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

1. Assembly and Equilibration of Plates

1. Ensure that both the compressor and the vacuum pump associated with the LiverChip platform are turned on. Perform the assembly and equilibration of the plates in a class II cabinet.
2. Aseptically assemble the microfluidic plates by placing a sterile membrane between the plate base and adding the well-containing top plate (Figure 1a).
3. Ensure that the sterile membrane smoothly rests on the two pins of the base plate, since uneven membrane placement compromises the microfluidic circulation.
4. Add a sterile plate lid and tighten the screws at the base of the plate using an automated precision torque to 33 lb using a spiral tightening sequence. Ensure that all screws are tightened to 35 lb using a manual torque. During this step, ensure the screws are tightened symmetrically (Figure 1a).
5. Prewarm hepatocyte seeding medium containing Williams E medium, primary hepatocyte thawing, and plating supplements, 5% fetal bovine serum (FBS), and 1 µM dexamethasone to 37 °C before priming.
6. Prime the completely assembled plate by placing it within the washing dock and adding 400 µL of hepatocyte seeding medium to the reservoir side of each well. Ensure the plate snaps into the washing dock completely.
7. Initiate flow in the upward direction for 3.5 min at 1 µL/s. The successful function of the microfluidic circulation can be ascertained through the red indicators at the side of the plate (Figure 1a).
8. Once the medium is pumped to the cell growth side of the plate, ensuring the correct assembly of the flow channel, add an additional 1.2 mL of hepatocyte seeding medium.
9. Carefully transfer the plate into the docking station within a humidified incubator at 37 °C and 5% CO₂ and initiate flow in the upward direction at a flow rate of 1 µL/s for 16 h (Figure 1c).
10. Transfer the plate to the washing dock and eliminate bubbles in the well by gently pipetting up and down.
11. Add one sterile, round filter paper, followed by a cell attachment scaffold and a retaining ring, to each well using sterile forceps. Press down each well with a sterile plunger to lock the retaining rings and scaffolds into place (Figure 1b).
12. Aspirate all medium and add 400 µL of prewarmed hepatocyte seeding medium gently over the scaffold and initiate flow in the downward direction for 3.5 min at 1 µL/s.
13. Aspirate all medium pumped out of the reservoir side of the plate. This step is necessary to replace the medium contained in the flow channel.
14. Add 1.4 mL of hepatocyte seeding medium to each well before returning the plate to the dock to complete the total volume per well. The volume per well is now 1.6 mL (1.4 mL in the well and 0.2 mL in the flow channel).

