Culture density contributes to hepatic functions of fresh human hepatocytes isolated from chimeric mice with humanized livers: Novel, long-term, functional two-dimensional in vitro tool for developing new drugs.

Chimeric mice with humanized livers are considered a useful animal model for predicting human (h-) drug metabolism and toxicity. In this study, the characteristics of fresh h-hepatocytes (cFHHs, PXB-cells®) isolated from chimeric mice (PXB-mice®) were evaluated in vitro to confirm their utility for drug development. cFHHs cultured at high density (2.13 × 105 cells/cm2) displayed stable production of h-albumin and cytochrome P450 (CYP) 3A activities for at least 21 days. The mRNA expression levels of 10 of 13 CYP, UDP-glucuronosyltransferase (UGT), and transporters were maintained at >10% of the levels of freshly isolated cFHHs after 21 days. From 1 week, many bile canaliculi were observed between cFHHs, and the accumulation of the multidrug resistance-associated protein and bile salt export pump substrates in these bile canaliculi was clearly inhibited by cyclosporin A. Microarray analysis of cFHHs cultured at high density and at low density (0.53 × 105 cells/cm2) revealed that high density culture maintained high expressions of some transcription factors (HNF4α, PXR, and FXR) perhaps involved in the high CYP, UGT and transporter gene expressions of cFHHs. These results strongly suggest that cFHHs could be a novel in vitro tool for drug development studies.

3-D culture and endothelial cells improve maturity of human pluripotent stem cell-derived hepatocytes.

Human-induced pluripotent stem cell (hiPSC)-derived hepatocytes (iHEP) offer an attractive alternative to primary human hepatocytes (PHH) for drug toxicity studies, as PHHs are limited in supply, vary in their metabolic activity between donors, and rapidly lose their functionality in vitro. However, one of the major drawbacks with iHEP cells in drug safety studies is their decreased phenotypic maturity, with lower liver specific enzyme activity compared with that of PHH. Here we evaluated the effects of 3D culture and non-parenchymal cells on the maturation of iHEPs. We describe a serum-free, chemically defined 3D in vitro model using iHEP cells, which is compatible with automation and conventional assay plates. The iHEP cells cultured in this model form polarized aggregates with functional bile canaliculi and strongly increased expression of albumin, urea and genes encoding phase I and II drug metabolism enzymes and bile transporters. Cytochrome P450-mediated metabolism is significantly higher in 3D iHEP aggregates compared to 2D iHEP culture. Furthermore, addition of human liver sinusoidal endothelial cells (sECs) and iPS-derived endothelial cells (iECs) improved mature hepatocyte function and CYP450 enzyme activity. Also, ECs formed endothelial networks within the hepatic 3D cultures, mimicking aspects of an in vivo architecture. Collectively, these results suggest that the iHEP/EC aggregates described here may have the potential to be used for many applications, including as an in vitro model to study liver diseases associated with sinusoidal endothelial cells. STATEMENT OF SIGNIFICANCE: iPS-derived hepatocytes provide an inexhaustible source of cells for drug screening, toxicology studies and cell-based therapies, but lack mature phenotype of adult primary human hepatocytes (PHH). Herein, we show that 3D culture of iPS-derived hepatocytes and their co-culture with human sinusoidal endothelial cells (sECs) to improve their maturity.

In vivo construction of liver tissue by implantation of a hepatic non-parenchymal/adipose-derived stem cell sheet.

Subcutaneous hepatocyte sheet implantation is an attractive therapeutic option for various liver diseases. However, this technique is limited by the availability of hepatocytes. Thus, the use of hepatic non-parenchymal cells (NPCs) containing small hepatocytes, which have the ability to proliferate more rapidly than mature hepatocytes, for transplantation has been suggested. The aim of our study was to construct liver tissue subcutaneously in rats by implanting NPC sheets co-cultivated with adipose-derived stem cells (ADSCs), which produce certain angiogenic factors. We crafted NPC-ADSC sheets on temperature-responsive culture dishes. NPCs formed functioning bile canaliculi and stored glycogen. In addition, their ability to produce albumin was not inferior to that of hepatocytes. Albumin production increased over time when co-cultivated with ADSCs. We then implanted the co-cultivated cell sheets subcutaneously. The co-cultivated sheets retained glycogen, formed bile canaliculi, showed signs of vascularization and survived subcutaneously without pre-vascularization. These results suggest that NPCs can be a viable option in cell therapy for liver diseases. This technique using co-cultivated cell sheets may be useful in the field of regenerative medicine. Copyright © 2017 John Wiley & Sons, Ltd.

