Three-dimensional HepaRG spheroids as a liver model to study human genotoxicity in vitro with the single cell gel electrophoresis assay

Abstract

Many efforts have been made in the last 30 years to develop more relevant in vitro models to study genotoxic responses of drugs and environmental contaminants. While 2D HepaRG cells are one of the most promising models for liver toxicology, a switch to 3D cultures that integrate both in vivo architecture and cell-cell interactions has occurred to achieve even more predictive models. Preliminary studies have indicated that 3D HepaRG cells are suitable for liver toxicity screening. Our study aimed to evaluate the response of HepaRG spheroids exposed to various genotoxic compounds using the single cell gel electrophoresis assay. HepaRG spheroids were used at 10 days after seeding and exposed for 24 and 48 hours to certain selected chemical compounds (methylmethansulfonate (MMS), etoposide, benzo[a]pyrene (B[a]P), cyclophosphamide (CPA), 7,12-dimethylbenz[a]anthracene (DMBA), 2-acetylaminofluorene (2-AAF), 4-nitroquinoline (4-NQO), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3-methylimidazo[4,5-f]quinolone (IQ), acrylamide, and 2-4-diaminotoluene (2,4-DAT)). After treatment, the comet assay was performed on single cell suspensions and cytotoxicity was determined by the ATP assay. Comet formation was observed for all compounds except IQ, etoposide and 2,4-DAT. Treatment of spheroids with rifampicin increased CYP3A4 activity, demonstrating the metabolic capacity of HepaRG spheroids. These data on genotoxicity in 3D HepaRG spheroids are promising, but further experiments are required to prove that this model can improve the predictivity of in vitro models to detect human carcinogens.

Figure 1

3D HepaRG spheroid culture and treatment schedule for the ATP and comet assays.

Figure 2

3D HepaRG spheroid. (A) HepaRG cells seeded at 2,000 cells per well in ULA 96-well plates. The spheroid-like structure was formed in 7 days. Scale bar = 100 µm. (B) Stability of size and morphology of spheroids from Day 10 to Day 21. Scale bar = 100 µm.

Figure 3

Cytotoxicity assay. Percentage of cell viability (ratio compared to negative control). Results were calculated from at least 3 independent experiments. *p < 0.05 (t-test).

Figure 4

Comet assay in 3D HepaRG spheroids. Level of DNA damage measured for 11 chemicals in 3D HepaRG spheroids with the comet assay. Blue point: % of tail DNA intensity (median value obtained in each experiment); red line: mean of medians of tail intensity. Results were calculated from at least 3 independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 (t-test).

Figure 5

Comparison of comet assay results obtained with 3D and 2D HepaRG cells. −:Negative result; +:positive result; LEC: lowest effecting concentration (mM) yielding a positive result in the assay, b highest concentration tested, c LEC was not determined in this model.