Increased fluorescence signal reflecting the accumulation of phospholipids was observed

at 5 μM concentrations ( Fig. Suppl. Fig. 5L and P), as compared to vehicle control ( Suppl. Fig. 5I and M). LDH release following CPZ exposure was found only at day 14 of treatment (5 μM, **p < 0.01; 10 μM, *p < 0.05) ( Fig. 6B), whereas Mrp2-mediated canalicular transport was reduced at day 3 at all concentrations used (1 μM, *p < 0.05; 5 μM and 10 μM, ****p < 0.0001). In addition, 10 μM CPZ enhanced the content of intracellular phospholipids after 7 and 14 days (*p < 0.05). Similarly, treatment of rat hepatocytes with TGZ resulted into inhibition of canalicular transport after 3 days at all concentrations (5 μM, *p < 0.05; 10 μM, ***p < 0.001; 25 μM, ***p < 0.001) ( Fig. 7A), whereas no cytotoxicity was observed over 14-day exposure. Exposure of RGZ resulted into accumulation of neutral lipids by day 14 at the highest concentration (50 μM, *p < 0.05) ( Fig. 6A; selleck kinase inhibitor Suppl. Fig. 6K, L, Q and P) which was associated at the same time with increased leakage of LDH. Cells treated with ACT and VPA

did not display any sign of cytotoxicity (Fig. 9A and B). In addition canalicular transport and lipid metabolism were not affected. MET 50 μM (Fig. 7B), FFB 25 μM (Fig. 8A) and IBU 50 μM (Fig. 8B) treatments were only associated with LDH release at day 10 and 14. The data presented here illustrate further improvement of the rat hepatocyte Collagen I-Matrigel™ sandwich in vitro model, where primary rat hepatocytes were maintained

in a functional state for Mitomycin C manufacturer a period of 14 days. The addition of multiple layers of Matrigel™ has been demonstrated to be a robust method for the maintenance of hepatocyte morphology and specific functions. Mrp2-mediated transport quantified by HCI was enhanced when hepatocytes received 4 layers of Matrigel™ over 2 weeks of culture. In these conditions, the expression of liver specific genes, such as transporters, nuclear receptor and CYPs was stable over the whole culture duration. However, in this setting the addition of a low percentage of serum as well as the presence of EGF in the cell culture medium did not improve the cells status, despite some published evidences showing enhancement these of long-term maintenance and survival of rat hepatocytes ( Farkas and Tannenbaum, 2005). ( Suppl. Fig. 1B and F). These culture conditions allowed mimicking chronic treatment by multiple exposures of hepatocytes to different hepatotoxicants. By combining a stable and reproducible in vitro culture system exposed daily to low non-cytotoxic concentrations for 14 days together with HCI technology, specific cellular responses associated with liver pathological features could be monitored and quantified. In some instances, drug withdrawals from market were the result of “Hy’s law” cases, indicated by a 2-fold bilirubin increase with a concomitant occurrence of 3-fold ALT increase in plasma.