Name: Liver-on-a-Chip Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection (Video) | JoVE Journal
Uploaded: 2019-02-19T13:00:00.000+00:00
Duration: 10 min 25 s
Description: Learn liver-on-a-chip culture of primary hepatocytes and Kupffer cells for hepatitis B virus infection studies. This 3D microfluidic platform enables longitudinal infection kinetics and drug testing in a physiological hepatic environment.

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5 minutes

This work was funded by a Starter grant from the European Research Council (637304), a Wellcome Trust Investigator Award (104771/Z/14/Z), and by CN Bio Innovations.

<ol><li>Verrier, E. R., Colpitts, C. C., Schuster, C., Zeisel, M. B., Baumert, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+Culture+Models+for+the+Investigation+of+Hepatitis+B+and+D+Virus+Infection%5BTitle%5D)+AND+%22Viruses%22%5BJournal%5D)">Cell Culture Models for the Investigation of Hepatitis B and D Virus Infection.</a> Viruses. 8 (9), (2016).</li>
<li>Elaut, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+mechanisms+underlying+the+dedifferentiation+process+of+isolated+hepatocytes+and+their+cultures%5BTitle%5D)+AND+%22Current+Drug+Metabolism%22%5BJournal%5D)">Molecular mechanisms underlying the dedifferentiation process of isolated hepatocytes and their cultures.</a> Current Drug Metabolism. 7 (6), 629-660 (2006).</li>
<li>Konig, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kinetics+of+the+bile+acid+transporter+and+hepatitis+B+virus+receptor+Na%2B%2Ftaurocholate+cotransporting+polypeptide+%28NTCP%29+in+hepatocytes%5BTitle%5D)+AND+%22Journal+of+Hepatology%22%5BJournal%5D)">Kinetics of the bile acid transporter and hepatitis B virus receptor Na+/taurocholate cotransporting polypeptide (NTCP) in hepatocytes.</a> Journal of Hepatology. 61 (4), 867-875 (2014).</li>
<li>Ortega-Prieto, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((3D+microfluidic+liver+cultures+as+a+physiological+preclinical+tool+for+hepatitis+B+virus+infection%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">3D microfluidic liver cultures as a physiological preclinical tool for hepatitis B virus infection.</a> Nature Communications. 9 (1), 682(2018).</li>
<li>Allweiss, L., Dandri, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Role+of+cccDNA+in+HBV+Maintenance%5BTitle%5D)+AND+%22Viruses%22%5BJournal%5D)">The Role of cccDNA in HBV Maintenance.</a> Viruses. 9 (6), (2017).</li>
<li>Guo, J. T., Guo, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metabolism+and+function+of+hepatitis+B+virus+cccDNA%3A+Implications+for+the+development+of+cccDNA-targeting+antiviral+therapeutics%5BTitle%5D)+AND+%22Antiviral+Research%22%5BJournal%5D)">Metabolism and function of hepatitis B virus cccDNA: Implications for the development of cccDNA-targeting antiviral therapeutics.</a> Antiviral Research. 122, 91-100 (2015).</li>
<li>Lucifora, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+antiviral+properties+of+TLR+ligands+against+HBV+replication+in+immune-competent+hepatocytes%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Direct antiviral properties of TLR ligands against HBV replication in immune-competent hepatocytes.</a> Scientific Reports. 8 (1), 5390(2018).</li>
