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Jul 2021

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Production of Recombinant Hepatitis B virus (HBV) and Detection of HBV in Infected Human Liver Organoids
重组乙型肝炎病毒 (HBV) 的产生和受感染的人类肝脏类器官中 HBV 的检测   

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Abstract

The absence of long term, primary untransformed in vitro models that support hepatitis B virus (HBV) infection and replication have hampered HBV pre-clinical research, which was reflected in the absence of a curative therapy until recently. One of the limitations for in vitro HBV research has been the absence of high titer and pure recombinant HBV stocks, which, as we describe here, can be generated using simple, and reproducible protocols. In addition to infection of more conventional in vitro and in vivo liver model systems, recombinant high titer purified HBV stocks can also be used to efficiently infect differentiated human liver organoids, whose generation, maintenance, and infection is discussed in detail in a companion organoid protocol. Here, we also describe the protocols for the detection of specific viral read-outs, including HBV DNA in the supernatant of the cultures, covalently closed circular DNA (cccDNA) from intracellular DNA preparations, and HBV viral proteins and viral RNA, which can be detected within the cells, demonstrating the presence of a complete viral replication cycle in infected liver organoids. Although an evolving platform, the human liver organoid model system presents great potential as an exciting new tool to study HBV infection and progression to hepatocellular carcinoma (HCC) in primary cells, when combined with the use of high-titer and pure recombinant HBV stock for infection.


Graphical abstract:



Keywords: HBV infection (乙肝病毒感染), Production of HBV (乙肝病毒的生产), Heparin purification (肝素纯化), Readouts for viral products (病毒产品读数)

Background

HBV is a member of the hepadnaviridae family, a group of viruses harboring a partially double stranded DNA genome, which is replicated through an RNA intermediate. Infectious and replication-competent recombinant hepatitis B virus was generated from HepG2.2.15 cells, a HepG2-derived cell line stably transfected with the full-length HBV genome. Recombinant HBV, released in the supernatant of HepG2.2.15 cells, needs to be concentrated before infection of organoids, to get the desired amount of viral particles in a smaller volume and achieve the desired multiplicity of infection (MOI) per organoid. This can be easily done, using a commercial PEG virus precipitation kit (detailed protocol is given below) (De Crignis et al., 2021). To generate a higher-titer virus stock, which is purified by heparin affinity chromatography to remove subviral particles and enrich for complete HBV, we are currently using a modified version of a previously published protocol that increases the infection efficiency of the organoids (Wettengel et al., 2021). In the previously published protocol by Wettengel et al. (2021), sedimentation centrifugation by the sucrose-gradient system was used to further concentrate the heparin-column purified eluate HBV stock. Therefore, the final HBV stock that is generated via Wettengel et al. (2021) contains a high percentage of sucrose solution, which makes it unsuitable for infection of liver organoids, as the presence of high sucrose concentrations in the culture media can cause changes in osmolarity that result in the disruption of the 3D structure of the liver organoids. In the protocol described below, centrifugal filter units are used to further concentrate the eluate HBV stock purified through a heparin column, instead of sedimentation centrifugation by sucrose-gradient. Therefore, the final HBV stocks generated using this protocol are in a solution of Ad+++ (see Recipes section), which makes them suitable for use to infect human liver organoids. For more information on this, please refer to the companion protocol paper, which describes the generation, maintenance, and infection of the human liver organoids with HBV (Romal et al., 2022).


Upon infection, specific viral products that are produced at specific viral replication steps can be detected, which confirms the presence of a complete viral replication cycle in infected human liver organoids. Infection of liver organoids with recombinant HBV results in the generation of two different forms of HBV DNA, that can be detected using polymerase chain reaction (PCR). These are: 1) relaxed circular DNA (rcDNA), which is the actual genomic DNA of the virus, and can be detected both in DNA preparation from the culture supernatant as well as intracellular DNA; 2) cccDNA, which is generated inside the host cell after successful infection, and can only be detected from the intracellular DNA preparation. Detailed procedures for production of recombinant HBV virus and detection of viral products in human liver organoids are mentioned below.

Materials and Reagents

  1. Sterile filter pipette tips (Greiner Bio-One, catalog numbers: 774288 [P20]; 739288 [P200]; 740288 [P1000])

  2. Low retention sterile pipette tips with filter (Biotix, catalog numbers: M-0010-9FC [P10]; M-0020-9FC [P20]; M-0200-9FC [P200]; M-1000-9FC [P1000])

  3. Disposable sterile serological pipette with filter (VWR, catalog numbers: 89130-910 [10 mL]; 89130-890 [25 mL])

  4. 15 mL Falcon tubes (Greiner, catalog number: 188285)

  5. 50 mL Falcon tubes (Greiner, catalog number: 227285)

  6. T175 cell culture flask (Thermo Fisher Scientific, catalog number: 159920)

  7. Sterile syringe filter, 0.45 μm (GE Healthcare, Whatman, catalog number: 6896-2504)

  8. Sterile syringe filter, 0.2 μm (GE Healthcare, Whatman, catalog number: 6900-2502)

  9. 1.5 mL microcentrifuge tubes, (Biotix, catalog number: MT-0150-BC)

  10. 24 well suspension plates (Greiner, catalog number: 662102)

  11. 48 well suspension plates (Greiner, catalog number: 677102)

  12. 100 mm Cell culture dish (Thermo Fisher Scientific, catalog number: 150464)

  13. HYPERFlask® cell culture vessels (Sigma-Aldrich, Corning, catalog number: CLS10030)

  14. 500 mL Vacuum Filter/Storage Bottle System, 0.45 µm Pore (Sigma-Aldrich, Corning, catalog number: CLS430770)

  15. HiTrap® Heparin High Performance, 5 mL (GE Healthcare, catalog number: GE17-0407-01)

  16. Amicon® Ultra-15 Centrifugal Filter Unit, 100 KDa (Sigma-Aldrich, MerckMillipore, catalog number: UFC9100)

  17. CryoTubesTM Vials (Thermo Scientific, Nunc, catalog number: 366656)

  18. HepG2.2.15 cell line (CCTCC, catalog number: CCTCC-GDC0141)

  19. DMEM (Dulbecco's Modified Eagle Medium) (Thermo Fisher Scientific, GibcoTM, catalog number: 41966029)

  20. Fetal Bovine Serum (FBS) (Capricorn Scientific, catalog number: FBS-12A)

  21. Penicillin-Streptomycin (10,000 U/mL) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122)

  22. Advanced DMEM/F-12 (Thermo Fisher Scientific, GibcoTM, catalog number: 12634028)

  23. 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1 M Buffer Solution in 0.85%

  24. NaCl (Lonza, catalog number: BE17-737E)

  25. UltraGlutamineTM I 200 mM in 0.85% NaCl Solution (Lonza, catalog number: BE17-605E/U1)

  26. Collagen R solution 0.2% (Serva, catalog number: 47254.02)

  27. Trypsin-EDTA solution (Sigma-Aldrich, catalog number: T3924)

  28. PEG Virus Precipitation Kit (Abcam, catalog number: ab102538)

  29. Phosphate Buffered Saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023)

  30. Sodium chloride (Honeywell, catalog number: 71380)

  31. Ethanol absolute (Sigma-Aldrich, MerckMillipore, catalog number: 1009832500)

  32. Cultrex Basement Membrane Extract, Type 2, Pathclear (R&D Systems, catalog number: 3533-010-02)

  33. Phenol:Chloroform:Isoamyl Alcohol 25:24:1, Saturated with 10 mM Tris, pH 8.0, 1 mM EDTA

  34. Chloroform (Sigma, catalog number: p3803)

  35. Isoamyl alcohol (Merck, catalog number: 100979)

  36. Chloroform stabilized with Ethanol (Boom, catalog number: 76025322.2500)

  37. 2-propanol (isopropanol) (Honeywell, catalog number: 33539)

  38. QIAamp MinElute Virus Spin Kit (Qiagen, catalog number: 57704)

  39. Proteinase K (Sigma, catalog number: P2308)

  40. Sodium acetate (Honeywell, catalog number: 32319)

  41. Nuclease free water (Promega, catalog number: P1193)

  42. DNeasy Blood & Tissue Kit (Qiagen, catalog number: 69504)

  43. T5 Exonuclease: (New England Biolabs, catalog number: NEB:M0363)

  44. Plasmid-Safe ATP-Dependent DNase (Epicentre, catalog number: E3101K)

  45. Ethylenediaminetetraacetic acid disodium salt dehydrate (EDTA) (Sigma, catalog number: E1644)

  46. QIAquick PCR Purification Kit (Qiagen, catalog number: 28104)

  47. Tris (hydroxymethyl) aminomethane (THAM) hydrochloride (Sigma, catalog number: PHG0002)

  48. Sodium Hydroxide (Sigma, catalog number: S8045)

  49. NP-40 Surfact-AmpsTM Detergent Solution (Thermo Scientific, catalog number: 85124)

  50. Sodium dodecyl sulfate (Sigma, catalog number: 71729)

  51. 2-Butanol (Honeywell, catalog number: 19440)

  52. Glycogen (20 mg/mL), molecular biology grade (Thermo Scientific, catalog number: R0561)

  53. Potassium Acetate (Honeywell, catalog number: 236497)

  54. Ammonium acetate (Honeywell, catalog number: 17836)

  55. Platinum Taq DNA Polymerase (Invitrogen, catalog number: 10966034)

  56. 100 mM Deoxynucleoside triphosphate (dNTP) set (Invitrogen, catalog number: 10297-117)

  57. 2× Light Cycler 480 probe master (Roche, catalog number: 04887301001)

  58. TRI Reagent (Trizol) (Sigma, catalog number: T9424)

  59. RealiaPrep RNA cell miniprep system (Promega, catalog number: Z6012)

  60. DNase I (ThermoFisher, Invitrogen, catalog number: 18047019)

  61. Superscript II Reverse Transcriptase (ThermoFisher, Invitrogen, catalog number:18064022)

  62. Random Primers (ThermoFisher, Invitrogen, catalog number:48190011)

  63. Formaldehyde, 16%, methanol free, Ultra-Pure (Polysciences, catalog number: 18814)

  64. Acetone (Honeywell, catalog number: 00585)

  65. Glycine (Sigma, catalog number: G7126)

  66. Monolisa Hepatitis B surface Antigen (HBsAg) ULTRA (Bio-Rad, catalog number: 72346)

  67. Human Hepatitis B e Antigen (HBeAg) ELISA Kit (Cusabio, catalog number: CSB-E13557h)

  68. Hoechst 33342 trihydrochloride, trihydrate (Invitrogen, catalog number: H3570)

  69. Anti-Hepatitis B Virus Core Antigen antibody, (Abcam, catalog number: ab115992)

  70. Alexa FluorTM 488 Phalloidin (actin stain) (Invitrogen, catalog number: A12379)

  71. TrypLE Express (1×), no Phenol Red (Invitrogen, catalog number: 12604-013)

  72. Triton X-100 (Sigma aldrich, catalog number: 10789704001)

  73. Dimethyl sulfoxide, DMSO (Sigma aldrich, catalog number: D9170)

  74. Sodium phosphate dibasic dihydrate, Na2HPO4·2H2O (Sigma aldrich, catalog number: 71643)

  75. Potassium phosphate monobasic, KH2PO4 (Sigma aldrich, catalog number: P9791)

  76. Potassium chloride, KCl (Sigma aldrich, catalog number: P9541)

  77. Sodium Bicarbonate, NaHCO3 (Fisher Scientific, catalog number: S233-500)

  78. DMEM ++ culture medium (500 mL) (see Recipes)

  79. Ad+++ culture medium (500 mL) (see Recipes)

  80. 70% Ethanol solution (100 mL) (see Recipes)

