Hepatitis B virus causes chronic infections in 250 million people worldwide. Chronic hepatitis B virus carriers are at risk of developing fibrosis, cirrhosis, and hepatocellular carcinoma. A prophylactic vaccine exists and currently available antivirals can suppress but rarely cure chronic infections. The study of hepatitis B virus and development of curative antivirals are hampered by a scarcity of models that mimic infection in a physiologically relevant, cellular context. Here, we show that cell-culture and patient-derived hepatitis B virus can establish persistent infection for over 30 days in a self-assembling, primary hepatocyte co-culture system. Importantly, infection can be established without antiviral immune suppression, and susceptibility is not donor dependent. The platform is scalable to microwell formats, and we provide proof-of-concept for its use in testing entry inhibitors and antiviral compounds.

In this study, a new 3D liver model was developed using biomimetic nanofiber scaffolds and co-culture system consisting of hepatocytes and fibroblasts for the maintenance of long-term liver functions. The chitosan nanofiber scaffolds were fabricated by the electrospinning technique. To enhance cellular adhesion and spreading, the surfaces of the chitosan scaffolds were coated with fibronectin (FN) by adsorption and evaluated for various cell types. Cellular phenotype, protein expression, and liver-specific functions were extensively characterized by immunofluorescent and histochemical stainings, albumin enzyme-linked immunosorbent assay and Cytochrome p450 detoxification assays, and scanning electron microscopy. The electrospun chitosan scaffolds exhibited a highly porous and randomly oriented nanofibrous structure. The FN coating on the surface of the chitosan nanofibers significantly enhanced cell attachment and spreading, as expected, as surface modification with this cell adhesion molecule on the chitosan surface is important for focal adhesion formation and integrin binding. Comparison of hepatocyte mono-cultures and co-cultures in 3D culture systems indicated that the hepatocytes in co-cultures formed colonies and maintained their morphologies and functions for prolonged periods of time. The 3D liver tissue model developed in this study will provide useful tools toward the development of engineered liver tissues for drug screening and tissue engineering applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2119-2128, 2017.

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Hepatitis B virus (HBV) remains a major global health problem with 257 million chronically infected individuals worldwide, of whom approximately 20 million are co‐infected with hepatitis delta virus (HDV). Progress toward a better understanding of the complex interplay between these two viruses and the development of novel therapies have been hampered by the scarcity of suitable cell culture models that mimic the natural environment of the liver. Here, we established HBV and HBV/HDV co‐infections and super‐infections in self‐assembling co‐cultured primary human hepatocytes (SACC‐PHHs) for up to 28 days in a 384‐well format and highlight the suitability of this platform for high‐throughput drug testing. We performed RNA sequencing at days 8 and 28 on SACC‐PHHs, either HBV mono‐infected or HBV/HDV co‐infected. Our transcriptomic analysis demonstrates that hepatocytes in SACC‐PHHs maintain a mature hepatic phenotype over time, regardless of infection condition. We confirm that HBV is a stealth virus, as it does not induce a strong innate immune response; rather, oxidative phosphorylation and extracellular matrix–receptor interactions are dysregulated to create an environment that promotes persistence. Notably, HDV co‐infection also did not lead to statistically significant transcriptional changes across multiple donors and replicates. The lack of innate immune activation is not due to SACC‐PHHs being impaired in their ability to induce interferon stimulated genes (ISGs). Rather, polyinosinic:polycytidylic acid exposure activates ISGs, and this stimulation significantly inhibits HBV infection, yet only minimally affects the ability of HDV to infect and persist. Conclusion: These data demonstrate that the SACC‐PHH system is a versatile platform for studying HBV/HDV co‐infections and holds promise for performing chemical library screens and improving our understanding of the host response to such infections.

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