Footprint-free human fetal foreskin derived iPSCs: A tool for modeling hepatogenesis associated gene regulatory networks.

Induced pluripotent stem cells (iPSCs) are similar to embryonic stem cells and can be generated from somatic cells. We have generated episomal plasmid-based and integration-free iPSCs (E-iPSCs) from human fetal foreskin fibroblast cells (HFF1). We used an E-iPSC-line to model hepatogenesis in vitro. The HLCs were characterized biochemically, i.e. glycogen storage, ICG uptake and release, UREA and bile acid production, as well as CYP3A4 activity. Ultra-structure analysis by electron microscopy revealed the presence of lipid and glycogen storage, tight junctions and bile canaliculi- all typical features of hepatocytes. Furthermore, the transcriptome of undifferentiated E-iPSC, DE, HE and HLCs were compared to that of fetal liver and primary human hepatocytes (PHH). K-means clustering identified 100 clusters which include developmental stage-specific groups of genes, e.g. OCT4 expression at the undifferentiated stage, SOX17 marking the DE stage, DLK and HNF6 the HE stage, HNF4α and Albumin is specific to HLCs, fetal liver and adult liver (PHH) stage. We use E-iPSCs for modeling gene regulatory networks associated with human hepatogenesis and gastrulation in general.

Hepatocytic parental progenitor cells of rat small hepatocytes maintain self-renewal capability after long-term culture.

The liver has a variety of functions for maintaining homeostasis, and hepatocytes play a major role. In contrast with the high regenerative capacity of mature hepatocytes (MHs) in vivo, they have not been successfully expanded ex vivo. Here we demonstrate that CD44-positive cells sorted from small hepatocyte (SH) colonies derived from a healthy adult rat liver can proliferate on a Matrigel-coated dish in serum-free chemically defined medium; in addition, a subpopulation of the cells can divide more than 50 times in a period of 17 weeks every 4-week-passage. The passage cells retained the capability to recover highly differentiated functions, such as glycogen storage, CYP activity and bile secretion. When Matrigel-treated cells from the third passage were transplanted into retrorsine/partial hepatectomy-treated rat livers, the cells engrafted to differentiate into MHs and cholangiocytes. These results suggest that long-term cultured CD44+ SHs retain hepatocytic characteristics in vitro and the capability to differentiate into hepatocytes and cholangiocytes in vivo. Thus, a newly identified subpopulation of MHs possessing the attributes of hepatocytic stem/progenitor cells can be passaged several times without losing hepatocytic characteristics.

Quantification of Drug-Induced Inhibition of Canalicular Cholyl-l-Lysyl-Fluorescein Excretion From Hepatocytes by High Content Cell Imaging.

We describe the use of a commercially available high content cell imaging algorithm (Cellomics Arrayscan Spot Detector) to quantify biliary excretion of the fluorescent probe substrate cholyl-l-lysyl-fluorescein (CLF) from rat hepatocytes cultured in collagen/matrigel sandwich configuration and to explore inhibition of this process by a variety of test compounds. The method provided robust, reproducible data. Twenty-nine pharmaceuticals inhibited biliary CLF efflux from hepatocytes and a broad range of potencies of inhibition were observed (IC50 values ranged between <1 and 794 µM). Thirteen drugs that inhibited CLF efflux also inhibited hepatocellular uptake of the probe substrate [(3)H]-taurocholate. Although no clear correlation between the potencies of inhibition of the 2 processes was evident, these data highlight the need to consider possible uptake transporter inhibition when interpreting hepatocyte CLF inhibition data. It has been reported that CLF is transported by MRP2. The CLF efflux inhibition data correlated closely with published data on inhibition by the drugs of the bile salt export pump (Bsep), which suggests that the tested drugs inhibit both Bsep and Mrp2. Calculation of the ratios between the maximum human plasma concentrations of the drugs and their CLF efflux inhibition IC50 values raised the possibility that for many, but not all, of them the in vitro effects may be functionally significant in vivo and that Mrp2 inhibition might be a drug-induced liver injury (DILI) risk factor. These data indicate that imaging hepatocyte CLF inhibition is a promising new method for quantification of biliary efflux inhibition by drugs, which could aid assessment of compound-related DILI risk.

