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Three-Dimensional (3D) HepaRG Spheroid Model with Physiologically Relevant Xenobiotic Metabolism Competence and Hepatocyte Functionality for Liver Toxicity Screening

Sreenivasa C. Ramaiahgari, Suramya Waidyanatha, Darlene Dixon, Michael J. DeVito, Richard S. Paules, Stephen S. Ferguson.
Toxicological Sciences (2017) DOI: PMID: 28633424



Effective prediction of human responses to chemical and drug exposure is of critical importance in environmental toxicology research and drug development. While significant progress has been made to address this challenge using invitro liver models, these approaches often fail due to inadequate tissue model functionality. Herein, we describe the development, optimization, and characterization of a novel three-dimensional (3D) spheroid model using differentiated HepaRG cells that achieve and maintain physiologically relevant levels of xenobiotic metabolism (CYP1A2, CYP2B6, and CYP3A4/5). This invitro model maintains a stable phenotype over multiple weeks in both 96- and 384-well formats, supports highly reproducible tissue-like architectures and models pharmacologically- and environmentally important hepatic receptor pathways (ie AhR, CAR, and PXR) analogous to primary human hepatocyte cultures. HepaRG spheroid cultures use 50–100× fewer cells than conventional two dimensional cultures, and enable the identification of metabolically activated toxicants. Spheroid size, time in culture and culture media composition were important factors affecting basal levels of xenobiotic metabolism and liver enzyme inducibility with activators of hepatic receptors AhR, CAR and PXR. Repeated exposure studies showed higher sensitivity than traditional 2D cultures in identifying compounds that cause liver injury and metabolism-dependent toxicity. This platform combines the well-documented impact of 3D culture configuration for improved tissue functionality and longevity with the requisite throughput and repeatability needed for year-over-year toxicology screening.


Figure 1. Morphology of HepaRG spheroids.

HepaRG cells seeded at 1000 cells per well form spheroids-like structures. Single spheroid per well in a 384-well plate (A); size of the spheroid is approximately 191.97 µm (B).

Figure 2. Immunohistochemical analysis of HepaRG spheroids.

HepaRG spheroids stained for H&E (haematoxylin and eosin) stain, PAS (periodic Schiff’s), Poly CEA (carcinoembryonic antigen), MRP2 transporter, cytokeratin 19 and CYP3A4 (Scale bar 100 µm). Merged image of synthetic bile acid analogue, cholyl-lysyl-fluorescein (CLF) (green) and nuclei (blue) in HepaRG spheroid (Scale bar 100 µm).

Figure 3. MRP2 localization in HepaRG spheroids.

Immunofluorescent staining of MRP2 (green) and nuclei (blue) on HepaRG spheroids (scale bar 100 µm).

Figure 4. CYP450 enzyme activity in 2D HepaRG cells, HepaRG spheroids and primary human hepatocytes.

LC-MS/MS analysis of metabolite formation rate of acetaminophen (CYP1A2); 1-hydroxybupropion (CYP2B6) and 1-hydroxymidazolam (CYP3A4). Data were the mean of three independent experiments (± SEM) from 10-day sandwich-cultured 2D HepaRG cells and 21-day cultured 3D HepaRG spheroids. Data from primary suspension cultures were from 212, 137 and 132 individual donor preparations for CYP1A2, CYP2B6 and CY3A4/5, respectively, and 52 individual donor preparations for 4-day sandwich–cultured primary hepatocytes.

Figure 5. AhR, CAR, PXR induction assay.

HepaRG spheroid cultures were treated with Prototypical inducers if AhR (Omeprazole); CAR (Phenobarbital) and PXR (Rifampin) for 72 h with fresh addition of compound every 24 h. Data were normalized to GAPDH, representative of two independent experiments (± SD).

Figure 6. Metabolism dependent toxicity in HepaRG spheroids.

HepaRG spheroid cultures at day 21 were treated with aflatoxin B1 in the presence and absence of 1 mM 1-Aminobenzotriazole (1-ABT) and cell viability was assessed after 24 h. Data are the means of three independent experiments ± SD.

Figure 7. Assessment of chemical induced toxicity.

24-h period single exposures on 2D HepaRG cells cultured for 10 days (▲); 3D HepaRG spheroids cultured for 21 days (•) and primary human hepatocytes cultured for 24 h (▼). Additionally, repeated dosing was performed on 3D HepaRG spheroids at days 22, 24 and 26 (▪) and cell viability assessed on day 28. Data are presented as the relative percentage cell viability compared with its vehicle-treated controls. Data are the means of three independent experiments ± SD.

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Toxicological Sciences (2017) DOI: PMID: 29077947