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

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.