  81. Wash solution (see Recipes)

  82. Elution buffer (100 mL) (see Recipes)

  83. Column wash buffer (100 mL) (see Recipes)

  84. Heparin column storage buffer (100 mL) (see Recipes)

  85. Cell lysis buffer (50 mL) (see Recipes)

  86. Alkali lysis buffer (50 mL) (see Recipes)

  87. PBSTD (50 mL) (see Recipes)

  88. 10× PBS solution (see Recipes)

Equipment

  1. Calibrated micropipettes (VWR, Gilson, catalog numbers: 613-5946 [P20]; 613-5948 [P200]; 613-5949 [P1000])

  2. Biosafety cabinet (Clean Air by Baker, Model name: BioVanguard Biological Safety cabinet-Class ll)

  3. Cell culture incubator with 5% CO2, 37°C (Panasonic catalog number: MCO-170AICUVH-PE)

  4. Eppendorf Centrifuge 5810 R for 15 mL Falcon tubes and cell culture plates (Eppendorf, model: A-4-62)

  5. Eppendorf Centrifuge 5417 R for 1.5 mL tubes (Eppendorf, catalog number: F45-30-11)

  6. Water bath 37°C (Grant, VFP)

  7. -80°C freezer (Panasonic, catalog number: MDF-794-PE)

  8. Freezing container (NalgeneTM Cryo, catalog number: 5100-0001)

  9. Refrigerator (Liebherr, catalog number: CP3523-22)

  10. Peristaltic Pump (Ismatec, model: ECOLINE VC-380)

  11. Eppendorf Thermomixer 5436 (Eppendorf, catalog number: T1442-1EA)

  12. Milli-Q® IQ 7000 Ultrapure Lab Water System (Merck)

  13. NanoDrop 2000/2000c spectrophotometers (Thermo Fisher Scientific, catalog number: ND2000LAPTOP)

  14. Bright field microscope: Leica DMIL microscope and a DFC420C camera

  15. Leica SP5 confocal

Software

  1. Leica LAS AF Lite software

Procedure

  1. Production of virus for PEG precipitation

    Day 1:

    1. Add 10 mL of 0.01% Collagen-R solution in sterile water to each 10-cm cell culture plate.

    2. Perform incubation at room temperature overnight.

    Day 2:

    1. Remove the collagen solution with a 10-mL pipette.

    2. Wash twice with 10 mL of PBS.

    3. Seed 3 × 106 HepG2.2.15 cells (up to passage 25) in 10 mL of DMEM ++ (see Recipes) per plate.

    4. Culture in the incubator at 37°C with 5% CO2 for 3-4 days (until the cells are 90–100% confluent).

    Day 5 (based on 90–100% confluency):

    1. Replace the culture medium to 10 mL of Ad+++ (see Recipes).

    2. Culture in the incubator at 37°C with 5% CO2 for 4 days.


    # Concentrating virus with PEG precipitation kit

    Day 8 (The complete procedure should be performed on ice):

    1. Collect the HBV-rich culture supernatant in a 50-mL Falcon tube.

    2. Centrifuge the culture supernatant at 3,200 × g and 4°C for 15 min, to remove cell-debris.

    3. Collect the supernatant in a new 50-mL Falcon tube.

    4. Add 2.5 mL of 5× PEG solution per 10 mL of HBV-rich culture supernatant and mix properly.

    5. Refrigerate the PEG-HBV culture supernatant mix overnight.

      Critical step: PEG-HBV is stable at 4°C for up to 2 days.

    Day 9 (The complete procedure should be performed on ice):

    1. Centrifuge the PEG-HBV culture supernatant mix at 3,200 × g and 4°C for 30 min.

    2. Carefully remove the supernatant with a 10-mL pipette. Do not touch the beige/white virus pellet.

    3. Resuspend the virus pellet that comes from 10 mL of culture supernatant in 100 μL of Ad+++, or proportionally adjust the volumes according to the initial volume of HBV-rich culture supernatant (e.g., if 50 mL of HBV-rich culture supernatant is used, then the final volume of Ad+++ to be used to resuspend the virus pellet will be 500 μL).

    4. Make small aliquots (100–500 μL) of virus stock and store at -80°C.

      Critical step: Avoid freeze/thaw cycles to maximize virus recovery.


  2. Production of high-titer purified and concentrated viral stock from HBV-rich HYPERFlask supernatant

    # Production of high-titer HBV-rich supernatant in HYPERFlask (The following protocol is designed for one HYPERFlask)

    Day 1:

    1. Add 20 mL of 0.01% Collagen-R solution in sterile water to each T175 cell culture flask.

    2. Prepare five T175 flasks for one HYPERFlask.

    3. Perform incubation at room temperature overnight.

    Day 2:

    1. Remove the collagen solution with a 10 mL pipette.

    2. Wash twice with 20 mL of PBS.

    3. Seed approximately 6 × 106 of HepG2.2.15 cells in 35 mL of DMEM++ per T175 flask.

    4. Culture in the incubator at 37°C with 5% CO2 until day 5.

    Day 4:

    1. Add 550 mL of 0.01% Collagen-R solution in sterile water to a 10-layer HYPERFlask cell culture vessel.

    2. Perform incubation at room temperature overnight.

    Day 5:

    # Preparation of HYPERFlask for cell seeding

    1. Remove the collagen solution by pouring it into a bottle.

      Optional step: Collect 0.01% Collagen R solution and store at 4°C for future HYPERflask collagen-coatings. The solution can be reused approximately five times.

    2. Wash twice with 300 mL of PBS.

    3. Label the two sides of the flask as A and B (Figure 1).



      Figure 1. Labelling of HYPERFlask.


    # Seeding of cells in the HYPERFlask

    1. Remove the culture media from the T175 flasks with a 10-mL pipette.

    2. Wash gently once with 20 mL of PBS.

    3. Add 10 mL of Trypsin-EDTA to each T175 flask and place it in the incubator, until all the cells are detached from the surface (the average time needed for the detachment of all the cells is 7–10 min).

    4. Stop the trypsin digestion by adding 20 mL of pre-warmed (at 37°C) DMEM++ culture media per T175 flask, which makes the final volume of liquid in each flask 30 mL.

    5. Collect 27 mL of cell suspension from each flask in a separate sterile bottle, and leave 3 mL.

    6. The remaining 3 mL of cell suspension can be cultured again. Add 27 mL of DMEM++ and culture in the incubator until day 8.

    7. Collect the total volume from step 17 for five T175 flasks (27 × 5 =135 mL) in a sterile bottle.

    8. Add 415 mL of pre-warmed DMEM++ media, to make the final volume 550 mL.

    9. Add the whole 550 mL of cell suspension (approximately 100 × 106 HepG2.2.15 cells) into the collagen coated HYPERFlask.

      Critical step: Remove all remaining air by applying pressure to the center of the HYPERFlask, enabling homogenous cell spreading, and guaranteeing space for medium extension during the 37°C incubation, to avoid a pressure burst of the HYPERFlask.

    10. Culture in the incubator at 37°C with 5% CO2 until day 8, with the A-labeled (step 12, Figure 1) side of the flask up.


    Day 8:

    1. Follow steps 13–16 again (# Seeding of cells in the HYPERFlask) for the five T175 flasks prepared at day 5, with the remaining 3 mL of cell suspension (step 17).

    2. Collect 30 mL of cell suspension from each flask in a separate sterile bottle.

    3. Collect the total volume from step 24 for five T175 flasks (30 × 5 =150 mL) in a sterile bottle.

    4. Add 400 mL of pre-warmed DMEM++ media, to make the final volume 550 mL.

    5. Remove the culture media from the HYPERFlask that was seeded at day 5.

    6. Wash gently once with 300 mL of PBS.

    7. Add 550 mL of cell suspension (from step 26) to the HYPERFlask (approximately 100 × 106 HepG2.2.15 cells).

    8. Culture in the incubator with the B-labeled (step 12, Figure 1) side up, which allows attachment and growth of cells on opposite sides of the HYPERFlask layers.

    9. Harvest the supernatant for purification in 4-day intervals (3–4 harvest cycles per HYPERFlask; see Notes).

      Critical step: Due to the presence of a high number of dead and non-attached cells, discard the first supernatant collection after each cell plating round.

      Notes:

      1. Do not harvest supernatant earlier than 2 days after changing the culture medium, to ensure that enough mature HBV virus particles are secreted in the supernatant. Cells will also start to detach and die after 5–6 days without media change. It is recommended to harvest supernatant and add fresh media in alternating 3–4 days interval.

      2. The titer of HBV in the HYPERFlask supernatant should be ≥107 copies/mL, to achieve the desired final concentration.

      3. Store cell culture supernatants at 4°C overnight, before purification to precipitate serum lipoproteins and cell debris.

      4. Do not store supernatant at 4°C for more than one day, as HBV infectivity drops with increasing storage time.

      5. Harvest supernatant 3–4 times (depending on cell attachment) from each HYPERFlask.

      6. After harvesting the supernatant four times, the HYPERFlask can be treated with Trypsin-EDTA, washed with PBS, and kept in a sterile environment, to reuse for production of subsequent rounds of HBV-rich supernatant. It is recommended to incubate the HYPERFlask with collagen-R solution before each round of cell seeding.

      7. After seeding the HYPERFlask, the HepG2.2.15 cells are cultured in DMEM++ medium, as the HepG2.2.15 cell growth is optimal in DMEM++. However, Ad+++ and not DMEM++ medium is optimal for culturing organoids. Therefore, in section A (Production of virus for PEG precipitation) step 7, the culture medium is changed into Ad+++, to ensure the absence of any residual DMEM in the concentrated virus stock, as the HBV-rich supernatant is directly used to precipitate virus with PEG. However, in the HYPERFlask, while HepG2.2.15 cells are cultured in DMEM++ to ensure their optimum growth, the HBV-rich supernatant from the HYPERFlask is passed through the heparin column after collection, to purify the virus stock. The purified virus is eluted with a high salt-containing buffer, the eluate is diluted four times with Ad+++, and it is then further concentrated using a centrifugal filter unit. Following these steps ensures the absence of residual DMEM++ in the purified and concentrated virus stock from the HYPERFlask.


    # Purification and concentration of high titer HBV stock (The complete procedure should be performed on ice or at 4°C)

    1. Cold HYPERFlask supernatant is centrifuged at 500 × g and 4°C for 5 min, to precipitate cell debris.

    2. Clear supernatant from the top is filtered through a 0.45 µm sterile filter, to remove remaining cell debris (see Notes).

    3. The silicone tubing is attached to a peristaltic pump (Figure 2). One end of the silicone tubing is connected to the heparin column, using a connector which is provided with the column. The remaining end of the silicone tubing is submerged in buffers or supernatant when required. Ensure that the flow direction of the peristaltic pump is from the buffers or supernatant end of the tubing to where the heparin column is attached.

    4. The whole tubing is flushed with 50 mL of 70% ethanol solution.



      Figure 2. Assembly of purification apparatus for perfusion of HYPERFlask supernatant (up) and elution of HBV particle (down).


    1. Afterward, the tubing is washed with 50 mL of 1× PBS.

      Critical step: Ensure that all the bubbles are removed and the tubing is completely filled with PBS, to prevent damage of the heparin columns.

    2. Serially connect two heparin columns (HiTrap heparin 5 mL) for every 550 mL of supernatant (Figure 2).

      Critical step: The heparin columns can also be connected in parallel using a stopcock. If connected in parallel, then the flow speed in step 7 can be 20 mL/min for two columns.

    3. Keep the filtered supernatant on ice, and perfuse it through the heparin columns at a flow rate of 10 mL/min.

      Critical step: A higher perfusion flow rate might reduce the lifetime of the columns.

    4. After a complete perfusion of the supernatant, remove the columns and flush the system with elution buffer.

      Critical step: Ensure that all the bubbles are removed and the tubing is completely filled with elution buffer.

    5. Re-attach one column at a time, and elute with elusion buffer with a 2 mL/min flow rate.

      Critical step: Before re-attaching the column, ensure that there is no air left in the system. Release a little bit of elution buffer in the connection point of the column, and then re-attach the column with the tubing.

      Critical Step: Two columns can be re-attached together in a serial connection. If re-attached serially, then discard the first 4 mL of the eluent, due to the dead volume, and collect the next 40 mL.

    6. Collect 20 mL of eluent per column in a sterile tube (40 mL from two columns).

      Critical Step: Keep the elution buffer at room temperature, and also collect the eluent and store at room temperature until step 11. Storing eluent on ice or using ice cold elution buffer may result in precipitation of HBV particles, due to the high salt concentration in the elution buffer.

    7. Immediately add 120 mL of Ad+++ to 40 mL of eluent (final volume =160 mL), to dilute the high salt concentration present in the elution buffer.

    8. Add 12–15 mL of Ad+++ to one Amicon® Ultra-15 Centrifugal Filter Unit of 100 KDa cutoff, and centrifuge at 3,000 × g and 4°C for 10 min, to wash the filter.

    9. Discard the flow through medium.

    10. Add 12–15 mL of the purified viral solution to one Amicon® Ultra-15 Centrifugal Filter Unit.

      Optional: Using multiple filter units will reduce the processing time.

    11. Centrifuge at a maximum speed of 3,000 × g and 4°C for 10 min.

    12. Discard the flow through filtrate from the bottom of the tube, and top up the remaining residual volume again to 12–15 mL with purified viral solution (step 11).

    13. Follow steps 13 and 14 again until the whole 160 mL (step 11) is filtered, and the remaining final volume of purified and concentrated HBV stock is 2 mL (starting from 550 mL of HYPERFlask supernatant).

      Critical Step: After 2–3 centrifugation rounds, the remaining viral solution starts to become sticky and might block the filter of the centrifugal unit. Pipetting up and down with a 1,000 μL micropipette during the topping up the centrifugal unit (step 16) helps to homogenize the solution and prevents blocking of the filter.

    1. Make 100–500 µL aliquots of virus stock and store it at -80°C for future use.

      Critical step: Avoid freeze/thaw cycles to maximize virus recovery and infectivity.

    2. Take 5 µL of purified and concentrated virus, and bring the volume up to 200 µL.

    3. Titer the virus stock after isolation of HBV DNA followed by qPCR (see section E).

      Note: In the HYPERFlask cell culturing system, a large number of cells are cultured in a single HYPERFlask, resulting in a generous amount of cell debris and proteins present in the supernatant, which can block the heparin column if not removed. To ensure the complete removal of cell debris and proteins, centrifugation of the HYPERFlask supernatant alone is not enough, and filtering with a 0.45 µm sterile filter is essential. However, in section A (Production of virus for PEG precipitation), only 10 mL of supernatant from a single 10 cm cell culture plate is processed in a single 50 mL Falcon tube, which contains limited amounts of cell debris for which centrifugation alone is sufficient to remove.


    # Maintenance of purification apparatus and heparin columns

    1. Flush the column with 20 mL of 10× PBS solution per heparin column, at a 5 mL/min flow rate.

    2. Flush again with 20 mL of column storage buffer, and store the columns in an air-tight manner (by tightening the screw caps provided with the column) at 4°C.

    3. Clean the tubing with 70% ethanol, to inactivate any remaining HBV.


  3. Production of heat-inactivated HBV

    1. Thaw the pre-tittered active recombinant HBV on ice.

    2. Transfer the virus to a 1.5-mL microcentrifuge tube.

    3. Centrifuge at maximum speed and 4°C for 10 min.

    4. Transfer the virus to a new 1.5-mL microcentrifuge tube.

    5. Boil the virus at 100°C for 30 min.

    6. Centrifuge at maximum speed and 4°C for 10 min.

    7. Use the supernatant as heat-inactivated virus.


  4. Brief description of the generation, maintenance, and HBV infection of human liver organoids

    There is a companion protocol paper that describes the culture method for human liver organoids, and their infection with recombinant HBV (Romal et al., 2022). A very brief description of that protocol paper is given below.

    1. Liver biopsies are dissociated into single cell level, mixed with BME/Ad+++, and cultured as a dome shaped drop, using a specialized culture medium that enables the growth of LGR5+ adult hepatic stem cells and formation of organoids.

    2. Organoids are cultured in an expansion medium that allows the continuous growth of human liver organoids.

    3. Culture medium is changed into differentiation medium, which halts the proliferation of the organoids and induces their differentiation into hepatocytes, as well as expression of differentiated hepatocyte markers and the HBV receptor NTCP, which is necessary for the infection of the organoids with HBV.

    4. Differentiated organoids are mixed with the appropriate amount of HBV and spin infected.

    5. After spin infection, infected organoids are cultured again in 24-well cell culture plates in dome shaped drops containing BME/Ad+++ (Section D, step 31 in Romal et al., 2022).


  5. Isolation and detection of HBV DNA from the supernatant

    HBV DNA from the supernatant can be isolated by either using available commercial kits or using the phenol-chloroform isolation method. A detailed protocol is described below:


    # Isolation of HBV DNA from supernatant with a commercial isolation kit:

    1. Take 200 µL of the culture supernatant (see section D, step 5).

    2. The HBV DNA from the supernatant is isolated using the QIAamp MinElute Virus Spin Kit, following the manufacturer’s instructions.


    # Isolation of HBV DNA from the supernatant using the phenol-chloroform isolation method:

    1. Take 200 µL of the supernatant.

    2. Add 300 µL of 1% SDS+ 0.1M NaHCO3 in MilliQ-water supplemented with 100 µg/mL proteinase K.

    3. Incubate the samples at 55°C for 1 h.

    4. Briefly spin down to collect all the components in the bottom of the tube.

    5. Add 500 µL of phenol-chloroform-isoamyl alcohol (PCI), and vortex thoroughly for 10 s.

    6. Centrifuge at maximum speed and room temperature for 5 min.

    7. Transfer the top aqueous phase to a new 1.5-mL microcentrifuge tube.

    8. Add 500 µL of 24:1 chloroform-isoamyl alcohol (CI), and vortex thoroughly for 10 s.

    9. Centrifuge at maximum speed and room temperature for 5 min.

    10. Transfer the top aqueous phase into a new 1.5-mL microcentrifuge tube, add 1 µL of glycogen from the stock (20 mg/mL), and 30 µL of 3M NaOAc pH 5.2. Vortex for 10 s, add 1 mL of 100% ethanol, and vortex again thoroughly for 10 s.

    11. Snap-freeze the microcentrifuge tubes in liquid nitrogen or freeze at -80°C for 30 min or overnight.

    12. Spin the DNA down at maximum speed and 4°C for 30 min.

    13. Wash the pellet with 500 µL of 70% ethanol.

    14. Spin down at maximum speed and 4°C for 15 min.

    15. Carefully remove the supernatant.

    16. Resuspend the DNA pellet in 30 µL of nuclease free-water.

    17. Store the DNA at -20°C.


    # Detection of HBV DNA

    Component Final concentration
    2× LightCycler480 Probes Master (Roche) 1× (12.5 µL)
    Forward Primer (100 µM) 0.5 µM
    Reverse Primer (100 µM) 0.5 µM
    Probe (50 µM) 0.1 µM
    DNA template 4 µL
    Nuclease free water x µL
    Total reaction volume 25 µL

    * Primer and probe sequences: Forward (5’-GCAACTTTTTCACCTCTGCCTA-3’)

                                                      Reverse (5’-AGTAACTCCACAGTAGCTCCAAATT-3’)

                                                      Probe (FAM-TTCAAGCCTCCAAGCTGTGCCTTGGGTGGC-BHQ1)

    1. Prepare the reaction mix and briefly centrifuge to collect all the components at the bottom of the PCR plate.

    2. Run the amplification, starting with 95°C for 10 min, followed by 45 cycles of 95°C for 10 s, 60°C for 30 s, and 72°C for 10 s.


    # Analysis and expected results:

    The qPCR reaction to detect HBV DNA from the supernatant included a standard curve made from dilutions of a plasmid containing the lenti-HBV construct with the 1.3mer HBV genome, ranging from 2 to 2 × 104 copies of plasmid. The number of HBV DNA copies is calculated using the standard curve. Figure 3A represents a PCR curve of different samples with the HBV specific primers and probe. For each PCR reaction, 4 µL of the DNA template is used and the starting volume of the DNA preparation of each sample condition is 30 µL. The culture supernatants from the uninfected organoids and the organoids infected with heat-inactivated HBV are used as negative control. The culture supernatant from the HepG2.2.15 cells is used as positive control. A standard curve is generated with 2, 20, 200, and 2000 copies of lenti-HBV plasmid construct containing 1.3 mer HBV genome. There are no amplifications observed in the uninfected organoids or the organoids infected with heat-inactivated HBV. The fluorescent signal curves for the infected organoids, HepG2.2.15 cells, and the standard plasmid dilutions are depicted in purple, red, and green, respectively. Figure 3B represents the individual and average (cycle threshold) Ct values for the different sample conditions. The standard curve (Figure 3C) is prepared by plotting the Ct values of the different standard plasmid dilutions in the y-axis, and the corresponding logarithm of the standard plasmid copies in the x-axis. The r2 value and the equation for the standard line are also mentioned in Figure 3C. Using the average Ct values for the infected organoids and HepG2.2.15 cells, and the standard line equation, the number of HBV DNA copies present per mililiter of the corresponding culture supernatants are calculated (Figure 3C). The bar graph in Figure 3D depicts the HBV DNA copy numbers present in the different sample conditions.



    Figure 3. Expected results for the detection of HBV DNA from the culture supernatants.

