Fig. 4

Evaluation of the in vitro zonal hepatotoxicity using the 3D hepatic zonation model. a Application of the 3D hepatic zonation model using imaging analysis after APAP treatment. The intact 3D HepaRG sample in the agarose hydrogel channel was removed from the polyolefin tube and then treated with 10 mM APAP for 2 days in a multi-well plate. Quantitative analysis of 3D hepatic zonal channels was performed using staining with dual live/dead dyes as well as CHIR for diffusion of CHIR, CellTracker Deep Red Dye for cellular distribution, and EthD-1 for membrane-damaged cytotoxic cells, and the ChemiDoc XRS+ Imaging System, and images were visualized and analysed with MATLAB. The red and blue colours indicate high and low intensity of the dyes, respectively. The relative concentration was quantified and plotted for the control and APAP-treated groups. The EthD-1⁺-damaged cells were predominantly observed in the high CHIR concentration region in response to 10 mM APAP treatment. b Molecular experimental approaches using the 3D hepatic zonation model. The 3D HepaRG sample was removed from the 3D hepatic zonal channel and then divided into 3 pieces corresponding to zone-1-like, zone-2-like and zone-3-like regions according to the guide. After treatment with 10 mM APAP for 2 days, total protein was extracted from the 3D HepaRG and western blot analysis was performed. The expression of β-catenin, CYP2E1, AIF, and cleaved PARP was analysed, and β-actin was used as an internal control