Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures

Abstract

Hepatitis C virus (HCV) remains a major public health problem, affecting approximately 130 million people worldwide. HCV infection can lead to cirrhosis, hepatocellular carcinoma, and end-stage liver disease, as well as extrahepatic complications such as cryoglobulinemia and lymphoma. Preventative and therapeutic options are severely limited; there is no HCV vaccine available, and nonspecific, IFN-based treatments are frequently ineffective. Development of targeted antivirals has been hampered by the lack of robust HCV cell culture systems that reliably predict human responses. Here, we show the entire HCV life cycle recapitulated in micropatterned cocultures (MPCCs) of primary human hepatocytes and supportive stroma in a multiwell format. MPCCs form polarized cell layers expressing all known HCV entry factors and sustain viral replication for several weeks. When coupled with highly sensitive fluorescence- and luminescence-based reporter systems, MPCCs have potential as a high-throughput platform for simultaneous assessment of in vitro efficacy and toxicity profiles of anti-HCV therapeutics.

Fig. 1.

Primary human hepatocytes in MPCCs form polarized cell layers, express HCV entry factors, and support HCV glycoprotein-mediated entry. Bright field images of primary hepatocytes in MPCCs (A) and in monocultures (B). Wide-field fluorescence images of fixed MPCCs stained for the canilicular marker MRP2 (C), and the basolateral marker CD26 (D). Nuclear (blue) and antigen-specific staining (green) for CD81 (E), SCARB1 (near edge of hepatocyte island) (F), CLDN1 (red) (G), OCLN (H) in MPCCs. (I) Merged image of primary hepatocytes stained for MRP2 (green), ZO1 (red), and nuclei (blue). (J) 3D rendering of boxed area in I. (K) Infection of MPCCs with retroviral pseudoparticles bearing HCV glycoproteins (HCVpp), vesicular stomatitis virus glycoprotein (VSVGpp), or no glycoproteins (Env^-pp) and containing an EGFP reporter gene. Representative images are shown for all experiments. (L) Anti-CD81 antibody blocks entry of HCVpp (dark bars), but not VSVGpp (white bars). Concentrations of antibody (μg/mL) are noted. Mean and SD are shown. Scale bars: 100 μm (a, b, k), 50 μm (c, d), 20 μm (e-i).

Fig. 2.

Primary human hepatocyte MPCCs are susceptible to HCV. (A) Persistent infection of primary human hepatocytes with HCVcc. Primary hepatocytes in MPCC were infected with Jc1FLAG2(p7-nsGluc2A). After 24 h, virus was removed and MPCC medium containing DMSO (0.1%) or the indicated inhibitors was added. All inhibitors were used at approximately 50× IC50 (polymerase inhibitor 2’CMA = 2.16 mM, protease inhibitor ITMN191 = 0.16 mM, IFN-α = 500 U/mL). Samples were taken daily and the media replaced with washing every 48 h. Accumulated luciferase activity in the supernatants is plotted. Arrows indicate the addition of fresh inhibitor. (B) Visualization of HCV infection in primary human hepatocytes. MPCCs were transduced with lentiviruses expressing wild-type (wt) or mutant (C508Y) RFP-NLS-IPS HCV reporter. Twenty-four hours after transduction, MPCCs were infected with Jc1FLAG2(p7-nsGluc2A) or plasma from HCV-infected patients in the presence of heparin (5 IU), CaCl₂ (9 mM), and MgCl₂ (6 mM). Twelve hours following infection, virus was removed and MPCC medium was added. Unfixed MPCCs were imaged by wide-field fluorescence microscopy at 48 h postinfection. Representative pseudocolored fluorescent images are shown; white arrow heads show nuclear RFP, indicative of HCV infection. Scale bar: 20 μm.

Fig. 3.

Utility of primary human hepatocyte MPCCs in antibody and small molecule screening. (A) Dose-dependent inhibition of HCVcc replication in MPCCs treated with antibodies against HCV glycoproteins (AP33, 3/11, CBH5, AR3A) or cellular CD81 (JS-81). Antibody concentrations are 0.1 (light gray), 1 (dark gray), and 10 (black) μg/mL (B) Dose-dependent inhibition of HCVcc replication in MPCCs treated with IFN-α (up to 0.13 μM) or small molecules (NS3-4A protease inhibitors, BILN2061 and ITMN191, or polymerase inhibitor, 2’CMA). HCVcc-infected MPCCs were pulse-treated for 2 days with compounds and supernatants were collected at days 2 and 4 (shown) postinhibitor treatment. (C) Drug-drug interactions lead to reduced efficacy of small molecules in HCVcc-infected MPCCs. Infected MPCCs were treated for 3 days with prototypical inducers of drug metabolism enzymes (12, 32), followed by treatment of cultures with small molecules for 2 days. In all experiments, HCVcc replication was monitored by luciferase secretion into the supernatants. Mean and standard error of the mean are shown.