High-throughput confocal imaging of differentiated 3D liver-like spheroid cellular stress response reporters for identification of drug-induced liver injury liability

Arch Toxicol. 2019 Oct;93(10):2895-2911. doi: 10.1007/s00204-019-02552-0. Epub 2019 Aug 27.


Adaptive stress response pathways play a key role in the switch between adaptation and adversity, and are important in drug-induced liver injury. Previously, we have established an HepG2 fluorescent protein reporter platform to monitor adaptive stress response activation following drug treatment. HepG2 cells are often used in high-throughput primary toxicity screening, but metabolizing capacity in these cells is low and repeated dose toxicity testing inherently difficult. Here, we applied our bacterial artificial chromosome-based GFP reporter cell lines representing Nrf2 activation (Srxn1-GFP and NQO1-GFP), unfolded protein response (BiP-GFP and Chop-GFP), and DNA damage response (p21-GFP and Btg2-GFP) as long-term differentiated 3D liver-like spheroid cultures. All HepG2 GFP reporter lines differentiated into 3D spheroids similar to wild-type HepG2 cells. We systematically optimized the automated imaging and quantification of GFP reporter activity in individual spheroids using high-throughput confocal microscopy with a reference set of DILI compounds that activate these three stress response pathways at the transcriptional level in primary human hepatocytes. A panel of 33 compounds with established DILI liability was further tested in these six 3D GFP reporters in single 48 h treatment or 6 day daily repeated treatment. Strongest stress response activation was observed after 6-day repeated treatment, with the BiP and Srxn1-GFP reporters being most responsive and identified particular severe-DILI-onset compounds. Compounds that showed no GFP reporter activation in two-dimensional (2D) monolayer demonstrated GFP reporter stress response activation in 3D spheroids. Our data indicate that the application of BAC-GFP HepG2 cellular stress reporters in differentiated 3D spheroids is a promising strategy for mechanism-based identification of compounds with liability for DILI.

Keywords: BAC-reporter cells; Cellular stress response; Drug-induced liver injury; HepG2 spheroids; High-throughput imaging; Liver transcription factors.

MeSH terms

  • Cell Differentiation
  • Chemical and Drug Induced Liver Injury / etiology
  • Chemical and Drug Induced Liver Injury / genetics
  • Chemical and Drug Induced Liver Injury / pathology*
  • DNA Damage / drug effects
  • Genes, Reporter / genetics
  • Green Fluorescent Proteins / genetics
  • Hep G2 Cells
  • Hepatocytes / drug effects*
  • Hepatocytes / pathology
  • High-Throughput Screening Assays / methods
  • Humans
  • Microscopy, Confocal / methods
  • Spheroids, Cellular / drug effects*
  • Spheroids, Cellular / pathology
  • Stress, Physiological / drug effects


  • Green Fluorescent Proteins