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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Culture+Models+for+the+Investigation+of+Hepatitis+B+and+D+Virus+Infection.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+underlying+the+dedifferentiation+process+of+isolated+hepatocytes+and+their+cultures.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+the+bile+acid+transporter+and+hepatitis+B+virus+receptor+Na+/taurocholate+cotransporting+polypeptide+(NTCP)+in+hepatocytes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+microfluidic+liver+cultures+as+a+physiological+preclinical+tool+for+hepatitis+B+virus+infection.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+cccDNA+in+HBV+Maintenance.">The Role of cccDNA in HBV Maintenance.</a> Viruses. 9 (6), (2017).</li><li>Guo, J. 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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+primary+human+hepatocyte+spheroids+as+a+model+system+for+drug-induced+liver+injury,+liver+function+and+disease.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constrained+spheroids+for+prolonged+hepatocyte+culture.">Constrained spheroids for prolonged hepatocyte culture.</a> Biomaterials. 80, 106-120 (2016).</li><li>Griffith, L. G., Wells, A., Stolz, D. 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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.

<table>
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>William&#39;s E Medium, no phenol red</td>
			<td>GIBCO</td>
			<td>A12176-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepatocyte Thaw Medium</td>
			<td>GIBCO</td>
			<td>CM7500</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary Hepatocyte Thawing and Plating Supplements</td>
			<td>GIBCO</td>
			<td>CM3000</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary Hepatocyte Maintenance Supplements</td>
			<td>GIBCO</td>
			<td>CM4000</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>GIBCO</td>
			<td>11320-033</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM</td>
			<td>GIBCO</td>
			<td>12491023</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, no calcium, no magnesium</td>
			<td>GIBCO</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids (NEAA) 100X</td>
			<td>GIBCO</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>GIBCO</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, USA origin, Heat Inactivated, sterile-filtered, suitable for cell 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&#160;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 Escherichia coli K12</td>
			<td>InvivoGen</td>
			<td>tlrl-eklps</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Kits/Consumables</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile membrane</td>
			<td>CN Bio innovations</td>
			<td>LC-SC</td>
			<td></td>
		</tr>
		<tr>
			<td>LiverChip Perfusion cell culture plate</td>
			<td>CN Bio innovations</td>
			<td>LC12</td>
			<td></td>
		</tr>
		<tr>
			<td>LiverChip culture plate lid</td>
			<td>CN Bio innovations</td>
			<td>LC-SC</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile round filter paper</td>
			<td>CN Bio innovations</td>
			<td>LC-SC</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell attachment scaffold</td>
			<td>CN Bio innovations</td>
			<td>LC-SC</td>
			<td></td>
		</tr>
		<tr>
			<td>Retaining ring</td>
			<td>CN Bio innovations</td>
			<td>LC-SC</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile plunger</td>
			<td>CN Bio innovations</td>
			<td>LC-ST</td>
			<td></td>
		</tr>
		<tr>
			<td>Dneasy blood &#38; tissue kit</td>