<li>Hosel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hepatitis+B+Virus+Activates+Signal+Transducer+and+Activator+of+Transcription+3+Supporting+Hepatocyte+Survival+and+Virus+Replication%5BTitle%5D)+AND+%22Cellular+and+Molecular+Gastroenterology+and+Hepatology%22%5BJournal%5D)">Hepatitis B Virus Activates Signal Transducer and Activator of Transcription 3 Supporting Hepatocyte Survival and Virus Replication.</a> Cellular and Molecular Gastroenterology and Hepatology. 4 (3), 339-363 (2017).</li>
<li>Mazza, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+production+of+human+liver+scaffolds+for+functional+tissue+engineering+by+high+shear+stress+oscillation-decellularization%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Rapid production of human liver scaffolds for functional tissue engineering by high shear stress oscillation-decellularization.</a> Scientific Reports. 7 (1), 5534(2017).</li>
<li>Hang, T. C., Lauffenburger, D. A., Griffith, L. G., Stolz, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lipids+promote+survival%2C+proliferation%2C+and+maintenance+of+differentiation+of+rat+liver+sinusoidal+endothelial+cells+in+vitro%5BTitle%5D)+AND+%22American+Journal+of+Physiology-Gastrointestinal+and+Liver+Physiology%22%5BJournal%5D)">Lipids promote survival, proliferation, and maintenance of differentiation of rat liver sinusoidal endothelial cells in vitro.</a> American Journal of Physiology-Gastrointestinal and Liver Physiology. 302 (3), G375-G388 (2012).</li>
<li>Hwa, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rat+liver+sinusoidal+endothelial+cells+survive+without+exogenous+VEGF+in+3D+perfused+co-cultures+with+hepatocytes%5BTitle%5D)+AND+%22The+FASEB+Journal%22%5BJournal%5D)">Rat liver sinusoidal endothelial cells survive without exogenous VEGF in 3D perfused co-cultures with hepatocytes.</a> The FASEB Journal. 21 (10), 2564-2579 (2007).</li>
<li>Khetani, S. R., Bhatia, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microscale+culture+of+human+liver+cells+for+drug+development%5BTitle%5D)+AND+%22Nature+Biotechnology%22%5BJournal%5D)">Microscale culture of human liver cells for drug development.</a> Nature Biotechnology. 26 (1), 120-126 (2008).</li>
<li>March, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Micropatterned+coculture+of+primary+human+hepatocytes+and+supportive+cells+for+the+study+of+hepatotropic+pathogens%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Micropatterned coculture of primary human hepatocytes and supportive cells for the study of hepatotropic pathogens.</a> Nature Protocols. 10 (12), 2027-2053 (2015).</li>
<li>Bell, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+primary+human+hepatocyte+spheroids+as+a+model+system+for+drug-induced+liver+injury%2C+liver+function+and+disease%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Characterization of primary human hepatocyte spheroids as a model system for drug-induced liver injury, liver function and disease.</a> Scientific Reports. 6, 25187(2016).</li>
<li>Tong, W. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Constrained+spheroids+for+prolonged+hepatocyte+culture%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Constrained spheroids for prolonged hepatocyte culture.</a> Biomaterials. 80, 106-120 (2016).</li>
<li>Griffith, L. G., Wells, A., Stolz, D. B. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4176555/">Engineering liver.</a> Hepatology. 60 (4), 1426-1434 (2014).</li>
<li>Domansky, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Perfused+multiwell+plate+for+3D+liver+tissue+engineering%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">Perfused multiwell plate for 3D liver tissue engineering.</a> Lab Chip. 10 (1), 51-58 (2010).</li>
</ol>

