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

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 micropatterned HepaRG monocultures (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 (HepaRG-RCCs) 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, while 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.

The methylimidazolium ionic liquid M8OI is detectable in human sera and is subject to biliary excretion in perfused human liver

“…Accordingly, the ability of perfused tissue and hepatocytes in culture to fully model M8OI metabolism in man is always in question. These issues are also relevant to HepaRG cells, which show reduced xenobiotic metabolising enzyme responses in the simple culture model used in our studies (see Ware et al, 2021 ). Finally, it should be noted that the concentrations of M8OI employed in perfusions and hepatocyte incubations were in considerable excess to the serum concentration detected in some individuals.…”

Study on the relationship between liver damage caused by anti-tuberculosis drug rifampicin and related metabolic enzyme gene polymorphisms

“…Therefore, studies based on haplotypes are better than studies based on single SNPs. The research can bring greater effect [10]. RIF is a macromolecular drug, which is mainly excreted via bile, and its own liver toxicity is relatively low.…”

In vitro models for non-alcoholic fatty liver disease: Emerging platforms and their applications

“…(2003) ; Green et al. (2020) ; Wilkening et al (2003) 3T3-L1 MBX Mouse fibroblast Adipocyte differentiation Robust response to insulin Present lipid droplets Low proliferation rate ATCC ; Berger and Géloën (2019) 3T3-J2 Embryonic mouse fibroblast Expression of genes present in liver Use in multicellular aggregates to enhance liver functions Stabilisation of liver phenotype without NPC is possible Non-liver source Fibroblast density modulates hepatic function in dose-dependent manner Bhatia et al (1998) ; Ware et al (2021) Liver sinusoidal endothelial cells (LSEC) Primary human cell line High endocytosis capacity High permeability Long life-span Low availability Difficult isolation Low proliferation rate Rapid loss of differentiation German and Madihally (2019) ; Poisson et al. (2017) Human umbilical vein endothelial cells (HUVEC) Human non-pathological tissue Generation of vascular-like endothelial network when cultured with hepatocytes in spheroids Easy access Established protocols for tissue culture in a serum-free medium Non-liver source Less migration of lymphocytes than LSEC Reduced viability when co-cultured with HepaRG than LSEC German and Madihally (2019) ; Medina-Leyte et al.…”