			<td>Qiagen</td>
			<td>69506</td>
			<td></td>
		</tr>
		<tr>
			<td>Rneasy mini kit</td>
			<td>Qiagen</td>
			<td>74106</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Magnetic Luminex assay</td>
			<td>R&#38;D Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1-Step Ultra TMB-ELISA Substrate Solution</td>
			<td>ThermoFisher Scientific</td>
			<td>34028</td>
			<td></td>
		</tr>
		<tr>
			<td>High Capacity cDNA Reverse Transcription Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>4368814</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAamp Viral RNA Mini Accessory Set</td>
			<td>Qiagen</td>
			<td>1048147</td>
			<td>Containing RNA carrier</td>
		</tr>
		<tr>
			<td>Millicell HY 5-layer cell culture flask, T-1000, sterile</td>
			<td>Millipore (Merck)</td>
			<td>PFHYS1008</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroAmp Optical 384-Well Reaction Plate with Barcode</td>
			<td>Life technologies</td>
			<td>4309849</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroAmp Optical Adhesive Film</td>
			<td>Life technologies</td>
			<td>4311971</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear Flat-Bottom Immuno Nonsterile 96-Well Plates</td>
			<td>ThermoFisher Scientific</td>
			<td>442404</td>
			<td></td>
		</tr>
		<tr>
			<td>Sealing Tape for 96-Well Plates</td>
			<td>ThermoFisher Scientific</td>
			<td>15036</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene 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&#160;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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Primary cells / Cell lines</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Primers/Probes/Standards</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HBV DNA forward primer</td>
			<td>Invitrogen</td>
			<td></td>
			<td>5&#39;-GTGTCTGCGGCGTTTTATCA-3&#39;</td>
		</tr>
		<tr>
			<td>HBV DNA reverse primer</td>
			<td>Invitrogen</td>
			<td></td>
			<td>5&#39;-GACAAACGGGCAACATACCTT-3&#39;</td>
		</tr>
		<tr>
			<td>HBV DNA probe</td>
			<td>Invitrogen</td>
			<td></td>
			<td>5&#39;FAM-CCTCTKCATCCTGCTGCTATGCCTCATC-3&#39;TAMRA</td>
		</tr>
		<tr>
			<td>pgRNA forward primer</td>
			<td>Invitrogen</td>
			<td></td>
			<td>5&#39;-GAGTGTGGATTCGCACTCC-3&#39;</td>
		</tr>
		<tr>
			<td>pgRNA reverse primer</td>
			<td>Invitrogen</td>
			<td></td>
			<td>5&#39;-GAGGCGAGGGAGTTCTTCT-3&#39;</td>
		</tr>
		<tr>
			<td>RPS11 forward primer</td>
			<td>Invitrogen</td>
			<td></td>
			<td>5&#39;-GCCGAGACTATCTGCACTAC-3&#39;</td>
		</tr>
		<tr>
			<td>RPS11 reverse primer</td>
			<td>Invitrogen</td>
			<td></td>
			<td>5&#39;-ATGTCCAGCCTCAGAACTTC-3&#39;</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Antibodies</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>#&#160;A-11072</td>
			<td>Lot 1431810</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</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 colspan="2">&#160;SAM12/01010101</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&#8482; 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 screwdrivers</td>
			<td>VTECH ltd</td>
			<td>FAB10RE/FR</td>
			<td></td>
		</tr>
		<tr>
			<td>KOLVER Power supply</td>
			<td>VTECH ltd</td>
			<td>EDU1FR</td>
			<td></td>
		</tr>
		<tr>
			<td>BAMBI VTS75D</td>
			<td>Air Equipment</td>
			<td>Discontinued</td>
			<td></td>
		</tr>
		<tr>
			<td>Integra Vacuboy</td>
			<td>INTEGRA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ViiA 7 Real-Time PCR System with 384-Well Block</td>
			<td>ThermoFisher Scientific</td>
			<td>4453536</td>
			<td></td>
		</tr>
	</tbody>
</table>