The authors have nothing to disclose.

The challenges in maintaining long-term cultures of PHH have driven the development of several culture models with increased functionality and longevity, each exhibiting differential advantages and disadvantages. It is now widely acknowledged that static 2D cultures of PHH are mimicking certain aspects of hepatocyte biology for very limited amounts of time. Thus, micropatterned cocultures12,13, spheroid cultures14,15, and 3D liver-on-a-chip cultures16,17 are rapidly replacing these more basic systems. Especially when studying infectious diseases, which have coevolved with their host to utilize specific microenvironments, the requirement for providing physiological environments is underpinned by the often challenging nature of culturing human-tropic infectious diseases, including hepatitis C virus, HBV, and malaria.
The most critical step in performing 3D liver-on-a-chip cultures is the quality of the initially sourced primary cell types. These cells should be tested for their adherence capacity and only plateable PHH lots should be used in order to ensure successful tissue formation and culture generation. Even though freshly isolated PHH can be used, their cryopreservation is usually complicated and requires special rate-controlled freezers.
In contrast to conventional static 2D cultures, the host genetic background is negligible in regard to susceptibility to HBV infection, and all thus-far tested hepatocyte donors are able to establish HBV infection4.
Even though patient-derived HBV establishes infections of 3D cultures, it is imperative to utilize PEG-precipitated and sucrose cushion-purified HBV whenever using inducible HBV producer cell lines for the generation of viral inocula. Cell culture supernatants directly applied to 3D liver-on-a-chip cultures, either through the presence of inhibitory factors or due to an incompatibility of present growth factors with hepatocytes, do not readily result in infection. Additionally, when selecting patient-derived viral inocula, only serum should be used, since plasma inevitably coagulates and clogs the microfluidic circulation of the culture platform.
Irrespective of the viral inoculum used, assuring cellular viability and differentiation, as well as ensuring complete removal of the initial HBV inoculum, is key to successful long-term infection studies. The most convenient way to do this is sampling cultures following the removal of the viral inoculum, as well as measuring human serum albumin levels throughout the culture period. Of note, similarly to all other described platforms, HBV infection, once established, does not readily spread to uninfected cells. The underlying mechanism for this remains elusive since HBV infection in vivo readily infects the majority of the hepatocytes within the liver.
In regard to cocultures of PHH and Kupffer cells, it is advisable to perform lot tests of Kupffer cells to evaluate IL6 and TNF&#945; secretion in response to LPS stimulation, since not all commercially available Kupffer cell donors have an equal responsiveness.
Importantly, for all drug treatments or initial infection of cultures with HBV, the total volume of the well (1.4 mL), as well as of the microfluidic channel (0.2 mL), must be taken into account for the calculation of drug or inoculum concentrations. In order to assure accurate dosing, one washing step with medium containing HBV or drugs is performed in order to prime the microfluidic channel.
The platform used utilizes 600,000 PHH per well, which ensures multilayered hepatocytes within the scaffolds. Even though the cell number can be varied, the chosen cell concentration ensures optimal results. The plate format holds a total of 12 scaffolds, which can be upgraded to 36 scaffolds. However, due to microfluidic requirements, scaling up to higher well numbers is not possible to date.
Using these approaches, cultures can be maintained with optimal cell performance for at least 40 days, which, thus far, offers unprecedented opportunities to evaluate novel drug candidates, as well as study the complex interplay between different hepatic cell populations during HBV infection.