2. Thawing and Seeding of Hepatocytes for Monocultures

1. Prewarm hepatocyte thawing medium and hepatocyte seeding medium to 37 °C prior to thawing one vial of PHH according to the suppliers' instructions. Use a centrifuge at room temperature during this step to avoid abrupt temperature changes. Perform the thawing and seeding of the hepatocytes in a class II cabinet.
2. Resuspend the cells in 1 mL of hepatocyte seeding medium and count the cells using trypan blue. Ensure that the viability of the cells is above 90%.
3. Keep the cells on ice until they are added to the wells.
4. Transfer the equilibrated and fully assembled plate to the washing dock and aspirate all medium from the wells.
5. Add 600,000 hepatocytes to each well in a 500 µL volume of hepatocyte seeding medium.
6. Initiate flow in the downward direction at a flow rate of 1 µL/s and bring the total volume in the well to 1.6 mL by adding 900 µL of hepatocyte seeding medium.
7. Transfer the plate to the docking station within a humidified incubator at 37 °C and with 5% CO₂.
8. Initiate flow in the downward direction at a flow rate of 1 µL/s for 8 h, followed by a flow reversal to the upward direction at a flow rate of 1 µL/s for 8 h.
9. Transfer the plate to the washing dock and aspirate all medium from the wells.
10. Add 400 µL of hepatocyte maintenance medium (Williams E medium supplemented with hepatocyte maintenance supplements and 100 nM dexamethasone) to each well and initiate flow in the downward direction at a flow rate of 1 µL/s for 3.5 min.
11. Aspirate all medium from the reservoir and add 1.4 mL of hepatocyte maintenance medium (Figure 1d).
12. Replace the medium with hepatocyte maintenance medium every 48 h. To ensure all medium in the well is replaced, perform a washing step before the addition of fresh hepatocyte maintenance medium.
13. For the washing step, transfer the plate to the washing dock, aspirate all medium from the wells, and add 400 µL of maintenance medium.
14. Initiate flow in the downward direction at 1 µL/s for 3.5 min. Aspirate all medium appearing on the reservoir side of the wells.
15. Add 1.4 mL of hepatocyte maintenance medium and, within a humidified incubator at 37 °C and with 5% CO₂, transfer the plate into the docking station and initiate flow in the upward direction at a flow rate of 1 µL/s for 48 h (Figure 1f).
    NOTE: For hepatocyte monocultures used as controls for cocultures, in order to ensure controlled conditions, a second type of maintenance medium is used, which is specific for use in the cocultures with primary human Kupffer cells. 48 h after replacing the hepatocyte seeding medium with hepatocyte maintenance medium (day 3 post-seeding), the regular hepatocyte maintenance medium will be replaced with coculture maintenance medium II, especially in monocultures of PHH when comparing to cocultures of both PHH and Kupffer cells, as the medium components differ slightly.

3. Thawing and Seeding of Kupffer Cells and Hepatocytes for Co-cultures

1. In order to ensure an accurate comparison of results, always compare PHH/Kupffer cell cocultures to PHH monocultures
2. For cocultures of PHH and Kupffer cells, thaw one vial of Kupffer cells in advanced Dulbecco's modified Eagle's medium (AdDMEM) without dexamethasone but supplemented with primary hepatocyte thawing and plating supplements (coculture seeding medium) according to the suppliers' instructions. Perform the thawing and seeding of the Kupffer cells and hepatocytes in a class II cabinet.
3. Resuspend the cells in 1 mL of coculture seeding medium and count the cells using trypan blue. Ensure that the viability of the cells is above 90%.
4. Keep the cells on ice prior to adding them to the wells to avoid cell adhesion.
5. Follow the instructions in steps 2.1–2.3 for the thawing of primary human hepatocytes.
6. Transfer the equilibrated and fully assembled plate to the washing dock and aspirate all medium from the wells.
7. Add 60,000 Kupffer cells and/or 600,000 hepatocytes to each well in a total volume of 250 µL each of coculture seeding medium.
8. Initiate flow in the downward direction at a flow rate of 1 µL/s and add 900 µL of coculture seeding medium to each well.
9. Transfer the plate into the docking station within a humidified incubator at 37 °C and with 5% CO₂.
10. Initiate flow in the downward direction at a flow rate of 1 µL/s for 8 h, followed by flow reversal to the upward direction at a flow rate of 1 µL/s for 8 h.
11. Transfer the plate to the washing dock and aspirate all medium from the wells.
12. Add 400 µL of coculture maintenance medium I (AdDMEM without dexamethasone but supplemented with hepatocyte maintenance supplements) to each well and initiate flow in the downward direction at a flow rate of 1 µL/s for 3.5 min.
13. Aspirate all medium from the reservoir side and add 1.4 mL of coculture maintenance medium I to each well.
14. Transfer the plate into the docking station within a humidified incubator at 37 °C and with 5% CO₂ and initiate flow in the upward direction at a flow rate of 1 µL/s for 48 h.
15. Transfer the plate to the washing dock and aspirate all medium from the wells. Add 400 µL of coculture maintenance medium II (Williams E medium without dexamethasone but supplemented with 100 nM hydrocortisone and hepatocyte maintenance supplements) and initiate flow in the downward direction at 1 µL/s for 3.5 min.
16. Aspirate all medium appearing on the reservoir side of the wells.
17. Add 1.4 mL of coculture maintenance medium II and transfer the plate into the docking station within a humidified incubator at 37 °C and with 5% CO₂.
18. Initiate flow in the upward direction at a flow rate of 1 µL/s for 48 h (Figure 1e).
19. Replace the medium every 48 h with coculture maintenance medium II. To ensure all medium in the well is replaced, perform a washing step before the addition of fresh medium.
20. To wash, transfer the plate to the washing dock, aspirate all medium from the wells, and add 400 µL of coculture maintenance medium II.
21. Initiate flow in the downward direction at 1 µL/s for 3.5 min. Aspirate all medium appearing on the reservoir side of the wells.
22. Add 1.4 mL of coculture maintenance medium II and transfer the plate into the docking station within a humidified incubator at 37 °C and with 5% CO₂, and initiate flow in the upward direction at a flow rate of 1 µL/s for 48 h (Figure 1f).