The importance of physiological oxygen concentrations in the sandwich cultures of rat hepatocytes on gas-permeable membranes.

Oxygen supply is a critical issue in the optimization of in vitro hepatocyte microenvironments. Although several strategies have been developed to balance complex oxygen requirements, these techniques are not able to accurately meet the cellular oxygen demand. Indeed, neither the actual oxygen concentration encountered by cells nor the cellular oxygen consumption rates (OCR) was assessed. The aim of this study is to define appropriate oxygen conditions at the cell level that could accurately match the OCR and allow hepatocytes to maintain liver specific functions in a normoxic environment. Matrigel overlaid rat hepatocytes were cultured on the polydimethylsiloxane (PDMS) membranes under either atmospheric oxygen concentration [20%-O2 (+)] or physiological oxygen concentrations [10%-O2 (+), 5%-O2 (+)], respectively, to investigate the effects of various oxygen concentrations on the efficient functioning of hepatocytes. In parallel, the gas-impermeable cultures (polystyrene) with PDMS membrane inserts were used as the control groups [PS-O2 (-)]. The results indicated that the hepatocytes under 10%-O2 (+) exhibited improved survival and maintenance of metabolic activities and functional polarization. The dramatic elevation of cellular OCR up to the in vivo liver rate proposed a normoxic environment for hepatocytes, especially when comparing with PS-O2 (-) cultures, in which the cells generally tolerated hypoxia. Additionally, the expression levels of 84 drug-metabolism genes were the closest to physiological levels. In conclusion, this study clearly shows the benefit of long-term culture of hepatocytes at physiological oxygen concentration, and indicates on an oxygen-permeable membrane system to provide a simple method for in vitro studies.

Cytokinesis defines a spatial landmark for hepatocyte polarization and apical lumen formation.

By definition, all epithelial cells have apical-basal polarity, but it is unclear how epithelial polarity is acquired and how polarized cells engage in tube formation. Here, we show that hepatocyte polarization is linked to cytokinesis using the rat hepatocyte cell line Can 10. Before abscission, polarity markers are delivered to the site of cell division in a strict spatiotemporal order. Immediately after abscission, daughter cells remain attached through a unique disc-shaped structure, which becomes the site for targeted exocytosis, resulting in the formation of a primitive bile canaliculus. Subsequently, oriented cell division and asymmetric cytokinesis occur at the bile canaliculus midpoint, resulting in its equal partitioning into daughter cells. Finally, successive cycles of oriented cell division and asymmetric cytokinesis lead to the formation of a tubular bile canaliculus, which is shared by two rows of hepatocytes. These findings define a novel mechanism for cytokinesis-linked polarization and tube formation, which appears to be broadly conserved in diverse cell types.

Protocols for staining of bile canalicular and sinusoidal networks of human, mouse and pig livers, three-dimensional reconstruction and quantification of tissue microarchitecture by image processing and analysis.