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 infection1. Furthermore, primary human hepatocytes are generally exceptionally fragile and rapidly dedifferentiate during conventional cultures2. This is mainly due to the fact that the flat and hard plastic surfaces do not mimic the natural extracellular environments found within the liver and the general lack of oxygenation of the cultures in the absence of microfluidic circulation. Conventional static hepatocyte cultures on collagen-coated plates rapidly dedifferentiate and lose their susceptibility to HBV infection3. Here, 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 days4. In this system, PHH are seeded on collagen-coated scaffolds, which are continually perfused with growth medium to supply oxygen and nutrients to the cells. 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-on-a-chip cultures remain the sole in vitro model system susceptible to 0.05 GE of HBV per cell4. This is additionally 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 systems4. Among the major hallmarks of HBV infection is the cccDNA pool, which acts as the transcriptional template for all de novo-produced virions5,6. Even though cccDNA can be detected in conventional hepatocyte cultures7,8, it remains unclear as to whether the regulation of cccDNA and any therapeutic approaches aimed at its elimination are recapitulated in partially or completely dedifferentiated hepatocytes. We have shown that cccDNA is functionally formed in 3D liver-on-a-chip cultures, responds to physiological stimuli, and can be targeted by interfering with the accessibility of the transcriptional machinery to the cccDNA genome4.
Host responses to HBV infection in 3D liver-on-a-chip mimic those observed in HBV-infected patients, enabling the identification of biomarkers for infection, as well as therapeutic success. Among the unique features of liver-on-a-chip cultures is the ability to evaluate long-term host responses between PHH and other nonparenchymal cells within the liver, including Kupffer cells4, stellate cells9, and liver sinusoidal endothelial cells10,11. This offers the unique opportunity to evaluate cell/cell interactions in a complex 3D microenvironment.
Additionally, the extended culture period of this&#160;platform&#160;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. Furthermore, 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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culture</td><td>SIGMA</td><td>12106C</td><td></td></tr><tr><td>Hydrocortisone</td><td>SIGMA</td><td>H0888</td><td></td></tr><tr><td>Trypan blue</td><td>Merck</td><td>T8154</td><td></td></tr><tr><td>Collagen from calf skin</td><td>Merck</td><td>C9791</td><td></td></tr><tr><td>G418</td><td>SIGMA</td><td>G418-RO</td><td></td></tr><tr><td>Tetracycline</td><td>SIGMA</td><td>T3258</td><td></td></tr><tr><td>Polyethylene glycol 8000</td><td>SIGMA</td><td>P2139</td><td></td></tr><tr><td>Sucrose</td><td>SIGMA</td><td>SO389</td><td></td></tr><tr><td>Sodium carbonate anhydrous</td><td>SIGMA</td><td>451614-25G</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>SIGMA</td><td>S5761</td><td></td></tr><tr><td>Sodium azide</td><td>SIGMA</td><td>S2002-5G</td><td></td></tr><tr><td>Sulfuric acid, 99.999%</td><td>SIGMA</td><td>339741</td><td></td></tr><tr><td>4% Paraformaldehyde</td><td>SIGMA</td><td>252549</td><td></td></tr><tr><td>Triton-X 100</td><td>SIGMA</td><td>X100</td><td></td></tr><tr><td>Tween 20</td><td>SIGMA</td><td>P1379</td><td></td></tr><tr><td>DAPI</td><td>SIGMA</td><td>D9564</td><td></td></tr><tr><td>Albumin (human)</td><td>SIGMA</td><td>A9731</td><td></td></tr><tr><td>Fisher BioReagent&amp;nbsp;Bovine Serum Albumin, Fraction V, Heat Shock Treated</td><td>Fisher Scientific</td><td>BP9701-100</td><td></td></tr><tr><td>ProLong Gold Antifade Mountant</td><td>Invitrogen</td><td>P36930</td><td></td></tr><tr><td>TaqMan Universal Master Mix II, no UNG</td><td>Applied Biosystems</td><td>4440040</td><td></td></tr><tr><td>SYBR Select Master Mix</td><td>Applied Biosystems</td><td>4472903</td><td></td></tr><tr><td>Lipopolysaccharide from &lt;em&gt;Escherichia coli&lt;/em&gt; K12</td><td>InvivoGen</td><td>tlrl-eklps</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog 