Name: "liver-on-a-chip" cultures of primary hepatocytes and kupffer cells for hepatitis b virus infection
Uploaded: 2019-02-19T13:00:00.000+00:00
Duration: 10 min 25 s
Description: 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

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. T., Guo, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+and+function+of+hepatitis+B+virus+cccDNA:+Implications+for+the+development+of+cccDNA-targeting+antiviral+therapeutics.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+antiviral+properties+of+TLR+ligands+against+HBV+replication+in+immune-competent+hepatocytes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+B+Virus+Activates+Signal+Transducer+and+Activator+of+Transcription+3+Supporting+Hepatocyte+Survival+and+Virus+Replication.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+production+of+human+liver+scaffolds+for+functional+tissue+engineering+by+high+shear+stress+oscillation-decellularization.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipids+promote+survival,+proliferation,+and+maintenance+of+differentiation+of+rat+liver+sinusoidal+endothelial+cells+in+vitro.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+liver+sinusoidal+endothelial+cells+survive+without+exogenous+VEGF+in+3D+perfused+co-cultures+with+hepatocytes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscale+culture+of+human+liver+cells+for+drug+development.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropatterned+coculture+of+primary+human+hepatocytes+and+supportive+cells+for+the+study+of+hepatotropic+pathogens.">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/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. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+liver.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfused+multiwell+plate+for+3D+liver+tissue+engineering.">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.

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>William&#039;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&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 Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Kits/Consumables&lt;/strong&gt;</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 &amp;amp; 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&amp;amp;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&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 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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

11.2K Views.  Imperial College. Deze methode kan helpen bij het beantwoorden van belangrijke vragen in hepatotropische infecties en leverziekte velden. Zoals de bijdrage van lever ingezeten celpopulaties, aan virale pathogenese, longitudinale infectiekinetiek, mechanismen van virale replicatie, immuuninvasie, in een fysiologisch modelsysteem en medicijnteststudies. Het belangrijkste voordeel van deze techniek is de mogelijkheid om een levermicro-omgeving samen te vatten.Die langere tijd kan worden gehandhaafd en van ...

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

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

Hepatitis B virus (HBV) infection has been considered a crucial risk factor for hepatocellular carcinoma. Current treatment can only lessen the viral load but not result in complete remission. An efficient hepatocyte model for HBV infection would offer a true-to-life viral life cycle that would be crucial for the screening of therapeutic agents. Most available anti-HBV agents target lifecycle stages post viral entry but not before viral entry. This protocol details the generation of a competent hepatocyte model capable of screening for therapeutic agents targeting pre-viral entry and post viral entry lifecycle stages. This includes the targeting of sodium taurocholate cotransporting polypeptide (NTCP) binding, cccDNA formation, transcription, and viral assembly based on imHC or HepaRG as host cells. Here, the HBV entry inhibition assay used curcumin to inhibit HBV binding and transporting functions via NTCP. The inhibitors were evaluated for binding affinity (KD) with NTCP using isothermal titration calorimetry (ITC)-a universal tool for HBV drug screening based on thermodynamic parameters.

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).

Hepatitis B virus (HBV) infection has been considered a crucial risk factor for hepatocellular carcinoma. Current treatment can only lessen the viral load ...

Biochemistry

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), ...

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the uremic milieu, oxidative stress, endotoxemia and the decreased clearance of circulating proinflammatory cytokines by producing a large number of acute-phase reactants. Experimental tools to study inflammation and the underlying role of hepatocytes are crucial to understand the regulation and contribution of hepatic cytokines to a systemic acute phase response and a prolonged pro-inflammatory scenario, especially in an intricate setting such as chronic kidney disease. Since studying complex mechanisms of inflammation in vivo remains challenging, resource-intensive and usually requires the usage of transgenic animals, primary isolated hepatocytes provide a robust tool to gain mechanistic insights into the hepatic acute-phase response. Since this in vitro technique features moderate costs, high reproducibility and common technical knowledge, primary isolated hepatocytes can also be easily used as a screening approach. Here, we describe an enzymatic-based method to isolate primary murine hepatocytes, and we describe the assessment of an inflammatory response in these cells using ELISA and quantitative real-time PCR.

