Human hepatic HepaRG co-culture model as a sensitive and non-invasive toxicological platform using ECIS biosensors

Introduction:
A robust HTS-compatible multiparametric platform for assessing drug hepatotoxicity would provide a new paradigm for in vitro toxicology. Using the highly-differentiated human hepatic cell line HepaRG as a surrogate to primary human hepatocytes, our aim was to monitor cell behaviour, by profiling real-time changes in cellular impedance, |Z|, in response to model hepatotoxins. The spectrum of drug-induced liver injury (DILI) was assessed, including: hepatocellular damage (amiodarone), cholestasis (Cyclosporine-A), and steatosis (paracetamol). Such dynamic screening allows capture and modeling of label-free multiparametric, high-content data using ECIS biosensors.

Methods:
HepaRG cells were cultured to confluence (terminally-differentiated Hepatocyte:Cholangiocyte co-culture) on 8-well (ECIS-8W10+) and 96-well (96-idf20) (250,000 cells/cm2) electrode arrays. Following 24h CYP450 induction (day 8), with appropriate inducers (eg Rifampicin for CYP3A4), time-/dose-response of model hepatotoxins [DMSO-vehicle as controls] was monitored with |Z| measurements taken at 180s intervals over a 500Hz to 64kHz frequency (f) range, for 96h post-induction. To evaluate correlation with |Z|-spectra modelling data, parallel, biochemical assays were performed: i) Cell viability: ATP-depletion (Promega-CellTiter©-Glo); Prestoblue (Invitrogen) hepatotoxicity assay; ii) Dose-response effects of: L-buthionine-(S,R)-sulfoximine (GSH synthesis inhibitor); Phorbol Ester (disrupts Tight Junction (TJ) integrity); and TrypLE™ (protease enzyme - disrupts cell adhesion).

Results:
|Z|-time curves showed highly-sensitive dose-related response to model hepatotoxins, such as paracetamol over the critical 24h period,, with decreasing |Z| values reflecting disruption of cell-cell/cell-substrate interactions (f=4KHz); whilst at f=64kHz, |Z| changes were indicative of loss of membrane integrity. Biochemical changes including HepaRG ATP content correlated with |Z| changes. |Z|-spectra modelling (utilizing ECIS algorithms) supported these observations, and provided further evidence of disruption of barrier function (TJs)/ cell-adhesion parameters prior to cell death.

Discussion / Conclusion:
Real-time high resolution tracking of morphological changes following hepatotoxic challenge was observed. The HepaRG-ECIS platform may offer a breakthrough technology for pre-clinical in vitro hepatotoxicology by quantifying cell behavior in response to DILI.