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In vitro Construction of the liver sinusoidal structure using artificial tissue is an important but worthwhile challenge, particularly for assessing the risk of diseases such as sinusoidal obstruction syndrome (SOS). Current models are unsuitable for evaluating the toxicity because of lacking sinusoidal capillary. In this study, we developed a vascularized hepatic tissue (VHT) using a unique tissue engineering technique, the cell assembled viscous tissue by sedimentation (CAViTs) method. The "viscous bodies" created using the CAViTs method exhibited significant self-assembly within 6 h after seeding, promoting cell-cell interaction. The level of albumin secreted by the VHT was four times higher than that of 2D-coculture and maintained for 1 month. The gene expression pattern of the VHT was closer to that of total human liver, compared with the 2D system. Quantitative evaluations of the vascular structure of VHT treated with two typical SOS-inducing compounds, monocrotaline and retrorsine, revealed higher sensitivity (IC 50 = 40.35 µM), 19.92 times higher than the cell-viability assay. Thus, VHT represents an innovative in vitro model that mimics the vessel network structure and could become a useful tool for the early screening of compounds associated with a risk of vascular toxicity. Mimicking sinusoidal structures in in vitro liver model is important to consider from the perspective of predicting hepatotoxicity such like sinusoidal obstruction syndrome (SOS). However, it was difficult to reconstruct the vascular structure within the hepatocyte-rich environment. In this study, we constructed a vascularized hepatic tissue in a high-throughput manner by a unique method using collagen and heparin, and evaluated its applicability to toxicity assessment. Vessel morphology analysis of the model treated by monocrotaline, which is a well-known SOS-inducing compound, could predict the toxicity with higher sensitivity. To the best of our knowledge, this is the first report to provide vascularized hepatic tissues using sinusoidal endothelial cells at least for demonstrating applicability to the evaluation of SOS induction risk. [Display omitted] [ABSTRACT FROM AUTHOR]

Cholestasis resulting from hepatic bile acid efflux transporter inhibition may contribute to drug-induced liver injury (DILI). This condition is a common safety-related reason for drug attrition and withdrawal. To screen for safety risks associated with efflux transport inhibition, we developed a high-throughput cellular assay for different drug discovery phases. Hepatocytes isolated from chimeric mice with humanized livers presented gene expression resembling that of the human liver and demonstrated apical membrane polarity when sandwiched between Matrigel and collagen. The fluorescent bile acid-derivative cholyl- l -lysyl-fluorescein (CLF) was used to quantify drug-induced efflux transport inhibition in hepatocytes. Cyclosporine inhibited CLF accumulation in the apical bile canalicular lumen in a concentration-dependent manner. The assay had equivalent predictive power to a primary human hepatocyte-based assay and greater predictive power than an assay performed with rat hepatocytes. Predictive power was tested using 45 pharmaceutical compounds, and 91.3% of the compounds with cholestatic potential (21/23) had margins (IC50/ C max) < 20. In contrast, 90.9% (20/22) of compounds without cholestatic potential had IC50/ C max>20. Assay sensitivity and specificity were 91.3% and 90.9%, respectively. We suggest that this improved assay performance could result from higher expression of efflux transporters, metabolic pathways, and/or species differences. Given the long-term supply of cells from the same donor, the humanized mouse-derived hepatocyte-based CLF efflux assay could be a valuable tool for predicting cholestatic DILI. [ABSTRACT FROM AUTHOR]

Hyperbaric oxygen therapy (HBO) has been used for many clinical treatments, including primary liver nonfunction. However, the cellular mechanism by which HBO treatment ameliorates liver function is not understood. Therefore, the purpose of this study was to elucidate this cellular mechanism using primary cultured rat hepatocytes in in vitro studies. Hepatocytes were treated with HBO at 1 day after plating, and the morphological and functional characteristics of bile canaliculi formed in cultured hepatocytes were observed by time-lapse microscopy. Multidrug resistance protein-2 localization was observed by confocal laser microscopy. In cultured hepatocytes, the labeling index in the HBO group at 2 days after treatment was significantly higher than that in the control group. In addition, the proliferating cellular nuclear antigen level in the HBO group was significantly higher than that in the control group. The contraction of the bile canaliculi in the HBO group was slower than in the control group and the dilatation of bile canaliculi in the HBO group was much larger than in the control group. Multidrug resistance protein-2 in the HBO group was localized at the apical membrane. These results show that HBO stimulates hepatocytes to proliferate and HBO normalizes Multidrug resistance protein-2 localization to the apical membrane, which could dilate bile canaliculi. [ABSTRACT FROM AUTHOR]

Here, we established a high-throughput in vitro assay system to predict reactive metabolite (RM) formation. First, we performed the glutathione (GSH) consumption assay to monitor GSH levels as an index of RM formation potential using HepaRG cells pretreated with 500 μM D,L-buthionine-(S,R)-sulfoximine (BSO) and then treated with ticlopidine and diclofenac. Both drugs, under GSH-reduced conditions, significantly decreased relative cellular GSH content by 70% and 34%, respectively, compared with that in cells not pretreated with BSO. Next, we examined the correlation between GSH consumption and covalent binding assays; the results showed good correlation (correlation coefficient = 0.818). We then optimized the test compound concentration for evaluating RM formation potential using 76 validation compound sets, and the highest sensitivity (53%) was observed at 100 μM. Finally, using HepG2 cells, PXB-cells, and human primary hepatocytes, we examined the cell types suitable for evaluating RM formation potential. The expression of CYP3A4 was highest in HepaRG cells, suggesting the highest sensitivity (56.4%) of the GSH consumption assay. Moreover, a co-culture model of PXB-cells and HepaRG cells showed high sensitivity (72.7%) with sufficient specificity (85.7%). Thus, the GSH consumption assay can be used to effectively evaluate RM formation potential in the early stages of drug discovery. • Cell-based assay was developed to predict reactive metabolite (RM) formation. • The optimum test compound concentration for evaluation of RM formation was 100 μM. • The optimum cell type was HepaRG cells, showing the highest sensitivity. • Co-culture of PXB and HepaRG cells showed high sensitivity and good specificity. • This assay is valuable for evaluation of RM formation potential in drug discovery. [ABSTRACT FROM AUTHOR]