Histological alterations often constitute a fingerprint of toxicity and diseases. The extent to which these alterations are cause or consequence of compromised organ function, and the underlying mechanisms involved is a matter of intensive research. In particular, liver disease is often associated with altered tissue microarchitecture, which in turn may compromise perfusion and functionality. Research in this field requires the development and orchestration of new techniques into standardized processing pipelines that can be used to reproducibly quantify tissue architecture. Major bottlenecks include the lack of robust staining, and adequate reconstruction and quantification techniques. To bridge this gap, we established protocols employing specific antibody combinations for immunostaining, confocal imaging, three-dimensional reconstruction of approximately 100-µm-thick tissue blocks and quantification of key architectural features. We describe a standard procedure termed 'liver architectural staining' for the simultaneous visualization of bile canaliculi, sinusoidal endothelial cells, glutamine synthetase (GS) for the identification of central veins, and DAPI as a nuclear marker. Additionally, we present a second standard procedure entitled 'S-phase staining', where S-phase-positive and S-phase-negative nuclei (stained with BrdU and DAPI, respectively), sinusoidal endothelial cells and GS are stained. The techniques include three-dimensional reconstruction of the sinusoidal and bile canalicular networks from the same tissue block, and robust capture of position, size and shape of individual hepatocytes, as well as entire lobules from the same tissue specimen. In addition to the protocols, we have also established image analysis software that allows relational and hierarchical quantifications of different liver substructures (e.g. cells and vascular branches) and events (e.g. cell proliferation and death). Typical results acquired for routinely quantified parameters in adult mice (C57Bl6/N) include the hepatocyte volume (5,128.3 ± 837.8 µm(3)) and the fraction of the hepatocyte surface in contact with the neighbouring hepatocytes (67.4 ± 6.7 %), sinusoids (22.1 ± 4.8 %) and bile canaliculi (9.9 ± 3.8 %). Parameters of the sinusoidal network that we also routinely quantify include the radius of the sinusoids (4.8 ± 2.25 µm), the branching angle (32.5 ± 11.2°), the length of intersection branches (23.93 ± 5.9 µm), the number of intersection nodes per mm(3) (120.3 × 103 ± 42.1 × 10(3)), the average length of sinusoidal vessel per mm(3) (5.4 × 10(3) ± 1.4 × 10(3)mm) and the percentage of vessel volume in relation to the whole liver volume (15.3 ± 3.9) (mean ± standard deviation). Moreover, the provided parameters of the bile canalicular network are: length of the first-order branches (7.5 ± 0.6 µm), length of the second-order branches (10.9 ± 1.8 µm), length of the dead-end branches (5.9 ± 0.7 µm), the number of intersection nodes per mm(3) (819.1 × 10(3) ± 180.7 × 10(3)), the number of dead-end branches per mm(3) (409.9 × 10(3) ± 95.6 × 10(3)), the length of the bile canalicular network per mm(3) (9.4 × 10(3) ± 0.7 × 10(3) mm) and the percentage of the bile canalicular volume with respect to the total liver volume (3.4 ± 0.005). A particular strength of our technique is that quantitative parameters of hepatocytes and bile canalicular as well as sinusoidal networks can be extracted from the same tissue block. Reconstructions and quantifications performed as described in the current protocols can be used for quantitative mathematical modelling of the underlying mechanisms. Furthermore, protocols are presented for both human and pig livers. The technique is also applicable for both vibratome blocks and conventional paraffin slices.

Peptide nanofiber hydrogel induces formation of bile canaliculi structures in three-dimensional hepatic cell culture.

Current hepatocyte models do not mimic the human liver morphology and functions properly and, therefore, drug metabolism, excretion, and toxicity in the liver are inadequately predicted. In this study, we established three-dimensional (3D) hepatic cell cultures in hydrogels of peptide nanofibers. The aim was to establish an improved 3D phenotype of HepG2 cells. In 3D hydrogel cultures, HepG2 cells formed multicellular spheroids that displayed filamentous actin accumulation and large tubular bile canalicular structures indicative of apicobasal cell polarity. Confocal imaging revealed the multidrug resistance-associated protein 2 (MRP2) and the multidrug resistance protein 1 (MDR1) localization on the bile canalicular membrane, and vectorial transport of fluorescent probes into bile canalicular structures. We conclude that 3D HepG2 cultures exhibited structural and functional polarity, suggesting that this model may be useful in drug research. This study shows the potential of 3D peptide nanofiber biomaterials in optimizing the cellular phenotype in organotypic cultures.

Complementary functions of the flippase ATP8B1 and the floppase ABCB4 in maintaining canalicular membrane integrity.