"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 TNF&#945;). 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="Figure 1" class="xfigimg" src="/files/ftp_upload/58333/58333fig1.jpg" /> 
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="Figure 2" class="xfigimg" src="/files/ftp_upload/58333/58333fig2.jpg" /> 
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 &#177; 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="Figure 3" class="xfigimg" src="/files/ftp_upload/58333/58333fig3.jpg" /> 
Figure 3: Kupffer cell functionality in 3-D cocultures. These panels show the secretion of (a) IL6 and (b) TNF&#945; 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="Figure 4" class="xfigimg" src="/files/ftp_upload/58333/58333fig4.jpg" /> 
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 &#177; 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>

Watch this Scientific Journal Video about "Liver-on-a-Chip" Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection at JoVE.com

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

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

11.2K Views.  Imperial College. The goal of this protocol is to provide a step-by-step guide to perform 3-D "liver-on-a-chip" infection experiments with the hepatitis B virus.

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

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Recombinant Retroviral Production and Infection of B Cells

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control of a heterologous expression system to facilitate the analysis of the resulting phenotype. This approach can be used to express a gene that is not normally found in the organism, to express a mutant form of a gene product, or to over-express a dominant-negative form of the gene product. It is particularly useful in the study of the hematopoetic system, where transcriptional regulation is a major control mechanism in the development and differentiation of B cells 1, reviewed in 2-4.
Mouse genetics is a powerful tool for the study of human genes and diseases. A comparative analysis of the mouse and human genome reveals conservation of synteny in over 90% of the genome 5. Also, much of the technology used in mouse models is applicable to the study of human genes, for example, gene disruptions and allelic replacement 6. However, the creation of a transgenic mouse requires a great deal of resources of both a financial and technical nature. Several projects have begun to compile libraries of knock out mouse strains (KOMP, EUCOMM, NorCOMM) or mutagenesis induced strains (RIKEN), which require large-scale efforts and collaboration 7. Therefore, it is desirable to first study the phenotype of a desired gene in a cell culture model of primary cells before progressing to a mouse model. 
Retroviral DNA integrates into the host DNA, preferably within or near transcription units or CpG islands, resulting in stable and heritable expression of the packaged gene of interest while avoiding transcriptional silencing 8 9. The genes are then transcribed under the control of a high efficiency retroviral promoter, resulting in a high efficiency of transcription and protein production. Therefore, retroviral expression can be used with cells that are difficult to transfect, provided the cells are in an active state during mitosis. Because the structural genes of the virus are contained within the packaging cell line, the expression vectors used to clone the gene of interest contain no structural genes of the virus, which both eliminates the possibility of viral revertants and increases the safety of working with viral supernatants as no infectious virions are produced 10. 
Here we present a protocol for recombinant retroviral production and subsequent infection of splenic B cells. After isolation, the cultured splenic cells are stimulated with Th derived lymphokines and anti-CD40, which induces a burst of B cell proliferation and differentiation 11. This protocol is ideal for the study of events occurring late in B cell development and differentiation, as B cells are isolated from the spleen following initial hematopoetic events but prior to antigenic stimulation to induce plasmacytic differentiation.

An efficient system of structure and function analysis of a gene in an ex vivo culture of splenic B-lymphocytes is described. This method takes advantage of recombinant retroviral production in a helper free, ecotrophic packaging cell line. Stable, heritable expression of a gene of interest within primary lymphocytes is achieved leading to generation of surface antibodies on B cells undergoing class switch recombination.

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control ...

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

A Protocol for Analyzing Hepatitis C Virus Replication

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped RNA virus belonging to the family Flaviviridae. Current treatment is not fully effective and causes adverse side effects. There is no HCV vaccine available. Thus, continued effort is required for developing a vaccine and better therapy. An HCV cell culture system is critical for studying various stages of HCV growth including viral entry, genome replication, packaging, and egress. In the current procedure presented, we used a wild-type intragenotype 2a chimeric virus, FNX-HCV, and a recombinant FNX-Rluc virus carrying a Renilla luciferase reporter gene to study the virus replication. A human hepatoma cell line (Huh-7 based) was used for transfection of in vitro transcribed HCV genomic RNAs. Cell-free culture supernatants, protein lysates and total RNA were harvested at various time points post-transfection to assess HCV growth. HCV genome replication status was evaluated by quantitative RT-PCR and visualizing the presence of HCV double-stranded RNA. The HCV protein expression was verified by Western blot and immunofluorescence assays using antibodies specific for HCV NS3 and NS5A proteins. HCV RNA transfected cells released infectious particles into culture supernatant and the viral titer was measured. Luciferase assays were utilized to assess the replication level and infectivity of reporter HCV. In conclusion, we present various virological assays for characterizing different stages of the HCV replication cycle.

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and ...

Kupffer Cell Isolation for Nanoparticle Toxicity Testing

The large majority of in vitro nanotoxicological studies have used immortalized cell lines for their practicality. However, results from nanoparticle toxicity testing in immortalized cell lines or primary cells have shown discrepancies, highlighting the need to extend the use of primary cells for in vitro assays. This protocol describes the isolation of mouse liver macrophages, named Kupffer cells, and their use to study nanoparticle toxicity. Kupffer cells are the most abundant macrophage population in the body and constitute part of the reticulo-endothelial system (RES), responsible for the capture of circulating nanoparticles. The Kupffer cell isolation method reported here is based on a 2-step perfusion method followed by purification on density gradient. The method, based on collagenase digestion and density centrifugation, is adapted from the original protocol developed by Smedsr&#248;d et al. designed for rat liver cell isolation and provides high yield (up to 14 x 106 cells per mouse) and high purity (&#62;95%) of Kupffer cells. This isolation method does not require sophisticated or expensive equipment and therefore represents an ideal compromise between complexity and cell yield. The use of heavier mice (35-45 g) improves the yield of the isolation method but also facilitates remarkably the procedure of portal vein cannulation. The toxicity of functionalized carbon nanotubes f-CNTs was measured in this model by the modified LDH assay. This method assesses cell viability by measuring the lack of structural integrity of Kupffer cell membrane after incubation with f-CNTs. Toxicity induced by f-CNTs can be measured consistently using this assay, highlighting that isolated Kupffer cells are useful for nanoparticle toxicity testing. The overall understanding of nanotoxicology could benefit from such models, making the nanoparticle selection for clinical translation more efficient.