4. Production of an Infectious Hepatitis B Virus for Infection Studies

1. Perform this section of the protocol in a containment level III lab. Do the seeding, medium changes, medium collection, and virus concentration in a class II cabinet.
2. Culture HBV-producing cells (e.g., HepDE19, HepAD38) in collagen-coated T1000 5-layer flasks in 120 mL of complete DMEM/F12 (10% FBS, penicillin/steptomycin, nonessential amino acids, 500 μg/mL G418, and 1 μg/mL tetracycline) until they reach 90% confluency.
3. Change the medium to induction medium (complete DMEM without tetracycline) to induce the HBV production.
4. Collect the complete medium volume every 48 h for 12 days post-withdrawal of tetracycline and store it at 4 °C.
5. Filter the collected medium through a 0.45 µm bottle top filter.
6. Add sterile PEG 8000 in phosphate-buffered saline (PBS) to the collected medium to a final concentration of 4% w/w, mix by inverting 8x–10x, and incubate at 4 °C for 16 h. Centrifuge at 10,000 x g for 1 h at 4 °C to collect the PEG-precipitated virus and resuspend the pellet in PBS containing 10% FBS.
7. Combine the PEG-precipitated virus from all harvesting time points and layer it on top of a 20% sucrose cushion. Centrifuge at 140,000 x g for 16 h at 4 °C using an SW28 rotor.
8. Aspirate the supernatant and resuspend the pellet in PBS supplemented with 10% FBS, and aliquot and store it at -80 °C.
9. Determine the HBV DNA copy number present in the supernatant by HBV DNA qPCR (step 6).

5. Infection of 3D Cultures with HBV

1. Perform infections in a class II cabinet within a containment level III lab.
2. 3 days after seeding the monocultures or cocultures, thaw the required number of HBV-containing aliquots at room temperature and dilute the required virus dose in 1.8 mL of hepatocyte maintenance medium or coculture maintenance medium II per well, respectively.
3. This 1.8-mL diluted virus is sufficient for the washing step (400 µL) and the replacement of medium in the well (1.4 mL). However, the required multiplicity of infection needs to be adjusted to account for the final culture volume of 1.6 mL.
4. Transfer the plate to the washing dock and aspirate all medium from the wells. Add 400 µL of HBV-containing medium and initiate flow in the downward direction at 1 µL/s for 3.5 min. Aspirate all medium appearing on the reservoir side of the wells.
5. Add 1.4 mL of HBV-containing maintenance medium/coculture maintenance medium II per well and transfer the plate into the docking station within a humidified incubator at 37 °C and with 5% CO₂. Initiate flow in the downward direction at a flow rate of 1 µL/s for 8 h, followed by a reversal to the upward direction at a flow rate of 1 µL/s.
6. 24 h following the addition of HBV, transfer the plate to the washing dock and aspirate all medium from the wells.
7. Wash each well in the plate 3x with the corresponding medium, according to the type of culture as outlined in steps 2.12–2.14, to eliminate leftover virus from the well. In contrast to steps 2.12–2.14, add 1.6 mL of medium in every well to account for the extra volume to be sampled to exclude inoculum carryover (Figure 1g).
8. Following these washing steps, collect 200 µL of medium from each well to confirm the complete removal of the HBV inoculum by quantification of extracellular HBV DNA.
9. Transfer the plate to the docking station within a humidified incubator at 37 °C and with 5% CO₂, and initiate flow in the upward direction at a flow rate of 1 µL/s for 48 h.
10. 48 h later, collect the complete well volume for downstream analysis, followed by three washes with hepatocyte maintenance medium as outlined in steps 2.12–2.14. Replace the medium and wash each well 3x every 48 h until experimental termination.

