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A 3D in vitro model of differentiated HepG2 cell spheroids with improved liver-like properties for repeated dose high-throughput toxicity studies

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Abstract

Immortalized hepatocyte cell lines show only a weak resemblance to primary hepatocytes in terms of gene expression and function, limiting their value in predicting drug-induced liver injury (DILI). Furthermore, primary hepatocytes cultured on two-dimensional tissue culture plastic surfaces rapidly dedifferentiate losing their hepatocyte functions and metabolic competence. We have developed a three-dimensional in vitro model using extracellular matrix-based hydrogel for long-term culture of the human hepatoma cell line HepG2. HepG2 cells cultured in this model stop proliferating, self-organize and differentiate to form multiple polarized spheroids. These spheroids re-acquire lost hepatocyte functions such as storage of glycogen, transport of bile salts and the formation of structures resembling bile canaliculi. HepG2 spheroids also show increased expression of albumin, urea, xenobiotic transcription factors, phase I and II drug metabolism enzymes and transporters. Consistent with this, cytochrome P450-mediated metabolism is significantly higher in HepG2 spheroids compared to monolayer cultures. This highly differentiated phenotype can be maintained in 384-well microtiter plates for at least 28 days. Toxicity assessment studies with this model showed an increased sensitivity in identifying hepatotoxic compounds with repeated dosing regimens. This simple and robust high-throughput-compatible methodology may have potential for use in toxicity screening assays and mechanistic studies and may represent an alternative to animal models for studying DILI.

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Acknowledgments

We thank the staff at pathology department of the Bronovo hospital for their technical assistance in performing histological analysis. This research was funded by the Netherlands Toxicogenomics Center (NTC) through support of the Netherlands Genomics Initiative.

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Authors and Affiliations

  1. Division of Toxicology, Leiden Academic Centre for Drug Research, Leiden University, Einsteinweg 55, P.O. Box 9502, 2300 RA, Leiden, The Netherlands

    Sreenivasa C. Ramaiahgari, Bram Herpers, Bob van de Water & Leo S. Price

  2. Division of Molecular Toxicology, AIMMS, Vrije University Amsterdam, Amsterdam, The Netherlands

    Michiel W. den Braver & Jan N. M. Commandeur

  3. Department of Pathology, Bronovo Hospital, Hague, The Netherlands

    Valeska Terpstra

  4. OcellO B.V., Leiden, The Netherlands

    Bram Herpers & Leo S. Price

Authors
  1. Sreenivasa C. Ramaiahgari
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  2. Michiel W. den Braver
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  3. Bram Herpers
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  4. Valeska Terpstra
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Corresponding authors

Correspondence to Bob van de Water or Leo S. Price.

Electronic supplementary material

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204_2014_1215_MOESM1_ESM.pdf

Schematic representation of 3D HepG2 spheroid culture. (A) Matrigel was added to the plates using the Cybi-Selma semiautomatic pipettor. A 96-well mother plate was prepared by manually pipetting Matrigel into the wells. Plates were then incubated for 30–45 min at 37°C for gelation before adding the required number of cells. (B) Number and area of spheroids after 28 days in 3D cell cultures (PDF 2137 kb)

204_2014_1215_MOESM2_ESM.tif

Urea production in 2D and 3D HepG2 cells. Data normalized to 6X104 cells. Data are representative of 2 independent experiments (TIFF 3598 kb)

Primer sequences used for real-time PCR analysis (PDF 77 kb)

Protocol used to analyze phase I metabolites at Pharmacelsus GmBH (DOCX 14 kb)

204_2014_1215_MOESM5_ESM.tif

Biotransformation of testosterone in 2D and 3D HepG2 cultures. Extracted ion chromatogram of androstenedione (m/z 287,20) in a 72-h 3D sample (A). Time curves of androstenedione formation after exposure of 2D/3D cell cultures, (B). Data are representative of two independent experiments. Extracted ion chromatogram of hydroxylated testosterone (m/z 305, 19) in a 72hr 3D sample, (C) Time curves of 1-β and 6β-hydroxy-testosterone formation and (D and E) after exposure. Quantification is based on UV and corrected for background. Data are representative of two independent experiments (TIFF 6531 kb)

Protocol used to analyze testosterone metabolism and phase II metabolism (Glucuronidation and sulfation) (DOC 28 kb)

List of compounds used for toxicity assessment (PDF 16 kb)

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Ramaiahgari, S.C., den Braver, M.W., Herpers, B. et al. A 3D in vitro model of differentiated HepG2 cell spheroids with improved liver-like properties for repeated dose high-throughput toxicity studies. Arch Toxicol 88, 1083–1095 (2014). https://doi.org/10.1007/s00204-014-1215-9

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