Micropatterned Coculture With 3T3-J2 Fibroblasts Enhances Hepatic Functions and Drug Screening Utility of HepaRG Cells

Brenton R Ware; Jennifer S Liu; Chase P Monckton; Kimberly R Ballinger; Salman R Khetani

doi:10.1093/toxsci/kfab018

Micropatterned Coculture With 3T3-J2 Fibroblasts Enhances Hepatic Functions and Drug Screening Utility of HepaRG Cells

Toxicol Sci. 2021 Apr 27;181(1):90-104. doi: 10.1093/toxsci/kfab018.

Authors

Brenton R Ware^{1

2}, Jennifer S Liu², Chase P Monckton², Kimberly R Ballinger^{1

3}, Salman R Khetani^{1

2

3}

Affiliations

¹ School of Biomedical Engineering, Colorado State University, Fort Collins, Colorado 80523, USA.
² Department of Bioengineering, University of Illinois at Chicago, Chicago, Illinois 60607, USA.
³ Department of Mechanical Engineering, Colorado State University, Fort Collins, Colorado 80523, USA.

PMID: 33590212
DOI: 10.1093/toxsci/kfab018

Abstract

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.

Keywords: CYP induction; DMSO; drug-induced liver injury; toxicity screening.

Publication types

Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Animals
Coculture Techniques
Drug Evaluation, Preclinical
Fibroblasts*
Hepatocytes*
Humans
Metabolic Clearance Rate
Mice