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.

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the ...

A Cell Culture Model for Producing High Titer Hepatitis E Virus Stocks

Hepatitis E virus is the leading cause of liver cirrhosis and liver failure with increasing prevalence worldwide. The single-stranded RNA virus is predominantly transmitted by blood transfusions, inadequate sanitary conditions and contaminated food products. To date the off-label drug ribavirin (RBV) is the treatment of choice for many patients. Nonetheless, a specific HEV treatment remains to be identified. So far, the knowledge about the HEV life cycle and pathogenesis has been severely hampered because of the lack of an efficient HEV cell culture system. A robust cell culture system is essential for the study of the viral life cycle which also includes the viral pathogenesis. With the method described here one can produce viral titers of up to 3 x 106 focus forming unit/mL (FFU/mL) of non-enveloped HEV and up to 5 x 104 FFU/mL of enveloped HEV. Using these particles, it is possible to infect a variety of cells of diverse origins including primary cells and human, as well as animal cell lines. The production of infectious HEV particles from plasmids poses an infinite source, which makes this protocol exceedingly efficient.

Described here is an effective method on how to produce high viral titer stocks of hepatitis E virus (HEV) to efficiently infect hepatoma cells. With the presented method, both non-enveloped, as well as enveloped viral particles can be harvested and used for inoculating various cell lines.

Hepatitis E virus is the leading cause of liver cirrhosis and liver failure with increasing prevalence worldwide. The single-stranded RNA virus is ...

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

HBV mainly infects human hepatocytes, but it has also been found to infect extrahepatic tissues such as kidney and testis. Nonetheless, cell-based HBV models are limited to hepatoma cell lines (such as HepG2 and Huh7) overexpressing a functional HBV receptor, sodium taurocholate co-transporting polypeptide (NTCP). Here, we used 293T-NE-3NRs (293T&#160;overexpressing human NTCP, HNF4&#945;, RXR&#945; and PPAR&#945;) and HepG2-NE (HepG2 overexpressing NTCP) as model cell lines.&#160;HBV infection in these cell lines was performed either by using concentrated HBV virus particles from HepG2.2.15 or co-culturing HepG2.2.15 with the target cell lines. HBcAg immunofluorescence for HBcAg was performed to confirm HBV infection. The two methods presented here will help us study HBV infection in non-hepatic cell lines.&#160;&#160;

This manuscript describes a detailed protocol for Hepatitis B virus (HBV) infection in novel engineered 293T cells (293T-NE-3NRs, expressing human NTCP, HNF4&#945;, RXR&#945; and PPAR&#945;) and traditional hepatic cells (HepG2-NE, expressing human NTCP).

HBV mainly infects human hepatocytes, but it has also been found to infect extrahepatic tissues such as kidney and testis. Nonetheless, cell-based HBV ...

Human Liver Spheroids from Peripheral Blood for Liver Disease Studies

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to their nature to cluster in the culture dishes with low or no adhesive characteristics, they form aggregates of multiple liver cells that are called human liver spheroids. The forming of 3D liver spheroids relies on the natural tendency of hepatic cells to aggregate in the absence of an adhesive substrate. These 3D structures possess better physiological responses than cells, which are closer to an in vivo environment. Using 3D hepatocyte cultures has numerous advantages when compared with classical two-dimensional (2D) cultures, including a more biologically relevant microenvironment, architectural morphology that reassembles natural organs as well as a better prediction regarding disease state and in vivo-like responses to drugs. Various sources can be used to generate spheroids, like primary liver tissue or immortalized cell lines. The 3D liver tissue can also be engineered by using human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs) to derive hepatocytes. We have obtained human liver spheroids using blood-derived pluripotent stem cells (BD-PSCs) generated from unmanipulated peripheral blood by activation of human membrane-bound GPI-linked protein and differentiated to human hepatocytes. The BD-PSCs-derived human liver cells and human liver spheroids were analyzed by light microscopy and immunophenotyping using human hepatocyte markers.

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

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to ...

