An Automated Multiplexed Hepatotoxicity and CYP Induction Assay Using HepaRG Cells in 2D and 3D

SLAS Discov. 2017 Jun;22(5):614-625. doi: 10.1177/2472555217701058. Epub 2017 Mar 27.


Drug-induced liver injury (DILI) and drug-drug interactions (DDIs) are concerns when developing safe and efficacious compounds. We have developed an automated multiplex assay to detect hepatotoxicity (i.e., ATP depletion) and metabolism (i.e., cytochrome P450 1A [CYP1A] and cytochrome P450 3A4 [CYP3A4] enzyme activity) in two-dimensional (2D) and three-dimensional (3D) cell cultures. HepaRG cells were cultured in our proprietary micromold plates and produced spheroids. HepaRG cells, in 2D or 3D, expressed liver-specific proteins throughout the culture period, although 3D cultures consistently exhibited higher albumin secretion and CYP1A/CYP3A4 enzyme activity than 2D cultures. Once the spheroid hepatic quality was assessed, 2D and 3D HepaRGs were challenged to a panel of DILI- and CYP-inducing compounds for 7 days. The 3D HepaRG model had a 70% sensitivity to liver toxins at 7 days, while the 2D model had a 60% sensitivity. In both the 2D and 3D HepaRG models, 83% of compounds were predicted to be CYP inducers after 7 days of compound exposure. Combined, our results demonstrate that an automated multiplexed liver spheroid system is a promising cell-based method to evaluate DILI and DDI for early-stage drug discovery.

Keywords: drug discovery; drug-induced liver injury; drug–drug interaction; scaffold-free; spheroid.

MeSH terms

  • Biological Assay / methods*
  • Cell Culture Techniques / methods*
  • Chemical and Drug Induced Liver Injury / prevention & control
  • Cytochrome P-450 Enzyme Inducers / pharmacology
  • Cytochrome P-450 Enzyme System / metabolism
  • Drug Interactions / physiology
  • Hepatocytes / drug effects
  • Hepatocytes / metabolism
  • Humans
  • Liver / drug effects*
  • Spheroids, Cellular / drug effects
  • Spheroids, Cellular / metabolism


  • Cytochrome P-450 Enzyme Inducers
  • Cytochrome P-450 Enzyme System