Liver macrophages, named Kupffer cells, are responsible for the capture of circulating nanoparticles. We describe here a method, of high cell purity and yield, for Kupffer cell isolation. The modified LDH assay is used here to measure the toxicity induced by carbon nanotubes in Kupffer cells.

The large majority of in vitro nanotoxicological studies have used immortalized cell lines for their practicality. However, results from nanoparticle ...

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

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible 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.

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally replicates through an episomal DNA intermediate; however, integration of HBV DNA fragments into the host genome has been observed, even though this form is not necessary for viral replication. The exact purpose, timing, and mechanism(s) by which HBV DNA integration occurs is not yet clear, but recent data show that it occurs very early after infection. Here, the in vitro generation and detection of HBV DNA integrations are described in detail. Our protocol specifically amplifies single copies of virus-cell DNA integrations and allows both absolute quantification and single-base pair resolution of the junction sequence. This technique has been applied to various HBV-susceptible cell types (including primary human hepatocytes), to various HBV mutants, and in conjunction with various drug exposures. We foresee this technique becoming a key assay to determine the underlying molecular mechanisms of this clinically relevant phenomenon.

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

We describe here the in vitro generation of HBV DNA via a Hepatitis B virus infection system and the highly sensitive detection of its (1&#8211;2 copies) integration using inverse nested PCR.

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally ...

Quantification of Antibody-dependent Enhancement of the Zika Virus in Primary Human Cells

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barr&#233; syndrome and microcephaly in infants, has brought serious public safety concerns. Among the risk factors, antibody-dependent enhancement (ADE) poses the most significant threat, as the recent re-emergence of the Zika virus (ZIKV) is primarily in areas where the population has been exposed and is in a state of pre-immunity to other closely related flaviviruses, especially dengue virus (DENV). Here, we describe a protocol for quantifying the effect of human serum antibodies against DENV on ZIKV infection in primary human cells or cell lines.

We describe a method to evaluate the effect of pre-existing immunity against dengue virus on the Zika virus infection by using human serum, primary human cells, and infection quantification by quantitative real-time polymerase chain reaction.

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barr&#233; syndrome and microcephaly in infants, has brought ...

Induction of Drug-Induced, Autoimmune Hepatitis in BALB/c Mice for the Study of Its Pathogenic Mechanisms

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients with autoimmune hepatitis. The overwhelming majority of patients with DIH are women. The underlying mechanisms of these sex differences in prevalence are unclear because of the paucity of animal models that mimic human disease. Even so, underlying mechanisms are widely believed to be associated with human leukocyte antigen haplotypes and sex hormones. In contrast, using a DIH mouse model, we have uncovered that IL-4 initiated&#160;CD4+ T cells directed against an epitope of cytochrome P450 2E1 induces influx of neutrophils, macrophages and mast cells into the livers of female BALB/c mice. Using this model, we have also shown that IL-33-induced FoxP3+regulatory T cells confer protection against DIH in female and male mice. This DIH model is induced by immunizing mice with an epitope of CYP2E1 that has been covalently altered with a drug metabolite that has been associated with DIH. This epitope is recognized by patients with DIH. Our method induces robust and reproducible hepatitis and autoantibodies that can be utilized to study the pathogenesis of DIH. While in vivo studies can cause undue pain and distress in mice when done improperly, the advantage of an in vivo model is the ability to evaluate the pathogenesis of disease in a large number of mice. Additionally, biological effects of the altered liver proteins can be studied using invasive procedures. The addition of in vitro studies to the experimental design allows rapid repetition and mechanistic analysis at a cellular level. Thus, we will demonstrate our model protocol and how it can be utilized to study in vivo and in vitro mechanisms of DIH.

We describe an in vivo immunization, translational hepatitis model in BALB/c mice that can be utilized to study the pathogenesis of drug-induced autoimmune hepatitis including sex differences seen in this disease. We will describe how this model demonstrates reproducible analyses using in vivo and in vitro experimental techniques.

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients ...