Recently, exciting clinical progress has been made in the study of hepatotropic pathogens in the context of liver-dependent infectious diseases. Tissue engineering has been applied to authentically recapitulate human liver biology, facilitating the study of host-pathogen interactions during the entire pathogen life cycle. This is crucial for the development and validation of therapeutic interventions, such as drug and vaccine candidates that may act on the liver cells. The engineered models range from two-dimensional (2-D) cultures of primary human hepatocytes (HH) and stem cell–derived progeny to three-dimensional (3-D) organoid cultures and humanized rodent models. A review by Nil Gural and colleagues, published in Cellular and Molecular Gastroenterology and Hepatology , described these unique models. Furthermore, the progress made in combining individual approaches and pairing the most appropriate model system and readout modality was discussed.

The major human hepatotropic pathogens include hepatitis C virus (HCV), hepatitis B virus (HBV), and the protozoan parasites Plasmodium falciparum and P. vivax. While HBV and HCV can cause chronic liver diseases such as cirrhosis and hepatocellular carcinoma, Plasmodium parasites cause malaria. The use of cancer cell lines and animal models to study host-pathogen interactions is limited by uncontrolled proliferation, abnormal liver-specific functions, and stringent host dependency of the hepatotropic pathogens. HHs are thus the only ideal system to study these pathogens, however, maintaining these cells ex vivo is challenging.

For instance, 2D monolayers of human hepatoma-derived cell lines (such as HepG2-A16 and HepaRG) are easier to maintain, to amplify for scaling up, and to use for drug screening, thus representing a renewable alternative to primary hepatocytes. These model systems have been useful to study short-term infections of human Plasmodium parasites (P. vivax and P. falciparum); other hepatotropic pathogens such as Ebola, Lassa, human cytomegalovirus, and dengue viruses; and to generate virion stocks (HCV, HBV). For long-term scientific analyses and cultures, as well as clinical isolates of pathogens that do not infect hepatoma cells, immortalized cell lines have been engineered to differentiate and maintain HH functions for a longer duration. Additionally, cocultivation of primary hepatocytes with nonparenchymal cells or hepatocytes with mouse fibroblasts preserves hepatocyte phenotype. The latter is a self-assembling coculture system that could potentially maintain an infection for over 30 days and be used for testing anti-HBV drugs. A micropatterned coculture system, in which hepatocytes are positioned in “islands” via photolithographic patterning of collagen, surrounded by mouse embryonic fibroblasts, can maintain hepatocyte phenotypes for 4-6 weeks, and remain permissive to P. falciparum, P. vivax, HBV, and HCV infections. Furthermore, micropatterned coculture systems support full developmental liver stages of both P. falciparum and P. vivax, with the release of merozoites from hepatocytes and their subsequent infection of overlaid human red blood cells.

Alternatively, embryonic stem cells and induced pluripotent stem cells of human origin can be differentiated into hepatocytelike cells that enable investigation of host genetics within the context of host-pathogen interactions, and can also be used for target identification for drug development. However, stem cell cultures require significant culture expertise and may not represent a fully differentiated adult hepatocyte phenotype.

Although 2D cultures offer ease of use and monitoring of infection, they often lack the complexity of the liver microenvironment and impact of different cell types on liver infections. A 3D radial-flow bioreactor (cylindrical matrix) was able to maintain and amplify human hepatoma cells (for example, Huh7 cells), by providing sufficient oxygen and nutrient supply, supporting productive HCV infection for months. Other 3D cultures of hepatoma cells using polyethylene glycol–based hydrogels, thermoreversible gelatin polymers, alginate, galactosylated cellulosic sponges, matrigel, and collagen have been developed and shown to be permissive to HCV or HBV infections. Although 3D coculture systems exhibit better hepatic function and differential gene expression profiles in comparison to 2D counterparts, they require a large quantity of cells and are a challenge to scale up. Recently, several liver-on-a-chip models have been created that mimic shear stress, blood flow, and the extracellular environment within a tissue, holding great potential for modeling liver-specific pathogens.

Humanized mouse models with ectopic human liver structures have been developed in which primary HHs are transplanted following liver injury. Chimeric mouse models including Alb-uPA/SCID (HHs transplanted into urokinase-type plasminogen activator-transgenic severe combined immunodeficient mice), FNRG/FRG (HHs transplanted into Fah[-/-], Rag2[-/-], and Il2rg[-/-] mice with or without a nonobese diabetic background), and TK-NOG (HHs transplanted into herpes simplex virus type-1 thymidine kinase mice) were validated for HCV, HBV, P. falciparum, and P. vivax infections. It is, however, laborious to create and maintain chimeric mouse models and monitor infection processes in them.

It is important to note that the selection of model system and the readout modality to monitor infection will vary based on the experimental question at hand. Tissue engineering has thus far made significant contributions to the knowledge of hepatotropic pathogens; a continued effort to develop better liver models is envisioned.