6. Quantification of Extracellular HBV DNA

1. Isolate total DNA from the culture supernatants according to the manufacturer's instructions with the addition of 1 µg of carrier RNA in a containment level III lab to ensure the virus inactivation in the samples prior to moving them to a different area.
2. Prepare a master mix containing quantitative PCR master mix, 600 nM forward primer, 600 nM reverse primer, and 300 nM of probe.
3. Add 7 µL of the master mix into each well of a 384-well plate.
4. Add 5 µL of DNA samples in duplicate, a no-template control, and duplicates of serially diluted HBV genome-containing plasmid-based standard (e.g., pCMV-HBV) ranging from 10⁹ copies per reaction to 10² copies per reaction to each well of the qPCR plate.
5. Place an adhesive cover over the plate and ensure that each well is sealed correctly.
6. Centrifuge the plate for 1 min at 300 x g.
7. Start the qPCR run according to the manufacturer's instructions. The cycle conditions for real-time PCR are 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min.
8. Quantify the number of HBV DNA copies within the unknown samples according to the standard curve.

7. Quantification of Intracellular HBV Pregenomic (pg)RNA

1. Isolate total RNA from the scaffolds according to the manufacturer's instructions. In order to ensure complete cell lysis, vortex each scaffold 3x for 30 s followed by centrifugation at 300 x g for 1 min between each vortexing. Perform the cell lysis in the containment level III lab to ensure the virus inactivation in the samples prior to moving them to a different area.
2. Transcribe cDNA from the isolated RNA according to the manufacturer's instructions.
3. The cycle conditions for the retrotranscription are 25 °C for 10 min, 37 °C for 120 min, and 85 °C for 5 min.
4. Keep the cDNA samples at 4 °C for short-term or at -20 °C for long-term storage.
5. Prepare master mixes for pgRNA and RPS11 containing quantitative PCR master mix and forward and reverse primers for pgRNA and RPS11 (used as housekeeping gene) at a final concentration of 0.2 µM.
6. Add 7.5 µL of the master mix and 2.5 µL of cDNA per well on a 384-well plate. Measure RPS11 and pgRNA of each sample in duplicate and include a no-template control well for both genes.
7. Place an adhesive cover over the plate and ensure that each well is sealed completely.
8. Centrifuge the plate for 1 min at 300 x g.
9. Insert the plate into the qPCR cycler and start the qPCR run using the standard quantitative PCR protocol according to the manufacturer's instructions. The cycle conditions for real-time PCR are 50 °C for 2 min, 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min.
10. Calculate the expression of pgRNA relative to RPS11.

