Development and Functional Characterization of 3D HepaRG Culture Models

Development and Functional Characterization of 3D HepaRG Culture Models with Physiologically-Relevant Levels of Xenobiotic Metabolism for Toxicology Screening


Sreenivasa C. Ramaiahgari




Sreenivasa C. Ramaiahgari, Suramya Waidyanatha, Darlene Dixon, Michael J. DeVito, Richard S. Paules and Stephen S. Ferguson


Current high-throughput in vitro assays employ two-dimensional (2D) tissue culture models that poorly predict xenobiotic exposure effects on humans. This is principally due to the poor differentiation status resulting in aberrant xenobiotic metabolism and cellular response. To overcome this, three-dimensional (3D) cell culture models are being developed that mimic and maintain biochemical aspects of a tissue to a higher extent than two-dimensional monolayer cultures.

We have developed a three-dimensional spheroid model of HepaRG cells that exhibits physiologically relevant levels of xenobiotic metabolism. HepaRG, is a bi-potent progenitor cell line that differentiates into co-cultures of hepatocyte- and cholangiocyte-like cells. When cultured as three-dimensional spheroids, they show several hallmarks of polarized hepatocytes with distinct apical/basolateral domains and robust levels of xenobiotic metabolism competence (with clinical substrates for CYP1A2, CYP2B6 and CYP3A4) near median levels observed over hundreds of preparations of primary human hepatocyte suspension cultures. Spheroid size, time in culture and culture media composition were important factors affecting basal xenobiotic metabolism, inducibility with activators of hepatic nuclear receptors AhR, CAR and PXR, and consequential metabolite profiles to xenobiotic metabolism transformations. Repeated exposure studies showed differential sensitivity in identifying compounds that cause metabolism-dependent toxicity. Pilot studies on high-throughput transcriptome analysis of Tox21 human sentinel 1500+ genes (~2700 genes) were performed to study molecular pathway responses to xenobiotic exposures that intrinsically incorporated xenobiotic metabolism functionality. These assays are implemented in 96 and 384- well formats for low cost and potential high-throughput toxicology screening.