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Rapid-Flow Sterile Disposable Bottle Top Filters with PES Membrane</td><td>ThermoFisher Scientific</td><td>295-3345</td><td></td></tr><tr><td>Fisherbrand&amp;nbsp;Microscopic Slides with Ground Edges, Twin Frost</td><td>Fisher Scientific</td><td>FB58628</td><td></td></tr><tr><td>Tube, Thinwall, Ultra-Clear, 38.5 mL, 25 x 89 mm</td><td>Beckman Coulter</td><td>344058</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Primary cells / Cell lines&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Human Plateable Hepatocytes, Transporter Qualified</td><td>Thermo Fisher Scientific</td><td>HMCPTS</td><td></td></tr><tr><td>Cryopreserved Human Kupffer Cells</td><td>Thermo Fisher Scientific</td><td>HUKCCS</td><td></td></tr><tr><td>HepDE19 cell line</td><td>Haitao Guo (Indiana University, IN, USA)</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Primers/Probes/Standards&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>HBV DNA forward primer</td><td>Invitrogen</td><td></td><td>5&#039;-GTGTCTGCGGCGTTTTATCA-3&#039;</td></tr><tr><td>HBV DNA reverse primer</td><td>Invitrogen</td><td></td><td>5&#039;-GACAAACGGGCAACATACCTT-3&#039;</td></tr><tr><td>HBV DNA probe</td><td>Invitrogen</td><td></td><td>5&#039;FAM-CCTCTKCATCCTGCTGCTATGCCTCATC-3&#039;TAMRA</td></tr><tr><td>pgRNA forward primer</td><td>Invitrogen</td><td></td><td>5&#039;-GAGTGTGGATTCGCACTCC-3&#039;</td></tr><tr><td>pgRNA reverse primer</td><td>Invitrogen</td><td></td><td>5&#039;-GAGGCGAGGGAGTTCTTCT-3&#039;</td></tr><tr><td>RPS11 forward primer</td><td>Invitrogen</td><td></td><td>5&#039;-GCCGAGACTATCTGCACTAC-3&#039;</td></tr><tr><td>RPS11 reverse primer</td><td>Invitrogen</td><td></td><td>5&#039;-ATGTCCAGCCTCAGAACTTC-3&#039;</td></tr><tr><td>pCMV-HBV</td><td>Professor Christoph Seeger (Fox Chase Cancer Centre, PA, USA)</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anti-Hepatitis B virus core antigen IgG fraction (polyclonal)</td><td>DAKO</td><td>discontinued</td><td>Lot 10102505</td></tr><tr><td>Human Albumin Antibody, A80-129A</td><td>Bethyl Laboratories. inc</td><td>A80-129A</td><td></td></tr><tr><td>Human Albumin cross-adsorbed Antibody, A80-229P</td><td>Bethyl Laboratories. inc</td><td>A80-229P</td><td></td></tr><tr><td>Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594</td><td>ThermoFisher Scientific</td><td>#&amp;nbsp;A-11072</td><td>Lot 1431810</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>LiverChip Vacuum pump</td><td>CN Bio innovations</td><td>LC-PN</td><td></td></tr><tr><td>LiverChip Pneumatic Hookup</td><td>CN Bio innovations</td><td>LC-PN</td><td></td></tr><tr><td>LiverChip platform</td><td>CN Bio innovations</td><td>LC-PN</td><td></td></tr><tr><td>LiverChip plate washing dock</td><td>CN Bio innovations</td><td></td><td></td></tr><tr><td>Autoclavable metal forceps</td><td>VWR</td><td>232-0106</td><td></td></tr><tr><td>Vortex Genie 2</td><td>Scientific industries</td><td>SKU: SI-0236</td><td></td></tr><tr><td>Optima XPN-80 Ultracentrifuge</td><td>Beckman Coulter</td><td>A95765</td><td></td></tr><tr><td>Heraeus Multifuge X3R Centrifuge</td><td>Thermo Scientific</td><td>75004500</td><td></td></tr><tr><td>SAM-12 Medical Suction High Vacuum High Flow</td><td>MGE worldwide</td><td>&amp;nbsp;SAM12/01010101</td><td></td></tr><tr><td>NUAIRE 5800 SERIES incubator</td><td>NUAIRE</td><td>NU-5841</td><td></td></tr><tr><td>Automated precision torgue</td><td>CN Bio innovations</td><td></td><td></td></tr><tr><td>Manual torque</td><td>CN Bio innovations</td><td></td><td></td></tr><tr><td>LiverChip compressor</td><td>CN Bio innovations</td><td></td><td></td></tr><tr><td>Luminex LX-200 Instrument with xPONENT 3.1</td><td>Luminex</td><td></td><td></td></tr><tr><td>Millipore Hand-Held Magnetic Separator Block</td><td>ThermoFisher Scientific</td><td>Millipore&amp;trade; 40-285</td><td></td></tr><tr><td>FluoStar Optima Plate Reader</td><td>BMG Labtech</td><td></td><td></td></tr><tr><td>KOLVER Precision electric 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"liver-on-a-chip" cultures of primary hepatocytes and kupffer cells for hepatitis b virus infection