BACKGROUND & AIMS: Progressive familial intrahepatic cholestasis can be caused by mutations in ABCB4 or ATP8B1; each encodes a protein that translocates phospholipids, but in opposite directions. ABCB4 flops phosphatidylcholine from the inner to the outer leaflet, where it is extracted by bile salts. ATP8B1, in complex with the accessory protein CDC50A, flips phosphatidylserine in the reverse direction. Abcb4(-/-) mice lack biliary secretion of phosphatidylcholine, whereas Atp8b1-deficient mice have increased excretion of phosphatidylserine into bile. Each system is thought to have a role protecting the canalicular membrane from bile salts. METHODS: To investigate the relationship between the mechanisms of ABCB4 and ATP8B1, we expressed the transporters separately and together in cultured cells and studied viability and phospholipid transport. We also created mice with disruptions in ABCB4 and ATP8B1 (double knockouts) and studied bile formation and hepatic damage in mice fed bile salts. RESULTS: Overexpression of ABCB4 was toxic to HEK293T cells; the toxicity was counteracted by coexpression of the ATP8B1-CDC50A complex. In Atp8b1-deficient mice, bile salts induced extraction of phosphatidylserine and ectoenzymes from the canalicular membrane; this process was not observed in the double-knockout mice. CONCLUSIONS: ATP8B1 is required for hepatocyte function, particularly in the presence of ABCB4. This is most likely because the phosphatidylserine flippase complex of ATP8B1-CDC50A counteracts the destabilization of the membrane that occurs when ABCB4 flops phosphatidylcholine. Lipid asymmetry is therefore important for the integrity of the canalicular membrane; ABCB4 and ATP8B1 cooperate to protect hepatocytes from bile salts.

Perfusion-based microfluidic device for three-dimensional dynamic primary human hepatocyte cell culture in the absence of biological or synthetic matrices or coagulants.

We describe a perfusion-based microfluidic device for three-dimensional (3D) dynamic primary human hepatocyte cell culture. The microfluidic device was used to promote and maintain 3D tissue-like cellular morphology and cell-specific functionality of primary human hepatocytes by restoring membrane polarity and hepatocyte transport function in vitro without the addition of biological or synthetic matrices or coagulants. A unique feature of our dynamic cell culture device is the creation of a microenvironment, without the addition of biological or synthetic matrices or coagulants, that promotes the 3D organization of hepatocytes into cord-like structures that exhibit functional membrane polarity as evidenced by the expression of gap junctions and the formation of an extended, functionally active, bile canalicular network.

Formation of hepatocyte spheroids with structural polarity and functional bile canaliculi using nanopillar sheets.

We developed a method for controlling the spheroid formation of adult rat primary hepatocytes simply by optimizing the pillar diameters and patterns of nanopillar sheets. To investigate the effects of the pillar parameters on the spheroid formation, rat primary hepatocytes were cultured on nanopillar sheets with pillars that had one of five different diameters and that had been precoated with a solution containing one of two different concentrations of type I collagen. Spheroids with a compact morphology that were adhesive to the substratum and had an optimal size (50 to 100 microm) were obtained using a sheet with a pillar diameter of 2.0 microm that was precoated with 100 ng/mL of type I collagen solution. Immunohistochemistry revealed that the spheroids had a structure similar to that of native liver tissue. We then assessed the effect of overlaying reconstituted spheroids with Matrigel with the aim of achieving a simulated in vivo environment. The mRNA expression levels of MRP2, albumin, and P450-3A3 for spheroids determined by semiquantitative real-time PCR were significantly higher than those for spheroids cultured without the Matrigel overlay or for hepatocytes cultured using a conventional two-dimensional method. The spheroids obtained exhibited higher structural polarity and functional bile canaliculi compared with hepatocytes cultured using a conventional two-dimensional method.

Regulatory subunit I-controlled protein kinase A activity is required for apical bile canalicular lumen development in hepatocytes.

Signaling via cAMP plays an important role in apical cell surface dynamics in epithelial cells. In hepatocytes, elevated levels of cAMP as well as extracellular oncostatin M stimulate apical lumen development in a manner that depends on protein kinase A (PKA) activity. However, neither the identity of PKA isoforms involved nor the mechanisms of the cross-talk between oncostatin M and cAMP/PKA signaling pathways have been elucidated. Here we demonstrate that oncostatin M and PKA signaling converge at the level of the PKA holoenzyme downstream of oncostatin M-stimulated MAPK activation. Experiments were performed with chemically modified cAMP analogues that preferentially target regulatory subunit (R) I or RII holoenzymes, respectively, in hepatocytes. The data suggest that the dissociation of RI- but not RII-containing holoenzymes, as well as catalytic activity of PKA, is required for apical lumen development in response to elevated levels of cAMP and oncostatin M. However, oncostatin M signaling does not stimulate PKA holoenzyme dissociation in living cells. Based on pharmacological and cell biological studies, it is concluded that RI-controlled PKA activity is essential for cAMP- and oncostatin M-stimulated development of apical bile canalicular lumens.

