Abstract
Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally infected chimpanzees, most in vitro models require several hundreds to thousands of viral genomes per cell in order to initiate a transient infection. Additionally, static 2D cultures of primary human hepatocytes (PHH) allow only short-term studies due to their rapid dedifferentiation. Here, we describe 3D liver-on-a-chip cultures of PHH, either in monocultures or in cocultures with other nonparenchymal liver-resident cells. These offer a significant improvement to studying long-term HBV infections with physiological host cell responses. In addition to facilitating drug efficacy studies, toxicological analysis, and investigations into pathogenesis, these microfluidic culture systems enable the evaluation of curative therapies for HBV infection aimed at eliminating covalently closed, circular (ccc)DNA. This presented method describes the set-up of PHH monocultures and PHH/Kupffer cell co-cultures, their infection with purified HBV, and the analysis of host responses. This method is particularly applicable to the evaluation of long-term effects of HBV infection, treatment combinations, and pathogenesis.
Highlights
The study of hepatitis B virus (HBV) has been complicated by the poor susceptibility of culture systems, requiring several hundreds to thousands of HBV genome copies per cell to initiate the infection[1]
This is underpinned by the necessity of using high concentrations of dimethyl sulfoxide (DMSO) and polyethylene glycol (PEG) to establish HBV infection in these cultures, which is dispensable for the infection of 3D liver-on-a-chip culture systems[4]
NOTE: For hepatocyte monocultures used as controls for cocultures, in order to ensure controlled conditions, a second type of maintenance medium is used, which is specific for use in the cocultures with primary human Kupffer cells. 48 h after replacing the hepatocyte seeding medium with hepatocyte maintenance medium, the regular hepatocyte maintenance medium will be replaced with coculture maintenance medium II, especially in monocultures of primary human hepatocytes (PHH) when comparing to cocultures of both PHH and Kupffer cells, as the medium components differ slightly
Summary
The study of HBV has been complicated by the poor susceptibility of culture systems, requiring several hundreds to thousands of HBV genome copies per cell to initiate the infection[1]. We describe the set-up and infection of PHH grown in 3D liver-on-a-chip cultures, which are vastly advantageous over conventional 2D static PHH cultures on collagen-coated plates due to their extended metabolic and functional competence, facilitating long-term cultures of at least 40 days[4]. Even though alternative culture systems for PHH based on complex cocultures of murine fibroblasts or 3D growth in spheroids have been validated and are susceptible to HBV infection using multiplicities of infection of 500 genome equivalents (GE) of HBV per cell, 3D liver-ona-chip cultures remain the sole in vitro model system susceptible to 0.05 GE of HBV per cell[4]. The extended culture period of this platform facilitates the evaluation of sequential drug treatments and their impact on HBV persistence, which are not possible using conventional hepatocyte culture systems This protocol describes how 3D liver-on-a-chip cultures are generated, either for monocultures of PHH or for cocultures of PHH with Kupffer cells. We describe the production of purified HBV for low-multiplicity-of-infection studies, as well as the subsequent analysis of host and viral responses
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