Development of an in vitro cholestatic drug-induced liver injury evaluation system using HepG2-hNTCP-C4 cells in sandwich configuration

The development of a predictive hepatotoxicity model is highly warranted, as the hepatotoxicity risk goes undetectable in nonclinical studies. Cholestatic drug induced liver injury (DILI) is a condition in which bile acids are not normally excreted into the capillary bile canaliculi and are accumulated in hepatocytes, caused by the inhibition of bile salt export pump (BSEP), a transporter that is mainly associated with excretion of bile acids. Although laboratory animals are used as predicting model, the use of human-derived cells is required due to species differences. Moreover, primary human hepatocytes (PHHs) show rapid loss of function, and difficulties in forming bile canaliculi in vitro. Therefore, we aimed to develop an in vitro culture method for efficient formation of bile canaliculi and the function of BSEP in PHHs. Here, PHHs were cultured from 1 h after thawing to day 2 with Z-VAD-FMK, a total caspase inhibitor, and RevitaCell™ supplement, an irreversible Rho -associated coiled-coil forming kinase (ROCK) inhibitor, in combination with RM-101. The PHHs were able to form bile canaliculi and show BSEP function on day 6 of culture. Therefore, these cultured PHHs may serve as an accurate predicting model for cholestatic DILI.

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“…5). This finding was consistent with the localization sites in human liver biopsies and rat hepatocytes [17][18][19] .…”

Section: Discussionsupporting

confidence: 91%

Culture methods focusing on bile canalicular formation using primary human hepatocytes in short time

Shibata

Matsumura

Iwao

et al. 2022

Preprint

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“…It was suggested that the established in vitro test system could be superior for DIC risk evaluation compared with the C ss /BSEP IC 50 (> 0.1) assessment system (Table 5). Alternatively, high-level studies have been conducted on the risk assessment of cholestasis DILI using rats (Susukida et al, 2015;Susukida et al, 2015;Oizumi et al, 2017) and human (Ogimura et al, 2017;Sakai et al, 2019a;Kawaguchi et al, 2020) hepatocytes, and various cell lines, including HepG2 (Sakai et al, 2019b;Kawaguchi et al, 2020) and HepaRG (Susukida et al, 2016), mainly in sandwich-culture. Although these bio-mimicking 3-D test systems cannot be directly compared with the present test system, the prediction accuracy of the present test system is considered to be comparable to that of reported risk predictions in spite of simply 2-D culture.…”

Section: Discussionmentioning

confidence: 99%

“…The disruption of the normal process of bile secretion results in cholestasis. Previously, the presence of quantified BAs in the human serum was reported (Scherer et al, 2009), which 12 BAs of them have been applied in sandwich-cultured test sys-tems for evaluating DIC using rat (Ogimura et al, 2011;Susukida et al, 2015;Oizumi et al, 2017) and human (Ogimura et al, 2017;Sakai et al, 2019a;Kawaguchi et al, 2020) hepatocytes, various cell lines including HepG2 (Sakai et al, 2019b;Kawaguchi et al, 2020) and Hep-aRG (Susukida et al, 2016). Several human hepatomaderived cell lines, in particular HepaRG cells, can differentiate from an epithelial phenotype to both canalicular-and hepatocyte-like cells under two-dimensional (2-D) culture condition (Parent et al, 2004), and are widely applied for testing enzyme induction (Turpeinen et al, 2009), transporter (Le et al, 2006), toxicology (Guillouzo et al, 2007;Sharanek et al, 2017), etc.…”

Section: Introductionmentioning

confidence: 99%

Establishment of an <i>in vitro</i> cholestasis risk assessment system using two-dimensional cultured HepaRG cells and 12 bile acids

2023

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Drug-induced liver injury (DILI) is a major cause of market withdrawal or drug-development discontinuation because of safety concerns. In this study, we focused on drug-induced cholestasis (DIC) to establish an in vitro cytotoxicity test system and analyze its sensitivity using two-dimensional (2-D) cultured HepaRG cells and 12 types of bile acids (BAs) present in the human serum. First, to detect the cytotoxicity associated with cholestasis effectively, non-toxic BA concentrations were investigated and determined to be 100-fold the human serum value (455 μM total BAs). Next, the cytotoxicity of 31 compounds that can inhibit the bile acid export pump (BSEP) and were categorized as no-DILI-concern, less-DILI-concern, and most-DILI-concern was examined. None of the no-DILI-concern compounds yielded cytotoxicity, whereas almost all less-DILI-concern compounds (with the exception of simvastatin) and most-DILI-concern compounds (with the exception of bosentan) exhibited cytotoxicity. An investigation of the cause of cytotoxicity using 3 H-taurocholic acid revealed that most-DILI-concern and less-DILI-concern compounds, but not no-DILI-concern compounds, triggered the accumulation of radioactivity in the cell lysates. Thus, the onset of cytotoxicity seemed to be associated with cholestasis. The established HepaRG cytotoxicity assessment system (sensitivity of 89%, specificity of 100%, and accuracy of 97%) was mostly superior to the C ss /BSEP IC 50 (> 0.1) assessment system (sensitivity of 83%, specificity of 100%, and accuracy of 72%). Therefore, the assay method using 2-D cultured HepaRG cells and 12 BAs established here can be widely applicable as a model for the in vitro potential assessment of DIC.

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Assessment of long-term functional maintenance of primary human hepatocytes to predict drug-induced hepatoxicity in vitro

Chen

Tang

et al. 2021

Arch Toxicol

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Development of an in vitro cholestatic drug-induced liver injury evaluation system using HepG2-hNTCP-C4 cells in sandwich configuration

Cited by 4 publications

References 24 publications

Culture methods focusing on bile canalicular formation using primary human hepatocytes in short time

Culture methods focusing on bile canalicular formation using primary human hepatocytes in short time

Establishment of an <i>in vitro</i> cholestasis risk assessment system using two-dimensional cultured HepaRG cells and 12 bile acids

Assessment of long-term functional maintenance of primary human hepatocytes to predict drug-induced hepatoxicity in vitro

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