    A) PCR Curve of the different sample conditions. B) Observed Ct values and average Ct values for the different sample conditions, C) Generation of the standard curve using the Ct values of different dilutions of the standard plasmids, and calculation of the copies of HBV DNA present in the infected organoids and the HepG2.2.15 cells. D) Bar graph of the HBV DNA copy numbers present in the different samples. The y-axis represents the HBV DNA copy numbers, and the x-axis represents different sample conditions.


  6. Isolation and detection of intracellular HBV DNA and cccDNA

    All the available techniques to detect cccDNA have their own sets of limitations. Therefore, using multiple techniques simultaneously is recommended. In our study, we have performed both treatment of whole genomic DNA with T5 exonuclease, and alkali lysis plasmid DNA isolation, followed by plasmid-safe digestion to differentiate cccDNA from rcDNA. Detailed protocols are described below:

    1. Prepare a 15-mL Falcon tube containing 10 mL of Ad+++.

    2. Remove the medium from each well of organoids (see section D, step 5).

    3. Add 1 mL of Ad+++ to each well of organoids, and collect 4–8 wells of a 24-well plate of organoids in each 15-mL Falcon tube.

    4. Incubate on ice for 30 min.

    5. Centrifuge at 200 × g and 4°C for 5 min.

    6. Remove the supernatant.

    7. Add 60–100 µL of TrypLE (a substitute for trypsin that is used to dissociate adherent cells and primary human cell cultures) to the sample, and mechanically digest for 30–60 s at room temperature.

    8. Wash the organoids with ice-cold PBS.

    9. Centrifuge at 200 × g and 4°C for 5 min.

    10. Remove the supernatant, leaving the pellet of digested organoids.


    # Whole genomic DNA isolation followed by T5 exonuclease digestion

    T5 exonuclease degrades all the linear and partially circular dsDNA. However, the enzyme does not degrade completely circular supercoiled dsDNA. T5 exonuclease is used to remove the rcDNA present in the whole genomic DNA preparation, which is partially double stranded, but will not degrade the completely circular and double stranded cccDNA.

    1. The pellet (section F, step 10) is used for whole genomic DNA isolation using the DNeasy Blood and Tissue kit.

    2. Continue the isolation, following the manufacturer’s instructions.

    3. Elute the final DNA from the column in 50 µL of nuclease free-water.

    4. Use 25 µL for T5 exonuclease digestion, and the remaining 25 µL for detection of intracellular HBV DNA.

    5. Follow the manufacturer’s instructions, to continue the digestion with T5 exonuclease.

    6. Inactivate the digestion by adding EDTA, to a final concentration of 11 mM.

    7. Purify the complete inactivated reaction volume using the QIAquick PCR Purification Kit.

    8. Continue the purification, following the manufacturer’s instructions.

    9. Elute the purified DNA from the column in 30 µL of nuclease free-water.


    # For the alkali lysis plasmid DNA-isolation followed by plasmid safe digestion

    1. Resuspend the pellet (section F, step 10) in 800 µL of ice-cold cell lysis buffer.

    2. Incubate on ice for 10 min.

    3. Add equal volume of alkali lysis buffer.

    4. Incubate the samples at 37°C for 30 min.

    5. Neutralize the DNA by adding 3 M potassium acetate (CH3COOK) (pH 5.0), to a final concentration of 0.6 M.

    6. Centrifuge at maximum speed and room temperature for 5 min.

    7. Transfer the supernatant to two new 1.5 mL microcentrifuge tubes, and add ~900 µL of phenol-chloroform-isoamyl alcohol (PCI). Vortex thoroughly for 10 s.

    8. Centrifuge at maximum speed and room temperature for 5 min.

    9. Transfer the top aqueous phase to a new 1.5 mL microcentrifuge tube.

    10. Repeat steps 7–9.

    11. Centrifuge at maximum speed and room temperature for 5 min.

    12. Add 500 µL of butanol:isopropanol (7:3), and vortex thoroughly for 10 s.

    13. Centrifuge at maximum speed and room temperature for 5 min.

    14. Transfer the bottom layer of each tube into two new 1.5 mL microcentrifuge tubes. (The bottom layers from two tubes will be distributed equally into four tubes.)

    15. Add 200 µL of 7.5 M Ammonium Acetate (CH3COONH4), 1 µL of glycogen stock (20 mg/mL), and 1 mL of 100% ethanol.

    16. Snap-freeze the microcentrifuge tubes in liquid nitrogen, or freeze at -80°C for 30 min or overnight.

    17. Spin the DNA down at maximum speed and 4°C for 30 min.

    18. Wash the pellet with 1 mL of 70% ethanol.

    19. Spin down at maximum speed and 4°C for 15min.

    20. Carefully remove the supernatant.

    21. Resuspend the DNA pellet in 50 µL of nuclease free-water.

      Pause step: DNA can be stored at -20°C for one year, until processing for further digestion.

    22. Take 25 µL of the DNA.

    23. Digest the samples using plasmid safe, following the manufacturer’s protocol.

    24. After digestion, top the volume up with PBS to 400 µL.

    25. Add 400 µL of phenol-chloroform-isoamyl alcohol (PCI). Vortex thoroughly for 10 s.

    26. Centrifuge at maximum speed and room temperature for 5 min.

    27. Take the top aqueous phase and transfer to a new 1.5 mL microcentrifuge tube.

    28. Add 400 µL of 24:1 Chloroform-isoamyl alcohol (CI). Vortex thoroughly for 10 s.

    29. Centrifuge at maximum speed and room temperature for 5 min.

    30. Transfer the top aqueous phase into a new 1.5 mL microcentrifuge tube, add 1 µL of glycogen from the stock (20 mg/mL), and 20 µL of 3M NaOAc pH 5.2. Vortex for 10 s, add 1 mL of 100% ethanol, and vortex thoroughly for 10 s.

    31. Snap-freeze the microcentrifuge tubes in liquid nitrogen, or freeze at -80°C for 30 min or overnight.

    32. Spin the DNA down at maximum speed and 4°C for 30 min.

    33. Wash the pellet with 500 µL of 70% ethanol.

    34. Spin down at maximum speed and 4°C for 15 min.

    35. Carefully remove the supernatant.

    36. Resuspend the DNA pellet in 30 µL of nuclease free-water.


    # Detection of intracellular HBV DNA

    Component Final concentration
    10× PCR Buffer 1× (2.5 µL)
    50 mM MgCl2 1.75 mM
    10 mM dNTP mix 400 µM
    Forward Primer (100 µM) 0.5 µM
    Reverse Primer (100 µM) 0.5 µM
    Probe (50 µM) 0.15 µM
    Platinum Taq DNA Polymerase 0.04 units/µL
    DNA template 7.5 µL
    Nuclease free water x µL
    Total reaction volume 25 µL

    * Primer and probe sequences: Forward (5’-GCAACTTTTTCACCTCTGCCTA-3’)

    Reverse (5’-AGTAACTCCACAGTAGCTCCAAATT-3’)

    Probe (FAM-TTCAAGCCTCCAAGCTGTGCCTTGGGTGGC-BHQ1)

    1. Prepare the reaction mix and briefly centrifuge, to collect all the components at the bottom of the PCR plate.

    2. Run the amplification starting with 95°C for 10 min, followed by 45 cycles of 95°C for 10 s, 60°C for 30 s, and 72°C for 10 s.


    # Detection of cccDNA

    Component Final concentration
    2× LightCycler480 Probes Master (Roche) 1× (10 µL)
    Forward Primer (100 µM) 1 µM
    Reverse Primer (100 µM) 1 µM
    Probe (50 µM) 0.2 µM
    DMSO 4%
    cccDNA template 4.2 µL
    Nuclease free water x µL
    Total reaction volume 20 µL

    * Primer and probe sequences: Forward (5’-GTCTGTGCCTTCTCATCTGC-3’)

    Reverse (5’-AGTAACTCCACAGTAGCTCCAAATT-3’)

    Probe (FAM-TTCAAGCCTCCAAGCTGTGCCTTGGGTGGC-BHQ1)

    1. Prepare the reaction mix and briefly centrifuge, to collect all the components at the bottom of the PCR plate.

    2. Run the amplification starting with 95°C for 10 min, followed by 50 cycles of 95°C for 15 s, and 61°C for 1 min, as mentioned in a previously published study (Winer et al., 2017).


    # Analysis and the expected results:

    Both PCRs to detect intracellular HBV DNA and cccDNA included a standard curve made from dilutions of a plasmid containing the lenti-HBV plasmid construct with 1.3mer HBV genome, ranging from 2 to 2 × 104 copies of plasmid. The number of intracellular HBV DNA and cccDNA copies were calculated using the standard curve. Figure 4A represents a PCR curve of the different samples, with the cccDNA specific primers and probe. For each PCR reaction, 4.2 µL of the DNA template is used, and the starting volume of the DNA preparation of each sample condition is 30 µL. DNA preparations from uninfected organoids and HepG2.2.15 cell culture supernatant, which should only contain rcDNA, are used as negative control. To generate a standard curve, 2, 20, 200, and 2000 copies of lenti-HBV plasmid construct containing 1.3 mer HBV genome are used. There is no amplification observed in the uninfected organoids (red) or HepG2.2.15 cell culture supernatant (navy blue). The fluorescent signal curves for the infected organoids and the standard plasmid dilutions are depicted in purple and green, respectively. Figure 4B represents the individual and average Ct values for the different sample conditions. The standard curve (Figure 4C) is prepared by plotting the Ct values of the different standard plasmid dilutions in the y-axis, and the corresponding logarithm of the standard plasmid copies in the x-axis. The r2 value and the equation for the standard line are also mentioned in Figure 4C. Using the average Ct value for the infected organoids and the standard line equation, the number of cccDNA copies present in the infected organoid sample is calculated (Figure 4C). The bar graph in Figure 4D depicts the cccDNA copy number present in the different samples.



    Figure 4. Expected results for the detection of cccDNA.

    A) PCR Curve of the different sample conditions. B) Observed Ct values and average Ct values for the different sample conditions, C) Generation of the standard curve using the Ct values of different dilutions of the standard plasmids, and calculation of the copies of cccDNA present in the infected organoids sample. D) Bar graph of the cccDNA copy numbers present in the different samples. The y-axis represents the cccDNA copy numbers, and the x-axis represents the different samples.


    Figure 5A represents a PCR curve of different samples with the HBV specific primers and probe. For each PCR reaction, 7.5 µL of the DNA template is used, and the starting volume of the DNA preparation for each sample is 30 µL. The uninfected organoids are used as negative control. The HepG2.2.15 cells are used as a positive control. To generate a standard curve, 2, 20, 200, and 2000 copies of lenti-HBV plasmid construct containing 1.3 mer HBV genome are used. There is no amplification observed in the uninfected organoids sample. The fluorescent signal curves for the infected organoids, HepG2.2.15 cells, and the standard plasmid dilutions are depicted in purple, red, and green, respectively. Figure 5B represents the individual and average Ct values for the different samples. The standard curve (Figure 5C) is prepared by plotting the Ct values of the different standard plasmid dilutions in the y-axis, and the corresponding logarithm of the standard plasmid copies in the x-axis. The r2 value and the equation for the standard line are also mentioned in Figure 5C. Using the average Ct values for the infected organoids and HepG2.2.15 cells, and the standard line equation, the number of intracellular HBV DNA copies present are calculated (Figure 5C). The bar graph in Figure 5D depicts the intracellular HBV DNA copy numbers present in the different samples.