8. Immunofluorescence Staining of Viral Antigen

1. Remove the retaining ring from the well and remove the scaffold with forceps in a class II cabinet in the containment level III lab.
2. Fix the cell-containing scaffolds with 4% paraformaldehyde in 1 mL of PBS for 30 min at room temperature in the containment level III lab. The following steps can be performed in a different area.
3. Wash the scaffolds 3x with 1 mL of PBS.
4. Permeabilize the cells using 0.1% Triton-X 100 in 1 mL of PBS for 1 h at room temperature.
5. Wash the scaffolds 3x with 1 mL of PBS.
6. Block non-specific binding by incubation of the scaffolds with 1% BSA in 1 mL of PBS for 16 h at 4 °C.
7. Wash the scaffolds 3x with 1 mL of PBS.
8. Perform primary antibody staining using rabbit anti-hepatitis B virus core antigen at a dilution of 1:200 in 1% BSA in 1 mL of PBS for 16 h at 4 °C.
9. Wash the scaffolds 1x with 0.1% Tween in 1 mL of PBS (PBS-Tween) and 3x with 1 mL of PBS.
10. Perform secondary antibody staining using goat anti-rabbit IgG (H+L) cross-adsorbed Alexa Fluor 594-conjugated secondary antibody at a dilution of 1:2,000 in 1% BSA in 1 mL of PBS for 16 h at 4 °C.
11. Wash the scaffolds 1x with 1 mL of 0.1% PBS-Tween and 3x with 1 mL of PBS.
12. Counterstain the scaffolds using DAPI in 1 mL of PBS at a concentration of 2 µg/mL for 10 min at room temperature.
13. Wash the scaffolds 1x with 1 mL of 0.1% PBS-Tween and 3x with 1 mL of PBS.
14. Transfer the scaffolds to a microscope slide and mount it for imaging.
15. Image the scaffolds using a fluorescence microscope.

9. Human Albumin ELISA

1. Perform this section of the protocol in a class II cabinet allocated in the containment level III lab if working with infectious material.
2. To evaluate the viability and metabolic functionality of PHH, evaluate human albumin production by ELISA.
3. Coat 96-well plates with 50 µL per well of goat anti-human antibody diluted 1:800 in coating buffer (100 mM bicarbonate/carbonate, pH 9.6). Cover the plates and incubate for 2 h at 37 °C or overnight at 4 °C.
4. Aspirate the coating antibody from the plate and wash it 4x with 200 µL of 0.05% PBS-Tween.
5. Add 200 µL of blocking buffer (1% BSA in PBS), cover the plates, and incubate them for 1 h at 37 °C or store them at 4 °C for 3 months. For long-term storage, add 0.05% sodium azide to the blocking buffer.
6. Aspirate the blocking buffer and wash 1x with 200 µL of 0.05% PBS-Tween.
7. Add 50 µL of previously diluted samples per well (1:100). The sample diluent contains 1% BSA in 0.05% PBS-Tween. Incubate for 1 h at 37 °C or overnight at 4 °C.
   NOTE: Incubate the standards at the same time as the samples. A concentration range of 500–0.488 ng/mL (1:2 serial dilutions) is recommended. Perform all serial dilutions of human albumin in sample diluent.
8. Aspirate the samples from the plate and wash it 4x with 200 µL of 0.05% PBS-Tween.
9. Add 50 µL of HRP-conjugated goat anti-human albumin antibody previously diluted 1:10,000 in sample diluent. Incubate for 2 h at 37 °C or overnight at 4 °C.
10. Aspirate the antibody from the plate and wash it 6x with 200 µL of 0.05% PBS-Tween.
11. Add 100 µL of TMB reagent and, as soon as the highest standards are fully developed, add 100 µL 1 M H₂SO₄ to stop the colorimetric reaction.
12. Read the absorbance at 450 nm on a 96-well plate reader for analysis.

10. Interleukin (IL)6 and Tumor Necrosis Factors (TNF)α Production in 3D Co-cultures

1. Perform this section of the protocol in a class II cabinet allocated in the containment level III lab if working with infectious material.
2. Quantify the IL6 and TNFα production to evaluate the functionality and viability of the primary Kupffer cells. To induce the production of these cytokines by Kupffer cells, treat cocultures with 1 µg/mL lipopolysaccharide (LPS) 9 d post-seeding in the coculture maintenance medium II for 48 h.
3. At day 11 post-seeding, harvest the medium from each well and store it at -80 °C.
4. Measure the IL6 and TNFα concentration in the culture medium by an appropriate assay and according to the manufacturer's instructions.