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.

Expression, localization, and inducibility by bile acids of hepatobiliary transporters in the new polarized rat hepatic cell lines, Can 3-1 and Can 10.

Sinusoidal and apical transporters are responsible for the uptake and biliary elimination of many compounds by hepatocytes. Few in vitro models are however available for analyzing such functions. The expression and bile-acid inducibility of 13 transporters and two nuclear receptors were investigated in the new rat polarized lines, Can 3-1 and Can 10, and in their unpolarized parent, Fao. The relative abundance of mRNA, the protein level, and their localization were examined by real-time quantitative PCR, Western blotting, immunofluorescence, and confocal microscopy. Compared with rat liver, mRNA levels of Fao cells were: negligible for Bsep/Abcb11; lower for the uptake transporters Ntcp and Oatps; similar for SHP, FXR, and Bcrp/Abcg2; and higher (four-fold to 160-fold) for the efflux pumps Mdr1b/Abcb1b, Mdr2/Abcb4, Mrp1/Abcc1, Mrp2/Abcc2, Mrp3/Abcc3, Abcg5, and Abcg8. This profile was mostly maintained (and improved for Bsep) in Can 10. Some transporters were less well expressed in Can 3-1. In both lines, sinusoidal (Ntcp, Mrp3) and canalicular transporters (Mdr-P-glycoproteins detected with C219 antibody, Mrp2) were localized at their correct poles. Bile-acid effects on polarity and mRNA levels of transporters were analyzed after a 6-day treatment with 50 microM taurocholic, chenodeoxycholic (CDCA), or ursodeoxycholic acid (UDCA). No polarization of Fao cells was induced; Can 10 and Can 3-1 polarity was maintained. CDCA and UDCA induced marked enhancement of the volume of Can 10 bile canaliculi. CDCA upregulated Bsep, Mdr2, SHP, Mdr1b, and Oatp2/1a4 in Can 10 (two- to seven-fold) and in Fao cells. Thus, Can 10 constitutes an attractive polarized model for studying vectorial hepatobiliary transport of endogenous and xenobiotic cholephilic compounds.

Lipid rafts establish calcium waves in hepatocytes.

BACKGROUND & AIMS: Polarity is critical for hepatocyte function. Ca(2+) waves are polarized in hepatocytes because the inositol 1,4,5-trisphosphate receptor (InsP3R) is concentrated in the pericanalicular region, but the basis for this localization is unknown. We examined whether pericanalicular localization of the InsP3R and its action to trigger Ca(2+) waves depends on lipid rafts. METHODS: Experiments were performed using isolated rat hepatocyte couplets and pancreatic acini, plus SkHep1 cells as nonpolarized controls. The cholesterol depleting agent methyl-beta-cyclodextrin (mbetaCD) was used to disrupt lipid rafts. InsP3R isoforms were examined by immunoblot and immunofluorescence. Ca(2+) waves were examined by confocal microscopy. RESULTS: Type II InsP3Rs initially were localized to only some endoplasmic reticulum fractions in hepatocytes, but redistributed into all fractions in mbetaCD-treated cells. This InsP3R isoform was concentrated in the pericanalicular region, but redistributed throughout the cell after mbetaCD treatment. Vasopressin-induced Ca(2+) signals began as apical-to-basal Ca(2+) waves, and mbetaCD slowed the wave speed and prolonged the rise time. MbetaCD had a similar effect on Ca(2+) waves in acinar cells but did not affect Ca(2+) signals in SkHep1 cells, suggesting that cholesterol depletion has similar effects among polarized epithelia, but this is not a nonspecific effect of mbetaCD. CONCLUSIONS: Lipid rafts are responsible for the pericanalicular accumulation of InsP3R in hepatocytes, and for the polarized Ca(2+) waves that result. Signaling microdomains exist not only in the plasma membrane, but also in the nearby endoplasmic reticulum, which in turn, helps establish and maintain structural and functional polarity.

SR-BI undergoes cholesterol-stimulated transcytosis to the bile canaliculus in polarized WIF-B cells.