    Figure 5. Expected results for the detection of intracellular HBV DNA from the culture supernatants.

    A) PCR Curve of the different sample conditions. B) Observed Ct values and average Ct values for the different sample conditions, C) Generation of the standard curve using the Ct values of different dilutions of the standard plasmids, and calculation of the copies of intracellular HBV DNA present in the infected organoids and the HepG2.2.15 cells. D) Bar graph of the intracellular HBV DNA copy numbers present in the different samples. The y-axis represents the intracellular HBV DNA copy numbers, and the x-axis represents the different sample conditions.


  7. Isolation and detection of HBV RNA

    Isolation of the HBV RNA can be performed using both a commercial kit and Trizol RNA isolation procedure. Detailed protocols are described below:

    1. Remove the medium from the organoids.

    2. Collect 1–2 wells of a 24-well plate of organoids (see section D; step 5) with 1 mL of Ad+++, mix by pipetting 4–5 times, and transfer to a 1.5-mL microcentrifuge tube.

      Critical Step: One well of a 24-well plate with 90% density, or if the density is low use two wells of a 24-well plate. The samples can be stored in -80°C for six months, or can be directly isolated.

    3. Incubate on ice for 10–20 min.

    4. Centrifuge the samples at 200 × g and 4°C for 5 min.

    5. Remove the supernatant, leaving the organoids pellet behind.


    # Isolation of RNA using commercial kit

    1. Resuspend the organoids pellet (see section G, step 5) with 500 µL of cell lysis buffer provided in the kit (RealiaPrep RNA Cell Miniprep System, Promega).

    2. Continue the isolation, following the manufacturer’s instructions.

    3. Elute the RNA in 40 µL of nuclease free-water.

    4. Store the RNA at -80°C, until further processing.


    # Isolation of RNA using Trizol RNA isolation protocol

    1. Lyse the organoid pellet (see section G, step 5) with 1 mL of Trizol, by pipetting up and down, and vortexing vigorously for 20–40 s.

    2. Incubate the sample at room temperature for 5 min.

    3. Add 200 μL of chloroform, vortex the samples vigorously for 20 s, and incubate at room temperature for 2–3 min.

    4. Centrifuge the samples at 12,000 × g and 4°C for 15 min.

    5. Carefully transfer the upper aqueous phase, without disturbing the interphase, into a new 1.5-mL microcentrifuge tube.

    6. Precipitate the RNA by adding 500 μL of isopropyl alcohol, inverting the tubes four to five times, and incubating the samples at room temperature for 10 min.

    7. Centrifuge the samples at 12,000 × g and 4°C for 10 min.

    8. Remove the supernatant.

      Critical Step: To avoid losing the RNA pellet, especially when the pellet is not visible or is very small, leave ~50 μL of the supernatant.

    9. Add 1 mL of 75% ethanol, and centrifuge at 7,500 × g and 4°C for 5 min.

      Critical Step: Do not vortex the RNA pellet.

    10. Repeat step 9, and remove the wash buffer completely.

      Critical Step: To make sure that the wash buffer is completely removed, briefly centrifuge the samples, to get rid of the remaining wash buffer from the edges of the tube.

    11. Air-dry the pellet for 10 min.

    12. Resuspend the pellet in 40 μL of nuclease free water.

    13. Store the RNA samples at -80°C.


    #DNase l treatment

    1. Use 1 μL of the sample to measure the RNA concentration using a NanoDrop 2000/2000c spectrophotometer.

    2. Use 300–1,000 ng of RNA for DNase l treatment.

    3. Complete the DNase treatment, following the manufacturer’s instruction.


    # cDNA synthesis

    1. Use the DNase l treated samples for cDNA synthesis with Superscript II Reverse Transcriptase kit.

    2. Complete the cDNA synthesis, following the manufacturer’s instructions, and using random primers.


    # Detection of HBV RNA

    Component Final concentration
    10× PCR Buffer 1× (2.5 µL)
    50 mM MgCl2 1.75 mM
    10 mM dNTP mix 400 µM
    Forward Primer (100 µM) 0.5 µM
    Reverse Primer (100 µM) 0.5 µM
    Probe (50 µM) 0.15 µM
    Platinum Taq DNA Polymerase 0.04units/µL
    cDNA template (diluted 1:2.5 or 1:5 in water, based on the starting amount of RNA used for cDNA synthesis) 4 µL
    Nuclease free water x µL
    Total reaction volume 25 µL

    * Primer and probe sequences (HBV): Forward (5’-GCAACTTTTTCACCTCTGCCTA-3’)

    Reverse (5’-AGTAACTCCACAGTAGCTCCAAATT-3’)

    Probe (FAM-TTCAAGCCTCCAAGCTGTGCCTTGGGTGGC-BHQ1)

    * Primer and probe sequences (Beta-2-microglobulin):

    Forward (5’- AGCGTACTCCAAAGATTCAGGTT-3’)

    Reverse (5’- ATGATGCTGCTTACATGTCTCGAT-3’)

    Probe (FAM- TCCATCCGACATTGAAGTTGACTTACTG-BHQ1)

    1. Prepare the reaction mix, and briefly centrifuge to collect all the components at the bottom of the PCR plate.

    2. Run the amplification starting with 95°C for 10 min, followed by 45 cycles of 95°C for 10 s, 60°C for 30 s, and 72°C for 10 s.


    # Analysis and the expected results:

    Beta-2-microglobulin (B2M) is used as a housekeeping control for analysis of cDNA sample expression. Fold increase is calculated using the 2-ΔΔCt method. Figure 6A represents PCR curves of different samples with the HBV specific primers and probe, and the B2M specific primers and probe. For each PCR reaction, 4 µl of the cDNA template is used. The uninfected organoids and the organoids infected with heat-inactivated HBV are used as negative control. The HepG2.2.15 cells are used as positive control. The fluorescent signal curves for the uninfected organoids, infected organoids, HepG2.2.15 cells, and the organoids infected with heat-inactivated HBV are depicted in blue, purple, red, and green, respectively. Figure 6B represents the individual and average Ct values for the different samples, as well as the calculation to determine the fold changes of the HBV RNA expression in the infected organoids and the HepG2.2.15 cells compared to the organoids infected with heat-inactivated HBV. There is no expression of HBV RNA in the uninfected organoids. Therefore, the uninfected organoids sample is excluded from the fold change calculation. The bar graph in Figure 6C depicts the fold changes of the HBV RNA expression in the different samples.



    Figure 6. Expected results for the detection of HBV RNA.

    A) PCR Curve of the different sample conditions with HBV specific (left) and B2M specific (right) primers and probes. B) Observed Ct values and average Ct values, as well as the HBV RNA fold change calculations for the different samples compared to the organoids infected with heat-inactivated HBV. C) Bar graph of the HBV RNA fold changes present in the different samples. The y-axis represents the fold changes, and the x-axis represents the different sample conditions.


  8. Immunofluorescence

    Critical step: Use an ultra-low retention pipette tip to reduce sample loss while triturating.

    First, pre-wet the pipette with the wash medium or buffer solution, and then resuspend the sample.

    1. Prepare a 15-mL Falcon tube with 10 mL of ice-cold Ad+++, and place it on ice.

    2. Remove the medium from all the wells (see section D, step 5).

    3. Add ~1 mL of ice-cold Ad+++ to each well.

    4. Collect the drops containing the organoids with a 10 mL pipette, by scrapping gently.

      Critical step: Avoid breaking the organoids.

    5. Mix three to four times by triturating with a 10-mL pipette.

    6. Incubate on ice for 30 min to 1 h.

      Critical step: Mix the organoids by inverting the Falcon tubes four to five times every 5 min. This will help the BME to dissolve efficiently.

    7. Pellet the organoids by centrifuging at 100 × g at 4°C for 5 min.

    8. Wash 1× with 10 mL of ice-cold Ad+++, mix by pipetting up and down four to five times with a 10 mL pipette.

    9. Centrifuge at 100 × g and 4°C for 5 min.

    10. Remove the medium and wash once with 10 mL of ice-cold PBS.

    11. Remove the medium and gently resuspend the pellet with 200 µL of freshly prepared fixative solution [either 4% PFA for HBcAg, Hepatocyte Nuclear Factor 4 Alpha (HNF4α), sodium taurocholate co-transporting polypeptide (NTCP), and Albumin, or 100% acetone for HBsAg].

      Critical step: Fixative reagent and concentration can differ depending on the antibodies.

    12. Incubate on ice for 30 min.

    13. Remove fixative solution and add 10 mL of ice-cold PBS.

    14. Centrifuge at 100 × g and 4°C for 5 min.

    15. Remove the PBS.

    16. Add 1 mL of freshly made 0.1 M glycine in PBS.

    17. Incubate at room temperature for 15–30 min.

      Critical step: Treating organoids with glycine after fixation will work as a quencher. This can help in blockage of unreacted aldehydes, which may can cause an increase in background fluorescence.

    18. Add 10 mL of PBS, and centrifuge at 100g × g for 5 min.

      Pause step: Fixed organoids can be stored at 4°C for 3–4 weeks.

    19. Remove the PBS.

    20. Permeabilize the organoids by adding 1 mL of 0.3% Triton in PBS (HBcAg, HNF4α, and NTCP), 1% Triton X-100 in PBS (Albumin), or 100% acetone (HBsAg).

    21. Incubate at room temperature for 30 min.

    22. Centrifuge at 100 × g for 5 min, and remove the permeabilizing solution.

    23. Wash once with 1 mL of 0.5% FBS in PBS at room temperature.

    24. Centrifuge at 100 × g and room temperature for 5 min.

    25. Add 1 mL of blocking buffer (PBSTD; PBS + 0.5% serum + 0.3% triton+ 1% DMSO for HBcAg, HNF4α, Albumin, and NTCP, or 10% BSA+ 0.5% serum in PBS for HBsAg), and incubate at room temperature for 2 h.

      Optional: Use goat serum for the blocking step, in case all the secondary antibodies are from goat.

    26. Centrifuge at 100 × g and room temperature for 5 min.

    27. Remove the blocking buffer.

    28. Incubate the organoids with primary antibody diluted in PBS + 10% blocking buffer (check the dilutions as recommended by the manufacturers).

    29. Incubate in an orbital shaker at 4°C overnight.

    30. Centrifuge at 100 × g and 4°C for 5 min.

    31. Remove the primary antibody mix.

    32. Add 10 mL of 0.5% FBS in PBS. Incubate in an orbital shaker at room temperature for 10 min. Remove the wash buffer and repeat this step at least three times.

    33. Add 200 μL of 0.5% FBS in PBS containing the appropriate secondary antibody, and incubate in an orbital shaker in the dark at room temperature for 2 h.