"Liver-on-a-Chip" Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection

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.

We describe a simple and versatile platform for the long-term culture of primary human Kupffer cells and/or hepatocytes and their infection with HBV. Primary human cells are seeded on collagen-coated polystyrene scaffolds within a microfluidic plate assembly, which continuously perfuses the cells with growth medium (Figure 1a).
PHH, which usually are only stable for a limited amount of time in conventional culture systems, can be functionally maintained for extended periods of time. Human albumin, which is secreted by functional hepatocytes and is considered the best marker for the evaluation of hepatic metabolism, is stably and highly expressed by 3D cultures until day 40 post-seeding (Figure 2). For cocultures, Kupffer cell functionality and viability can be evaluated by the secretion of specific cytokines (e.g., IL6 and TNFα). To measure cytokine production, the use of capture-based detection means upon LPS-stimulation of cocultures is recommended (Figure 3).
Cells form hepatic microtissues, usually within 3 days of seeding of PHH, demonstrating functional bile canaliculi and complete cell polarization (Figure 2). In addition to retaining their physiological cellular metabolism, these cultures become exceptionally susceptible to HBV infection. HBV DNA and other viral markers, in contrast to other culture systems, become readily detectable from day 2 post-infection (Figure 4). In addition to secreted markers of viral infection, hepatocyte-containing scaffolds can be retrieved from the cultures and used for the immunofluorescence detection of viral antigens (e.g., HBsAg, HBcAg) (Figure 4). Where conventional hepatocyte cultures require inoculation with at least 500 HBV GE per cell and the addition of 2% DMSO and 4% PEG, as few as 0.05 HBV GE are able to initiate infection in 3-D cultures without the requirement of DMSO or PEG (Figure 4).
<img alt="Static equilibrium diagram; sterility setup with membranes; microtissue seeding, HBV infection process." class="xfigimg" src="/files/ftp_upload/58333/58333fig1.jpg" data-altupdatedat="1788250015000" data-alt="Figure 1"> 
Figure 1: Set-up of 3-D liver-on-a-chip cultures. (a) This is a schematic layout for the assembly of the culture plate in order to ensure the establishment of microfluidic circulation. (b) This panel shows a close-up view of the culture wells, including the filter paper, scaffold, and retaining ring. (c) This panel shows the process of plate equilibration prior to seeding the cultures. The next two panels show the process of seeding for (d) hepatocyte monocultures and (e) hepatocyte/Kupffer cell cocultures. (f) This panel shows the washing steps involved in medium changes. (g) This panel shows the HBV infection set-up, including the removal of inoculum. S.M. = seeding medium, M.M. = maintenance medium. <a href="https://www.jove.com/files/ftp_upload/58333/58333fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="3D cell culture development with albumin production; microscopy images, fluorescence, chart analysis." class="xfigimg" src="/files/ftp_upload/58333/58333fig2.jpg" data-altupdatedat="1788250015000" data-alt="Figure 2"> 
Figure 2: Hepatic microtissue formation and hepatocyte viability. (a) This panel shows longitudinal brightfield images of 3D hepatocyte monocultures demonstrating microtissue formation following seeding. (b) This panel shows immunofluorescence imaging of cultures for nuclei (blue) and human albumin (green). (c) This panel shows longitudinal total albumin, as well as per cell adjusted albumin production, during 40 days of hepatocyte monocultures, as determined by ELISA. The data shown are mean ± SD. This figure is adapted from Ortega-Prieto et al.4. <a href="https://www.jove.com/files/ftp_upload/58333/58333fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="IL-6 and TNFα levels bar chart; monoculture vs coculture; LPS stimulation, immune response." class="xfigimg" src="/files/ftp_upload/58333/58333fig3.jpg" data-altupdatedat="1788250015000" data-alt="Figure 3"> 
Figure 3: Kupffer cell functionality in 3-D cocultures. These panels show the secretion of (a) IL6 and (b) TNFα in hepatocyte monocultures and hepatocyte/Kupffer cell cocultures 11 days post-seeding in response to exogenously added LPS at day 9 post seeding, as determined using Human Magnetic Luminex assay. This figure is adapted from Ortega-Prieto et al.4. <a href="https://www.jove.com/files/ftp_upload/58333/58333fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="HBV infection analysis: microscopy images (a), DNA copies graph (b), transcript levels chart (c)." class="xfigimg" src="/files/ftp_upload/58333/58333fig4.jpg" data-altupdatedat="1788250015000" data-alt="Figure 4"> 
Figure 4: HBV infection in liver-on-a-chip cultures. (a) This panel shows the immunofluorescence microscopy detection of HBcAg (red), HBsAg (green), and nuclei (blue) 10 days following the infection of the cultures with HBV. (b) This panel shows the susceptibility of the cultures to HBV infection using different multiplicities of infection, as determined by a quantification of HBV DNA in the culture supernatants. (c) This panel shows the quantification of the longitudinal accumulation of HBV pgRNA relative to the housekeeping gene RPS11. The data shown are mean ± SD. This figure is adapted from Ortega-Prieto et al.4. <a href="https://www.jove.com/files/ftp_upload/58333/58333fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

The goal of this protocol is to provide a step-by-step guide to perform 3-D &#34;liver-on-a-chip&#34; infection experiments with the hepatitis B virus.