The scavenger receptor BI (SR-BI) is highly expressed in hepatocytes, where it mediates the uptake of lipoprotein cholesterol, promotes the secretion of cholesterol into bile, and protects against atherosclerosis. Despite a strong correlation between the hepatic expression of SR-BI and biliary cholesterol secretion, little is known about SR-BI trafficking in response to changes in sterol availability. Using a well characterized polarized hepatocyte cell model, WIF-B, we determine that in cholesterol-depleted cells, SR-BI is extensively located on the basolateral surface, where it can access circulating lipoproteins. However, in response to cholesterol loading, SR-BI undergoes a slow transcytosis to the apical bile canaliculus independently of lipoprotein binding and new protein synthesis. In cholesterol-replete WIF-B cells, SR-BI that resides on the canalicular membrane is dynamically associated with defined microdomains and does not rapidly recycle to and from the subapical or basolateral regions. Taken together, these data demonstrate that hepatic SR-BI transcytosis is regulated by cholesterol and suggest that SR-BI has a stationary function on the bile canaliculus.

Taurolithocholate impairs bile canalicular motility and canalicular bile secretion in isolated rat hepatocyte couplets.

AIM: To investigate the effects of taurolithocholate (TLC) on the canalicular motility in isolated rat hepatocyte couplets (IRHC). METHODS: TLC was added to IRHC at concentrations of 10 and 50 mumol/L, respectively. In each group, five time-lapse movies containing 3 representative bile canaliculi were taken under phase-contrast microscopy for 12 h. The number of bile canalicular contractions and the intervals between consecutive canalicular contractions were calculated. Furthermore, the effects of TLC on IRHC were examined by transmission electron microscopy. RESULTS: The bile canalicular contractions were spontaneous and forceful in the controls. Active vesicular movement was observed in the pericanalicular region. Immediately after the addition of TLC, the bile canaliculi were deformed, and canalicular bile was incorporated into the vacuoles. The canaliculi were gradually dilated, and canalicular contractions were markedly inhibited by TLC. The vesicular movements became extremely slow in the pericanalicular region. The number of canalicular contractions significantly decreased in the TLC-treated groups, as compared with that in the controls. The time intervals were prolonged, as the TLC dosage increased, indicating that bile secretion into the canaliculi was impaired with TLC. Transmission electron microscopy revealed the lamellar transformation of the canalicular membranes in IRHC treated with TLC. CONCLUSION: TLC impairs both the bile canalicular contractions and the canalicular bile secretion, possibly by acting directly on the canalicular membranes in TLC-induced cholestasis.

Use of cryopreserved human hepatocytes in sandwich culture to measure hepatobiliary transport.

Fresh hepatocytes cultured in a sandwich configuration allow for the development of intact bile canaliculi and the ability to measure hepatic uptake and biliary clearance. A disadvantage of this model is its dependence upon hepatocytes from fresh tissue. Therefore, the ability to use cryopreserved human hepatocytes in this model would be a great advantage. Multiple variables were tested, and the recommended conditions for culturing cryopreserved human hepatocytes in a sandwich configuration in 24-well plates are as follows: BioCoat plates, a cell density of 0.35 x 10(6) cells/well in 500 microl, an overlay of Matrigel and InVitroGRO media. These conditions resulted in good hepatocyte morphology and the formation of distinct bile canaliculi. The function of multiple uptake and efflux transporters was tested in multiple lots of cryopreserved and fresh human hepatocytes. For taurocholate [Na+ taurocholate cotransporting polypeptide/organic anion transporting polypeptide (OATP) uptake/bile salt export pump efflux], the average apparent uptake, apparent intrinsic biliary clearance, and biliary excretion index among five cryopreserved hepatocyte lots was high, ranging from 11 to 17 pmol/min/mg protein, 5.8 to 10 microl/min/mg protein, and 41 to 63%, respectively. The corresponding values for digoxin (OATP-8 uptake/multidrug resistance protein 1 efflux) were 0.69 to 1.5 pmol/min/mg protein, 0.60 to 1.5 microl/min/mg protein, and 37 to 63%. Both substrates exhibited similar results when fresh human hepatocytes were used. In addition, substrates of breast cancer resistance protein and multidrug resistance-associated protein 2 were also tested in this model, and all cryopreserved lots showed functional transport of these substrates. The use of cryopreserved human hepatocytes in 24-well sandwich culture to form intact bile canaliculi and to exhibit functional uptake and efflux transport has been successfully demonstrated.