    34. Remove the secondary antibody.

    35. Wash by adding 10 mL of 0.5% FBS in PBS. Incubate in an orbital shaker at room temperature for 10 min.

    36. Centrifuge at 100 × g and room temperature for 5 min.

    37. Repeat steps 35–36 at least four to five times.

    38. Add 200 μL of 0.5% FBS in PBS containing 5 μg/mL of Hoechst.

    39. Incubate in an orbital shaker in the dark at room temperature for 15–20 min.

    40. Remove the Hoechst and wash with 1 mL of 0.5% FBS in PBS.

    41. Incubate in an orbital shaker in the dark at room temperature for 10 min.

    42. Centrifuge at 100 × g and room temperature for 5 min.

    43. Remove the supernatant using a plastic Pasteur pipette, and transfer the organoids to the slide.

    44. Add one drop of anti-fade solution, and apply the coverslip.

    45. Store the slides in the dark at 4°C.

    46. Images are taken and processed using Leica LAS AF Lite software (Leica SP5 confocal) (Figure 7).



      Figure 7. Immunofluorescence staining showing the expression of HBV core antigen (HBcAg) (magenta) together with actin (green), performed in healthy donor liver organoids six days post HBV infection.


  9. Detection of HBsAg and HBeAg from the supernatant

    1. Collect the organoid supernatant at 3–6 days post infection (see section D, step 5).

    2. Spin the samples down at 200 × g and 4°C for 5 min.

    3. Use 50 μL of the supernatant for detection of HBsAg or HBeAg.

    4. Use the HBeAg (Human Hepatitis Be Antigen ELISA Kit, Cusabio) or HBsAg (Monolisa HBs Ag ULTRA, Bio-Rad) specific kits for the detection of HBeAg and HBsAg, respectively.


    # Analysis and the expected results:

    Commercially available ELISA kits are used to detect HBsAg and HBeAg from the culture supernatants. Figure 8A represents the absorbances to detect HBsAg, measured as the optical densities (OD) at a wavelength of 450 nm for the different samples, calculation of the corrected cut-off value using absorbances of the negative samples provided by the kit manufacturer, and the signal to cut-off values for the different samples, as well as the bar diagram depicting the expressions of HBsAg in the different samples. Figure 8B represents the absorbances to detect HBeAg measured as OD450 for the different samples, calculation of the corrected cut-off value using absorbances of the negative samples provided by the kit manufacturer, and the signal to cut-off values for the different samples, as well as the bar diagram depicting the expressions of HBeAg in the different samples.



    Figure 8. Expected results for the detection of HBsAg and HBeAg.

    A) Absorbances to detect HBsAg, measured as OD450 for the different samples, calculation of the corrected cut-off value using absorbances of the negative samples provided by the kit manufacturer, and the signal to cut-off values for the different samples, as well as the bar diagram depicting the expressions of HBsAg in the different samples. The y-axis represents the OD at 450 nm S/CO and the x-axis represents the different samples. B) Absorbances to detect HBeAg measured as OD450 for the different samples, calculation of the corrected cut-off value using absorbances of the negative samples provided by the kit manufacturer, and the signal to cut-off values for the different samples, as well as the bar diagram depicting the expressions of HBeAg in the different samples. The y-axis represents the OD at 450 nm S/CO, and the x-axis represents the different samples.

Recipes

  1. DMEM ++ culture medium (500 mL)

    445 mL of DMEM

    50 mL of FBS

    5 mL of Penicillin-Streptomycin

  2. Ad+++ culture medium (500 mL)

    485 mL of DMEM

    5 mL of HEPES

    5 mL of Ultraglutamine

    5 mL of Penicillin-Streptomycin

  3. 70% Ethanol solution (100 mL)

    70 mL of Ethanol absolute

    30 mL of Milli-Q treated Water

  4. Wash solution

    500 mL of 1× PBS, pH 7.4

  5. Elution buffer (100 mL)

    25 mL of 10× PBS, pH 7.4

    75 mL of Milli-Q treated Water

  6. Column wash buffer (100 mL)

    40 mL of 5M NaCl

    60 mL of Milli-Q treated Water

  7. Heparin column storage buffer (100 mL)

    13 mL of 10× PBS, pH 7.4

    20 mL of Ethanol absolute

    67 mL of Milli-Q treated Water

  8. Cell lysis buffer (50 mL)

    100 μL of 0.5M EDTA (pH 8.0)

    250 μL of 1 M Tris HCl (pH 7.5)

    250 μL of 10% Nonidet P-40

    49.4 mL of Milli-Q treated Water

  9. Alkali lysis buffer (50 mL)

    5 mL of 1 M NaOH

    30 mL of 10% SDS

    15 mL of Milli-Q treated Water

  10. PBSTD (50 mL)

    250 μL of FBS

    150 μL of Triton X-100

    500 μL of DMSO

    49.1 mL of PBS

  11. 10× PBS (500 mL)

    8.9 g of Na2HPO4·2H2O

    1.2 g of KH2PO4

    40 g of NaCl

    1 g of KCl

    Adjust the final volume to 500 mL with Milli-Q treated Water.

Acknowledgments

Protocol for the generation of high-titer HBV stock has been adapted from a recently published protocol by Wettengel et al. (2021) and necessary modifications have been added to make the virus stock suitable to infect the human liver organoids. Protocol for the detection of the different HBV specific viral products upon infection of the human liver organoids with recombinant HBV has been adapted from the previously published work by the authors (De Crignis et al., 2021).

Competing interests

The authors declare no conflict of interest.

References

  1. De Crignis, E., Hossain, T., Romal, S., Carofiglio, F., Moulos, P., Khalid, M. M., Rao, S., Bazrafshan, A., Verstegen, M. M. and Pourfarzad, F. (2021). Application of human liver organoids as a patient-derived primary model for HBV infection and related hepatocellular carcinoma. Elife 10: e60747.
  2. Wettengel, J. M., Linden, B., Esser, K., Laue, M., Burwitz, B. J. and Protzer, U. (2021). Rapid and Robust Continuous Purification of High-Titer Hepatitis B Virus for In Vitro and In Vivo Applications. Viruses 13(8): 1503.
  3. Winer, B. Y., Huang, T. S., Pludwinski, E., Heller, B., Wojcik, F., Lipkowitz, G. E., Parekh, A., Cho, C., Shrirao, A., Muir, T. W., et al. (2017). Long-term hepatitis B infection in a scalable hepatic co-culture system. Nat Commun 8(1): 125.
  4. Romal, S., Hossain, T. and Mahmoudi, T. (2022). Generation, Maintenance and HBV Infection of Human Liver Organoids. Bio-protocol 12(6): e4358.

简介

[摘要]缺乏支持乙型肝炎病毒 (HBV) 感染和复制的长期、原始的未转化体外模型阻碍了 HBV 的临床前研究,这反映在直到最近还没有治愈性疗法。体外HBV 研究的限制之一是缺乏高滴度和纯重组 HBV 储备,正如我们在此描述的,可以使用简单且可重复的方案生成。除了感染更常规的体外和体内肝脏模型系统外,重组高效价纯化的 HBV 原种也可用于有效感染分化的人类肝脏类器官,其生成、维持和感染在配套类器官协议中进行了详细讨论.在这里,我们还描述了检测特定病毒读数的协议,包括培养物上清液中的 HBV DNA, 来自细胞内 DNA 制剂的共价闭合环状 DNA ( cccDNA ) 以及可在细胞内检测到的 HBV 病毒蛋白和病毒 RNA,表明在受感染的肝脏类器官中存在完整的病毒复制周期。虽然是一个不断发展的平台,但当与高滴度和纯重组 HBV 原液结合使用时,人类肝脏类器官模型系统作为研究原代细胞中 HBV 感染和进展为肝细胞癌 (HCC) 的令人兴奋的新工具具有巨大潜力感染。


图文摘要:

[背景] 乙肝病毒是其中的一员 嗜肝DNA病毒科 家族,一组病毒,含有部分双链 DNA 基因组,通过 RNA 中间体进行复制。 HepG2.2.15 细胞是一种稳定转染全长 HBV 基因组的 HepG2 衍生细胞系,可产生感染性和复制能力强的重组乙型肝炎病毒。在 HepG2.2.15 细胞的上清液中释放的重组 HBV 需要在感染类器官之前进行浓缩,以在更小的体积内获得所需数量的病毒颗粒,并达到所需的每个类器官的感染复数 (MOI)。这可以使用商业 PEG 病毒沉淀试剂盒轻松完成(详细方案如下) (De Crignis等人,2021) 。为了产生更高滴度的病毒原液,通过肝素亲和层析纯化以去除亚病毒颗粒并富集完整的 HBV,我们目前正在使用先前发布的协议的修改版本,以提高类器官的感染效率(Wettengel等人. , 2021) . 在Wettengel之前发布的协议中 等人。 (2021 年),蔗糖梯度系统的沉降离心被用于进一步浓缩肝素柱纯化的洗脱液 HBV 储备液。因此,通过Wettengel生成的最终 HBV 库存 等人。 (2021) 含有高百分比的蔗糖溶液,这使得它不适合感染肝脏类器官,因为培养基中高浓度蔗糖的存在会导致渗透压的变化,从而导致肝脏类器官的 3D 结构被破坏.在下面描述的协议中,离心过滤装置用于进一步浓缩通过肝素柱纯化的洗脱液 HBV 库存,而不是通过蔗糖梯度进行沉降离心。因此,使用该协议生成的最终 HBV 库存在 Ad+++ 的解决方案中(参见食谱部分),这使得它们适合用于感染人类肝脏器官。有关这方面的更多信息,请参阅配套协议文件,该文件描述了人类肝脏类器官的产生、维持和感染 HBV ( Romal 等人, 2022)。
感染后,可以检测到在特定病毒复制步骤产生的特定病毒产物,这证实了受感染的人类肝脏器官中存在完整的病毒复制周期。用重组 HBV 感染肝脏类器官会导致产生两种不同形式的 HBV DNA,这可以使用聚合酶链式反应 (PCR) 进行检测。它们是: 1) 松弛环状 DNA ( rcDNA ),它是病毒的实际基因组 DNA,既可以在从培养上清液制备的 DNA 中检测到,也可以在细胞内 DNA 中检测到; 2) cccDNA ,成功感染后在宿主细胞内产生,只能从细胞内DNA制剂中检测到。下文提到了重组 HBV 病毒的生产和人类肝脏类器官中病毒产物检测的详细程序。