Bioengineering

Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for systemic energy homeostasis. Primary hepatocytes isolated from the murine liver constitute a valuable biological tool to understand the functional properties or alterations occurring in the liver. Herein we describe a method for the isolation and culture of primary mouse hepatocytes by performing a two-step collagenase perfusion technique and discuss their utilization for investigating protein metabolism. The liver of an adult mouse is sequentially perfused with ethylene glycol-bis tetraacetic acid (EGTA) and collagenase, followed by the isolation of hepatocytes with the density gradient buffer. These isolated hepatocytes are viable on culture plates and maintain the majority of endowed characteristics of hepatocytes. These hepatocytes can be used for assessments of protein metabolism including nascent protein synthesis with non-radioactive reagents. We show that the isolated hepatocytes are readily controlled and comprise a higher quality and volume stability of protein synthesis linked to energy metabolism by utilizing the chemo-selective ligation reaction with a Tetramethylrhodamine (TAMRA) protein detection method and western blotting analyses. Therefore, this method is valuable for investigating hepatic nascent protein synthesis linked to energy homeostasis. The following protocol outlines the materials and methods for the isolation of high-quality primary mouse hepatocytes and detection of nascent protein synthesis.

Here, we present a protocol for the isolation of healthy and functional primary mouse hepatocytes. Instructions for detecting hepatic nascent protein synthesis by non-radioactive labeling substrate were provided to help understand the mechanisms underlying protein synthesis in the context of energy-metabolism homeostasis in the liver.

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for ...

Medicine

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

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the &#34;gold standard&#34; for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine1. Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique.
Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation.
Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models.

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.

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic ...

Biology

Establishment and Genetic Manipulation of Murine Hepatocyte Organoids

The liver is the largest organ in mammals. It plays an important role in glucose storage, protein secretion, metabolism and detoxification. As the executor for most of the liver functions, primary hepatocytes have limited proliferating capacity. This requires the establishment of ex vivo hepatocyte expansion models for liver physiological and pathological research. Here, we isolated murine hepatocytes by two steps of collagenase perfusion and established a 3D organoid culture as the 'mini-liver' to recapitulate cell-cell interactions and physical functions. The organoids consist of heterogeneous cell populations including progenitors and mature hepatocytes. We introduce the process in detailed to isolate and culture the murine hepatocytes or fetal hepatocyte to form organoids within 2-3 weeks and show how to passage them by mechanically pipetting up and down. In addition, we will also introduce how to digest the organoids into single cells for lentivirus infection of shRNA/ectopic construction, siRNA transfection and CRISPR-Cas9 engineering. The organoids can be used for drug screens, disease modelling, and basic liver research by modeling liver biology and pathobiology.

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.

The liver is the largest organ in mammals. It plays an important role in glucose storage, protein secretion, metabolism and detoxification. As the ...

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

Primary hepatocytes are a commonly used tool for in vitro liver-related studies. However, the maintenance of these cells has always been a challenge due to the rapid loss of morphology, viability, and functionality in culture. A recent approach to long-term culture is the generation of three-dimensional (3D) organoids, an in vitro tool that can recapitulate tissues in a dish based on the marvelous ability of the liver to regenerate itself. Published protocols have been designed to obtain long-term functional 3D organoids from primary adult hepatocytes (Hep-Orgs). The 3D organoid cutting-edge tool requires the ability to isolate cells from adult tissue, and this initial step is crucial for a high-quality final result. The two-step collagenase perfusion, introduced in the 1970s, is still a valid procedure to obtain single hepatocytes. The present article aims to describe all the crucial steps of the surgical procedure, thereby optimizing the primary hepatocytes isolation procedure in the rat model. Moreover, particular attention is paid to the PREPARE guidelines to increase the likelihood of successful procedures and ensure high-quality results. A detailed protocol allows researchers to speed up and optimize the downstream work to establish 3D organoids from primary adult rat hepatocytes. Compared to 2D hepatocytes, Hep-Orgs were still viable and in active proliferation at Day 15, demonstrating a long-term potential.

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.

Primary hepatocytes are a commonly used tool for in vitro liver-related studies. However, the maintenance of these cells has always been a challenge due ...

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

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video