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally ...

liver-on-chip-cultures-primary-hepatocytes-kupffer-cells-for

Research

JoVE Journal

Immunology and Infection

A technique to isolate human hepatocytes and non-parenchymal liver cells from the same donor is described. The different liver cell types build the basis for functional liver models and tissue engineering. This new method aims to isolate liver cells in a high yield and viability.

Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Here, we show an enzymatic approach to isolate primary hepatocytes from adult mice, and we describe the quantification of an inflammatory response using ELISA and real-time PCR.

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

Presented here is a protocol for the effective suppression of hepatitis B virus (HBV) replication in mice by utilizing adoptive cell transfer (ACT) of stem cell-derived viral antigen (Ag)-specific T lymphocytes. This procedure may be adapted for potential ACT-based immunotherapy of HBV infection.

Stem Cell-Derived Viral Ag-Specific T Lymphocytes Suppress HBV Replication in Mice

This manuscript describes a detailed protocol for Hepatitis B virus (HBV) infection in novel engineered 293T cells (293T-NE-3NRs, expressing human NTCP, HNF4α, RXRα and PPARα) and traditional hepatic cells (HepG2-NE, expressing human NTCP).

Modeling Hepatitis B Virus Infection in Non-Hepatic 293T-NE-3NRs Cells

This protocol details the use of a special intravenous catheter, standardized sterile disposable tubing, temperature control complemented by real-time monitoring, and an alarm system for two-step collagenase perfusion procedure to improve the consistency in the viability, yield, and functionality of isolated primary rat hepatocytes.

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

A long-term ex vitro 3D organoid culture system was established from mouse hepatocytes. These organoids can be passaged and genetically manipulated by lentivirus infection of shRNA/ectopic construction, siRNA transfection and CRISPR-Cas9 engineering.

Establishment and Genetic Manipulation of Murine Hepatocyte Organoids

We present a protocol to screen anti-hepatitis B virus (HBV) compounds targeting pre- and post-viral entry lifecycle stages, using isothermal titration calorimetry to measure binding affinity (KD) with host sodium taurocholate cotransporting polypeptide. Antiviral efficacy was determined through the suppression of viral lifecycle markers (cccDNA formation, transcription, and viral assembly).

A Competent Hepatocyte Model Examining Hepatitis B Virus Entry through Sodium Taurocholate Cotransporting Polypeptide as a Therapeutic Target

Here we present a non-genetic method to generate human autologous liver spheroids using mononuclear cells isolated from steady-state peripheral blood.

Human Liver Spheroids from Peripheral Blood for Liver Disease Studies

Here, we describe a platform that allows noninvasive in vivo imaging of liver spheroids engrafted in the anterior chamber of the mouse eye. The workflow spans from generating spheroids from primary liver cells to transplantation into the mouse eye and in vivo imaging at cellular resolution by confocal microscopy.

In Vivo Imaging of Liver Spheroids Engrafted in the Anterior Chamber of the Mouse Eye

This protocol presents an optimized two-step collagenase liver perfusion technique in a rat model and shows the use of isolated hepatocytes for in vitro long-term culture of 3D organoids.

Rat Liver Perfusion and Primary Hepatocytes Isolation: An Old Procedure Crucial for Cutting-Edge 3D Organoids Culture

Learn liver-on-a-chip culture of primary hepatocytes and Kupffer cells for hepatitis B virus infection studies. This 3D microfluidic platform enables longitudinal infection kinetics and drug testing in a physiological hepatic environment.

"Liver-on-a-Chip" Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection

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