关键字:乙肝病毒感染, 乙肝病毒的生产, 肝素纯化, 病毒产品读数



材料和试剂


1.无菌过滤器吸头(Greiner Bio-One,目录号:774288 [P20];739288 [P200];740288 [P1000])
2.带过滤器的低保留无菌移液器吸头( Biotix ,目录号:M-0010-9FC [P10];M-0020-9FC [P20];M-0200-9FC [P200];M-1000-9FC [P1000])
3.带过滤器的一次性无菌血清移液管(VWR,目录号:89130-910 [10 mL];89130-890 [25 mL])
4.15 mL Falcon 管(Greiner,目录号:188285)
5.50 mL Falcon 管(Greiner,目录号:227285)
6.T175细胞培养瓶(Thermo Fisher Scientific,目录号:159920)
7.无菌注射器过滤器,0.45 μm (GE Healthcare,Whatman,目录号:6896-2504)
8.无菌注射器过滤器,0.2 μm (GE Healthcare,Whatman,目录号:6900-2502)
9.1.5 mL微量离心管,(Biotix,目录号:MT-0150-BC)
10.24孔悬浮板(Greiner,目录号:662102)
11.48孔悬浮板(Greiner,目录号:677102)
12.100 mm 细胞培养皿(Thermo Fisher Scientific,目录号:150464)
13.HYPERFlask ®细胞培养容器(Sigma-Aldrich,Corning,目录号:CLS10030)
14.500 mL 真空过滤器/储存瓶系统,0.45 µm 孔(Sigma-Aldrich,Corning,目录号:CLS430770)
15.HiTrap ® Heparin High Performance,5 mL(GE Healthcare,目录号:GE17-0407-01)
16.Amicon ® Ultra-15 离心过滤装置,100 KDa(Sigma-Aldrich,MerckMillipore,目录号:UFC9100)
17.CryoTubes TM Vials (Thermo Scientific,Nunc,目录号:366656)
18.HepG2.2.15细胞系(CCTCC,目录号:CCTCC-GDC0141)
19.DMEM(Dulbecco's Modified Eagle Medium)(Thermo Fisher Scientific, Gibco TM ,目录号:41966029)
20.胎牛血清(FBS)(Capricorn Scientific,目录号:FBS-12A)
21.青霉素-链霉素(10,000 U/mL)(Thermo Fisher Scientific, Gibco TM ,目录号:15140122)
22.Advanced DMEM/F-12(Thermo Fisher Scientific, Gibco TM ,目录号:12634028)
23.4-(2-羟乙基)-1-哌嗪乙磺酸 (HEPES),1 M 缓冲溶液,0.85%
24.NaCl(Lonza,目录号:BE17-737E)
25.UltraGlutamine TM I 200 mM 溶于 0.85% NaCl 溶液(Lonza,目录号:BE17-605E/U1)
26.胶原蛋白R溶液0.2%( Serva ,目录号:47254.02)
27.胰蛋白酶-EDTA 溶液(Sigma-Aldrich,目录号:T3924)
28.PEG病毒沉淀试剂盒(Abcam,目录号:ab102538)
29.磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific, Gibco TM ,目录号:10010023)
30.氯化钠(霍尼韦尔,目录号:71380)
31.无水乙醇(Sigma-Aldrich, MerckMillipore ,目录号:1009832500)
32.Cultrex基底膜提取物,2 型, Pathclear (R&D Systems,目录号:3533-010-02)
33.苯酚:氯仿:异戊醇 25:24:1,用 10 mM Tris 饱和,pH 8.0,1 mM EDTA
34.氯仿(Sigma,目录号:p3803)
35.异戊醇(Merck,目录号:100979)
36.用乙醇稳定的氯仿(Boom,目录号:76025322.2500)
37.2-丙醇(异丙醇)(Honeywell,目录号:33539)
38.QIAamp MinElute Virus Spin Kit (Qiagen,目录号:57704)
39.蛋白酶K(Sigma,目录号:P2308)
40.乙酸钠(Honeywell,目录号:32319)
41.无核酸酶水(Promega,目录号:P1193)
42.DNeasy Blood & Tissue Kit(Qiagen,目录号:69504)
43.T5核酸外切酶:(New England Biolabs,目录号: NEB:M0363)
44.质粒安全的ATP依赖性DNase( Epicenter ,目录号:E3101K)
45.乙二胺四乙酸二钠盐脱水(EDTA)(Sigma,目录号:E1644)
46.QIAquick PCR Purification Kit(Qiagen,目录号:28104)
47.三(羟甲基)氨基甲烷(THAM)盐酸盐(Sigma,目录号:PHG0002)
48.氢氧化钠(Sigma,目录号:S8045)
49.NP-40 Surfact-Amps TM洗涤剂溶液(Thermo Scientific,目录号:85124)
50.十二烷基硫酸钠(Sigma,目录号:71729)
51.2-丁醇(霍尼韦尔,目录号:19440)
52.糖原(20 mg/mL),分子生物学级(Thermo Scientific,目录号:R0561)
53.乙酸钾(Honeywell,目录号:236497)
54.乙酸铵(Honeywell,目录号:17836)
55.Platinum Taq DNA 聚合酶(Invitrogen,目录号:10966034)
56.100 mM脱氧核苷三磷酸(dNTP)组(Invitrogen,目录号:10297-117)
57.2 × Light Cycler 480 probe master(Roche,目录号:04887301001)
58.TRI试剂( Trizol )(Sigma,目录号:T9424)
59.RealiaPrep RNA 细胞小量制备系统(Promega ,目录号:Z6012)
60.DNase I( ThermoFisher , Invitrogen,目录号:18047019)
61.Superscript II 逆转录酶( ThermoFisher ,Invitrogen,目录号:18064022)
62.随机引物( ThermoFisher ,Invitrogen,目录号:48190011)
63.甲醛,16%,无甲醇,超纯( Polysciences ,目录号:18814)
64.丙酮(霍尼韦尔,目录号:00585)
65.甘氨酸(Sigma,目录号:G7126)
66.Monolisa乙型肝炎表面抗原( HB sAg )ULTRA(Bio-Rad,目录号: 72346 )
67.人乙型肝炎e抗原(HBeAg)ELISA试剂盒( Cusabio ,目录号:CSB-E13557h)
68.Hoechst 33342三盐酸盐,三水合物(Invitrogen,目录号:H3570)
69.抗乙型肝炎病毒核心抗原抗体,(Abcam,目录号:ab115992)
70.Alexa Fluor TM 488 Phalloidin(肌动蛋白染色剂)(Invitrogen,目录号:A12379)
71.TrypLE Express(1 × ),无酚红(Invitrogen,目录号:12604-013)
72.Triton X-100(Sigma aldrich,目录号:10789704001)
73.二甲基亚砜,DMSO(Sigma aldrich,目录号:D9170)
74.磷酸氢二钠二水合物, Na 2 HPO 4 · 2H 2 O (Sigma aldrich,目录号:71643)
75.磷酸二氢钾, KH 2 PO 4 (Sigma aldrich,目录号:P9791)
76.氯化钾,KCl(Sigma aldrich,目录号:P9541)
77.碳酸氢钠,NaHCO 3 (Fisher Scientific,目录号:S233-500)
78.DMEM ++ 培养基(500 mL)(参见食谱)
79.Ad+++ 培养基(500 mL)(参见食谱)
80.70% 乙醇溶液 (100 mL)(参见食谱)
81.洗涤液(见配方)
82.洗脱缓冲液 (100 mL)(参见配方)
83.柱洗涤缓冲液 (100 mL)(参见配方)
84.肝素柱储存缓冲液(100 mL)(参见配方)
85.细胞裂解缓冲液(50 mL) (参见配方)
86.碱裂解缓冲液(50 mL)(参见食谱)
87.PBSTD(50 毫升) (见食谱)
88.10 × PBS 溶液(见配方)


设备


1.校准的微量移液器(VWR,Gilson,目录号: 613-5946 [ P 20];613-5948 [P200];613-5949 [P1000] )
2.生物安全柜(贝克清洁空气,型号名称: BioVanguard生物安全柜-II级)
3.细胞培养箱,5% CO 2 ,37°C(Panasonic 目录号:MCO-170AICUVH-PE)
4.Eppendorf Centrifuge 5810 R 适用于 15 mL Falcon 管和细胞培养板(Eppendorf,型号:A-4-62)
5.用于1.5 mL管的Eppendorf Centrifuge 5417 R(Eppendorf,目录号:F45-30-11)
6.水浴 37°C (Grant, VFP)
7.-80°C冰箱(Panasonic,目录号:MDF-794-PE)
8.冷冻容器( Nalgene TM 冷冻,目录号:5100-0001)
9.冰箱(Liebherr,目录号:CP3523-22)
10.蠕动泵( Ismatec ,型号:ECOLINE VC-380)
11.Eppendorf Thermomixer 5436(Eppendorf,目录号:T1442-1EA)
12.Milli-Q ® IQ 7000 超纯实验室水系统(默克)
13.NanoDrop 2000/2000c 分光光度计(Thermo Fisher Scientific,目录号:ND2000LAPTOP)
14.明场显微镜:Leica DMIL 显微镜和 DFC420C 相机
15.徕卡 SP5 共焦


软件


1.徕卡 LAS AF Lite 软件


程序


A.PEG沉淀病毒生产
第 1 天:
1.在每个 10 cm 细胞培养板中加入 10 mL 的 0.01% Collagen-R 无菌水中的溶液。
2.在室温下过夜孵育。
第 2 天:
3.用 10 mL 移液器去除胶原蛋白溶液。
4.用 10 mL 的 PBS 清洗两次。
5.种子 3 × 10 6 HepG2.2.15 细胞(直至第 25 代)在每板 10 mL DMEM ++(参见食谱)中。
6.在 37°C 和 5% CO 2的培养箱中培养3-4 天(直到细胞达到 90 – 100% 融合)。
第 5 天(基于 90–100% 融合):
7.将培养基更换为 10 mL 的 Ad+++(参见食谱)。
8.在 37°C 和 5% CO 2的培养箱中培养4 天。


# 用 PEG 沉淀试剂盒浓缩病毒
第 8 天(完整的程序应在冰上进行):
9.在 50 mL Falcon 管中收集富含 HBV 的培养上清液。
10.以 3,200 离心培养上清液 × g和 4°C 15 分钟,以去除细胞碎片。
11.将上清液收集到一个新的 50 mL Falcon 管中。
12.每 10 mL 富含 HBV 的培养上清液加入 2.5 mL 的 5 × PEG 溶液,并适当混合。
13.将 PEG-HBV 培养上清液混合物冷藏过夜。
 关键步骤: PEG-HBV 在 4°C 下可稳定长达 2 天。
第 9 天(完整的程序应在冰上进行):
14.将 PEG-HBV 培养上清液混合物在 3,200 × g和 4°C 下离心 30 分钟。
15.用 10 mL 移液器小心取出上清液。不要触摸米色/白色病毒颗粒。
16.将来自 10 mL 培养上清液的病毒颗粒重悬于 100 μL Ad+++ 中,或根据富含 HBV 的培养上清液的初始体积按比例调整体积(例如,如果使用 50 mL 的富含 HBV 的培养上清液,那么用于重悬病毒颗粒的 Ad+++ 的最终体积将为 500 μL )。
17.制作小份 (100–500 μL ) 病毒原液并储存在 -80°C。
 关键步骤:避免冷冻/解冻循环以最大限度地恢复病毒。


B.从富含 HBV 的HYPERFlask上清液中生产高效价纯化和浓缩的病毒原液
# 在HYPERFlask中生产高滴度富含 HBV 的上清液(以下方案是为一个HYPERFlask设计的)
第 1 天:
1.在每个 T175 细胞培养瓶中加入 20 mL 的 0.01% 胶原蛋白-R 溶液在无菌水中。
2.HYPERFlask准备五个 T175 烧瓶。
3.在室温下过夜孵育。
第 2 天:
4.用 10 mL 移液器去除胶原蛋白溶液。
5.用 20 mL 的 PBS 清洗两次。
6.播种约 6 × 10 6个 HepG2.2.15 细胞。
7.在 37°C 和 5% CO 2的培养箱中培养至第 5 天。
第 4 天:
8.在无菌水中加入 550 mL 的 0.01% 胶原蛋白-R 溶液到 10 层HYPERFlask细胞培养容器中。
9.在室温下过夜孵育。
第 5 天:
# 准备用于细胞接种的HYPERFlask
10.将胶原蛋白溶液倒入瓶子中,取出胶原蛋白溶液。
 可选步骤:收集 0.01% Collagen R 溶液并储存在 4°C 以备将来使用 HYPERflask胶原蛋白涂层。该溶液可重复使用大约五次。
11.用 300 mL 的 PBS 清洗两次。
12.将烧瓶的两侧标记为 A 和 B(图 1)。


