured in the collagen-sandwich with serum-free medium showed the least variation in expression values over time. Importantly, stress markers were not induced in the serum-free sandwich culture, in contrast to the monolayer and the serum-containing sandwich cultures. Additionally, expression of the investigated cytochrome P450 genes was maintained in the serum-free monolayer and the sandwich cultures. In conclusion, culturing primary rat hepatocytes in a sandwich between two layers of gelled collagen and in a serum-free medium formulation, appears to be most suitable for long-term in vitro hepatotoxicity screening.

Effects of ethanol on the function of cultured hepatocytes

To develop a novel culture system to investigate the effects of ethanol on the function of cultured hepatocytes. Sandwich configuration was used to culture hepatocytes and the effects of ethanol on functions of bile excretion and protein synthesis as well as the morphology of cultured hepatocytes were observed. Bile canaliculi-like structures decreased and anastomatic networks disappeared in ethanol treated hepatocytes. The ability for hepatocytes to internalize, metabolize and excrete compounds into bile was indicated by FDA metabolizing in the hepatocytes. In hepatocytes without ethanol, the bile excretion was showed clearly, but in ethanol-interfered hepatocytes, no bile excretion was observed. After ethanol was given, the level of protein secretion decreased and with the time going, it became lower and lower. Hepatocytes can be seriously damaged by ethanol. The study provides a new model to investigate the mechanism of some liver diseases caused by ethanol.

1108 GP130 dependent signalling is protective after bile duct ligation in mice

Human hepatocytes cultured in a monolayer configuration represent a well-established in vitro model in liver toxicology, notably used in drug transporter studies. Polarized status of drug transporters, i.e., their coordinated location at sinusoidal or canalicular membranes, remains however incompletely documented in these cultured hepatocytes. The present study was therefore designed to analyze transporter expression and location in such cells. Most of drug transporters were first shown to be present at notable mRNA levels in monolayer-cultured human hepatocytes. Cultured human hepatocytes, which morphologically exhibited bile canaliculi-like structures, were next demonstrated, through immunofluorescence staining, to express the influx transporters organic anion transporting polypeptide (OATP) 1B1, OATP2B1 and organic cation transporter (OCT) 1 and the efflux transporter multidrug resistance-associated protein (MRP) 3 at their sinusoidal pole. In addition, the efflux transporters P-glycoprotein and MRP2 were detected at the canalicular pole of monolayer-cultured human hepatocytes. Moreover, canalicular secretion of reference substrates for the efflux transporters bile salt export pump, MRP2 and P-glycoprotein as well as sinusoidal drug transporter activities were observed. This polarized and functional expression of drug transporters in monolayer-cultured human hepatocytes highlights the interest of using this human in vitro cell model in xenobiotic transport studies.

Reversible induction of rat hepatoma cell polarity with bile acids.

A dynamic model for inducing and isolating polarized cell colonies from differentiated rat hepatoma was established with chenodeoxycholic acid (CDCA). Cells were treated with 75 microM CDCA in a 1% solvent mix (DMSO/ethanol: 0.5%/0.5%) for 11 days and positive Fao-BA1 and C2rev7-BA1 clones were isolated, respectively, from Fao and C2rev7. Cell polarization in these two clones was demonstrated by (i) the detection of (gamma)-glutamyl transpeptidase activity (gamma)-GT) and the presence of specific proteins, namely aminopeptidase N (APN), bile acid export pump (Bsep), multidrug resistance-associated protein 2 (Mrp2) at the canalicular pole, (ii) the expression of tight junction (ZO-1) and basolateral (1-18) marker proteins, (iii) the presence of regular microvilli in the cavities sealed by tight junctions, and (iv) functional bile canaliculi-like structures with the capacity to metabolise and secrete carboxyfluorescein diacetate dye. The polarized phenotype was maintained for more than 200 cell generations in the presence of CDCA and could be modulated by cell density or omitting the inducing agent. Hence this cellular model is well suited for studies on hepatic differentiation, polarization and bile salt trafficking with therapeutic implications.