Micropatterned Coculture With 3T3-J2 Fibroblasts Enhances Hepatic Functions and Drug Screening Utility of HepaRG Cells
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Human liver models are useful for assessing compound metabolism/toxicity; however, primary human hepatocyte (PHH) lots are limited and highly variable in quality/viability. In contrast, cell lines, such as HepaRG, are cheaper and more reproducible surrogates for initial compound screening; however, hepatic functions and sensitivity for drug outcomes need improvement. Here, we show that HepaRGs cocultured with murine embryonic 3T3-J2 fibroblasts, previously shown to induce PHH functions, could address such limitations. We either micropatterned HepaRGs or seeded them “randomly” onto collagen-coated plates before 3T3-J2 coculture. Micropatterned cocultures (HepaRG-MPCCs) secreted 2- to 4-fold more albumin and displayed more stable cytochrome P450 activities than HepaRG conventional confluent monocultures (HepaRG-CCs) and HepaRG micropatterned hepatocytes (HepaRG-MPHs) for 4 weeks, even when excluding dimethyl sulfoxide from the medium. Furthermore, HepaRG-MPCCs had the most albumin-only positive cells (hepatic), lowest cytokeratin 19 (CK19)-only positive cells (cholangiocytic), and highest mean albumin intensity per cell than HepaRG random cocultures and monocultures; however, 80%–84% of HepaRGs remained bipotential (albumin+/CK19+) across all models. The 3T3-J2s also induced higher albumin in HepaRG spheroids than HepaRG-only spheroids. Additionally, although rifampin induced CYP3A4 in HepaRG-MPCCs and HepaRG-CCs, only HepaRG-MPCCs showed the dual omeprazole-mediated CYP1A2/3A4 induction as with PHHs. Lastly, when treated for 6 days with 47 drugs and evaluated for albumin and ATP to make binary hepatotoxicity calls, HepaRG-MPCCs displayed a sensitivity of 54% and specificity of 100% (70%/100% in PHH-MPCCs), whereas HepaRG-CCs misclassified several hepatotoxins. Ultimately, HepaRG-MPCCs could be a more cost-effective and reproducible model than PHHs for executing a tier 1 compound screen.
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Brenton et al. co-cultured murine embryonic fibroblasts with hepatocytes on collagen-micropatterned plates, achieving superior CYP450 stability, diminished keratin-19 expression, and maximal per-cell albumin output relative to conventional monolayers.
“Brenton et al. co-cultured murine embryonic fibroblasts with hepatocytes on collagen-micropatterned plates, achieving superior CYP450 stability, diminished keratin-19 expression, and maximal per-cell albumin output relative to conventional monolayers [218].”
From Constructing biomimetic microenvironments for liver regeneration (2025) · cites this paper as [218]
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- Authors
- Ware, B. R., Liu, J. S., Monckton, C. P., Ballinger, K. R., & Khetani, S. R.
- Affiliations
- University of Illinois Chicago (US)
- Colorado State University (US)
- Corresponding author
- Khetani, Salman R
- Venue
- Toxicological Sciences
- Publisher
- Oxford University Press (OUP)
- Published
- 2021-02-16
- Volume
- 181
- Issue
- 1
- Pages
- 90-104
- ISSN
- 1096-0929 (ISSN-L)
- 1096-6080
- OpenAlex
- W3131024762
- Cited by
- 25
- References
- 41
- Citations by year
- 2026: 2 · 2025: 5 · 2024: 5 · 2023: 6 · 2022: 2 · 2021: 5
- FWCI
- 4.12
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model (vor)
- Locations
- Toxicological Sciences (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Science Foundation (CBET-1351909, CBET-1933540)
- Topics
- Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Pharmacology › Drug-Induced Hepatotoxicity and Protection
- Physical Sciences › Engineering › Biomedical Engineering › 3D Printing in Biomedical Research
- Health Sciences › Medicine › Hepatology › Liver physiology and pathology
- Keywords
- HepaRG cells, hepatocyte differentiation, albumin secretion, cytochrome P450 induction, hepatotoxicity screening, drug screening, spheroids, cholangiocytes, CYP3A4, CYP1A2
- MeSH terms
- Animals, Drug Evaluation, Preclinical, Fibroblasts, Humans, Metabolic Clearance Rate, Coculture Techniques, Hepatocytes, Mice
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