HepaRG spheroid models for predicting liver toxicity

Cholestatic drug-induced liver injury (cDILI) is a frequent reason for drug failure and withdrawal during premarketing and postmarketing stages of drug development. Strategies for reliable detection of cDILI in early drug development are therefore urgently needed. The drug-induced cholestasis index (DICI) concept was previously introduced as a tool for assessing the cholestatic potential of drug candidates. DICI is calculated as the ratio between the viability values obtained in drug-treated liver cells in the presence and absence of bile acids. The present in vitro study was set up to investigate the applicability of DICI in a novel high-throughput and large sample setting. Furthermore, the improvement of the predictivity of the DICI by introduction of advanced modeling was explored. Fifty-eight well-documented drugs were selected and categorized as drugs inducing cDILI, non-cholestatic DILI (ncDILI), and not inducing DILI (non-DILI).

Differentiated HepaRG cells were handled according to the manufacturer’s instructions. Briefly, thawed cells were centrifuged and resuspensed in MHTAP medium. Cells were seeded in ultra-low attachment plates. Plates were maintained for 1 day on a tilting stand to facilitate the aggregation of the cells and improve uniform spheroid formation at 37 °C under a humidified atmosphere containing 5 % CO2. Cell culture medium was replaced with MHPIT medium and cultured for 7 additional days with cell culture medium renewal every 2–3 days until spheroid formation and maturation. Morphological features and viability of the fully formed spheroids were assessed by phase-contrast imaging at days 0 following spheroid generation (i.e., 8 days after seeding). The average diameter of the spheroids at day 0 after spheroid generation was 189.1 ± 9.4 µm. HepaRG spheroids showed BSEP protein expression as an indicator of their suitability to investigate cholestatic liability.