 
图 1. HYPERFlask的标签。


# 在HYPERFlask中播种细胞
13.用 10 mL 移液器从 T175 烧瓶中取出培养基。
14.用 20 mL 的 PBS 轻轻清洗一次。
15.在每个 T175 烧瓶中加入 10 mL 的胰蛋白酶-EDTA,并将其放入培养箱中,直到所有细胞从表面分离(所有细胞分离所需的平均时间为 7-10 分钟)。
16.通过在每个 T175 烧瓶中添加 20 mL 预热(37°C)的 DMEM++ 培养基来停止胰蛋白酶消化,这使得每个烧瓶中的液体最终体积为 30 mL。
17.从每个烧瓶中收集 27 mL 的细胞悬浮液,放入单独的无菌瓶中,并留下 3 mL。 
18.剩余的 3 mL 细胞悬液可再次培养。在孵化器中加入 27 mL 的 DMEM++ 和培养物,直到第 8 天。
19.从步骤 17 中收集五个 T175 烧瓶(27 × 5 = 135 mL)的总体积,放入无菌瓶中。
20.添加 415 mL 的预热 DMEM++ 介质,使最终体积为 550 mL。 
21.将整个 550 mL 的细胞悬浮液(约 100 × 10 6 HepG2.2.15 细胞)加入胶原蛋白涂层的HYPERFlask中。
关键步骤:通过向HYPERFlask的中心施加压力来去除所有剩余的空气,从而实现均匀的细胞扩散,并在 37°C 孵育期间保证培养基延伸的空间,以避免HYPERFlask的压力爆裂。
22.在 37°C 和 5% CO 2的培养箱中培养直到第 8 天,烧瓶的 A 标记(步骤 12,图 1)侧朝上。


第 8 天:
23.对于第 5 天准备的五个 T175 烧瓶,再次执行步骤 13-16(# 在HYPERFlask中接种细胞),剩余 3 mL 细胞悬液(步骤 17)。
24.从每个烧瓶中收集 30 mL 的细胞悬浮液,放入单独的无菌瓶中。
25.收集步骤 24 中五个 T175 烧瓶(30 × 5 = 150 mL)的总体积。
26.添加 400 mL 的预热 DMEM++ 介质,使最终体积为 550 mL。
27.第 5 天播种的HYPERFlask中取出培养基。
28.用 300 mL 的 PBS 轻轻清洗一次。
29.将 550 mL 的细胞悬浮液(来自步骤 26)添加到HYPERFlask (大约 100 × 10 6 HepG2.2.15 细胞)。
30.在培养箱中培养,B 标记(步骤 12,图 1)朝上,这允许细胞在HYPERFlask层的相对两侧附着和生长。
31.HYPERFlask 3-4 个收集周期;见注释)。
 关键步骤:由于存在大量死亡和未附着的细胞,在每个细胞电镀轮后丢弃第一个上清液收集。
 笔记:
a.不要在更换培养基后 2 天之前收集上清液,以确保上清液中分泌足够的成熟 HBV 病毒颗粒。细胞也将在 5-6 天后开始分离并死亡,无需更换培养基。建议每隔 3-4 天收集上清液并添加新鲜培养基。
b.HYPERFlask上清液中的 HBV 滴度应≥10 7拷贝/mL,以达到所需的最终浓度。
c.将细胞培养上清液在 4°C 下储存过夜,然后进行纯化以沉淀血清脂蛋白和细胞碎片。
d.不要将上清液在 4°C 下储存超过 1 天,因为 HBV 感染性会随着储存时间的延长而下降。
e.HYPERFlask中收集上清液 3-4 次(取决于细胞附着) 。
f.收获上清液四次后,可以用胰蛋白酶-EDTA 处理HYPERFlask ,用 PBS 洗涤,并保持在无菌环境中,以重复用于生产后续轮次富含 HBV 的上清液。建议在每轮细胞接种前用胶原蛋白-R 溶液孵育HYPERFlask 。
g.接种HYPERFlask后,HepG2.2.15 细胞在 DMEM++ 培养基中培养,因为 HepG2.2.15 细胞在 DMEM++ 中的生长是最佳的。然而,Ad+++ 而不是 DMEM++ 培养基最适合培养类器官。因此,在 A 部分(PEG 沉淀的病毒生产)步骤 7 中,将培养基更换为 Ad+++,以确保浓缩的病毒原液中没有任何残留的 DMEM,因为富含 HBV 的上清液直接用于沉淀病毒与 PEG。然而,在HYPERFlask中,而 HepG2.2.15 细胞 在 DMEM++ 中培养以确保其最佳生长,来自HYPERFlask的富含 HBV 的上清液在收集后通过肝素柱,以纯化病毒原液。纯化的病毒用高盐缓冲液洗脱,洗脱液用Ad+++稀释四倍,然后用离心过滤器进一步浓缩。遵循这些步骤可确保HYPERFlask中纯化和浓缩的病毒储备中不存在残留的 DMEM++ 。


# 高效价 HBV 原液的纯化和浓缩(整个过程应在冰上或 4°C 下进行)
1.冷的HYPERFlask上清液在 500 × g和 4°C 下离心 5 分钟,以沉淀细胞碎片。
2.顶部的透明上清液通过 0.45 µm 无菌过滤器过滤,以去除剩余的细胞碎片(见注释)。
3.硅胶管连接到蠕动泵(图 2)。硅胶管的一端连接到肝素柱,使用与柱一起提供的连接器。硅胶管的剩余端在需要时浸入缓冲液或上清液中。确保蠕动泵的流动方向是从管道的缓冲液或上清液端到肝素柱的连接位置。
4.用 50 mL 的 70% 乙醇溶液冲洗整个管道。


 
图 2.用于灌注HYPERFlask上清液(上)和洗脱 HBV 颗粒(下)的纯化装置组装。


5.之后,用 50 mL 的 1 × PBS 清洗管道。
 关键步骤:确保去除所有气泡并完全充满 PBS,以防止肝素柱损坏。
6.每 550 mL 上清液连续连接两个肝素柱( HiTrap肝素 5 mL)(图 2)。
 关键步骤:肝素柱也可以使用活塞并联。如果并联,则步骤 7 中的流速可以为两根柱子的 20 mL/min。
7.将过滤后的上清液保持在冰上,并以 10 mL/min 的流速通过肝素柱灌注。
 关键步骤:较高的灌注流速可能会缩短色谱柱的使用寿命。
8.上清液完全灌注后,取出柱子并用洗脱缓冲液冲洗系统。
 关键步骤:确保去除所有气泡,并且管道完全充满洗脱缓冲液。
9.一次重新连接一根柱子,用洗脱缓冲液以 2 mL/min 的流速洗脱。
 关键步骤:在重新连接色谱柱之前,确保系统中没有空气残留。在色谱柱的连接点释放一点洗脱缓冲液,然后用管子重新连接色谱柱。
 关键步骤:两列可以在串行连接中重新连接在一起。如果连续重新连接,则由于死体积而丢弃前 4 mL 的洗脱液,并收集下一个 40 mL。 
10.在无菌管中每列收集 20 mL 的洗脱液(从两列中收集 40 mL)。
 关键步骤:将洗脱缓冲液保持在室温,同时收集洗脱液并在室温下储存直至步骤 11。将洗脱液储存在冰上或使用冰冷的洗脱缓冲液可能会导致 HBV 颗粒沉淀,因为其中的盐浓度很高。洗脱缓冲液。
11.立即将 120 mL Ad+++ 添加到 40 mL 洗脱液(最终体积 =160 mL)中,以稀释洗脱缓冲液中的高盐浓度。
12.将 12–15 mL Ad+++ 添加到一个截断为 100 KDa的Amicon ® Ultra-15 离心过滤器单元中,并在 3,000 × g和4°C 下离心 10 分钟,以清洗过滤器。
13.丢弃通过介质的流量。
14.将 12–15 mL 纯化的病毒溶液添加到一台Amicon ® Ultra-15 离心过滤装置中。
 可选:使用多个过滤器单元将减少处理时间。
15.× g的最大速度和 4°C 离心 10 分钟。
16.从管底部丢弃流过滤液,并用纯化的病毒溶液再次将剩余的残留量加满至 12–15 mL(步骤 11)。
17.再次执行步骤 13 和 14,直到整个 160 mL(步骤 11)被过滤,纯化和浓缩 HBV 库存的剩余最终体积为 2 mL(从 550 mL 的HYPERFlask上清液开始)。
 关键步骤:在 2-3 轮离心后,剩余的病毒溶液开始变得粘稠,可能会堵塞离心单元的过滤器。在加满离心单元(步骤 16)期间,用 1,000 μL微量移液器上下移液有助于使溶液均匀化并防止过滤器堵塞。
18.制作 100–500 µL病毒原液等分试样,并将其储存在 -80°C 以备将来使用。
 关键步骤:避免冷冻/解冻循环,以最大限度地提高病毒恢复率和传染性。
19.取 5 μL纯化浓缩病毒,使体积达到 200 μL 。
20.在分离 HBV DNA 后对病毒原液进行滴度,然后进行 qPCR(参见 E 部分)。


 注意:在HYPERFlask细胞培养系统中,大量细胞在单个HYPERFlask中培养,导致上清液中存在大量细胞碎片和蛋白质,如果不去除可能会阻塞肝素柱。为确保完全去除细胞碎片和蛋白质,仅离心HYPERFlask上清液是不够的,必须使用 0.45 µm 无菌过滤器进行过滤。然而,在 A 部分(用于 PEG 沉淀的病毒生产)中,仅来自单个 10 cm 细胞培养板的 10 mL 上清液在单个 50 mL Falcon 管中处理,其中包含有限量的细胞碎片,仅离心就足够了去除。


# 纯化装置和肝素柱的维护
1.柱用 20 mL 的 10 × PBS 溶液以 5 mL/min 的流速冲洗柱子。
2.再次用 20 mL 柱储存缓冲液冲洗,并在 4°C 下以气密方式(通过拧紧色谱柱随附的螺帽)储存色谱柱。
3.用 70% 乙醇清洁油管,以灭活任何剩余的 HBV。


C.热灭活HBV的产生
1.在冰上解冻预先滴定的活性重组 HBV。
2.将病毒转移到 1.5 mL 微量离心管中。
3.以最大速度和 4°C 离心 10 分钟。
4.将病毒转移到新的 1.5 mL 微量离心管中。
5.将病毒在 100 ° C 下煮沸 30 分钟。
6.以最大速度和4°C 离心 10 分钟。
7.将上清液用作热灭活病毒。


D.人肝类器官的产生、维持和HBV感染简述
有一篇配套的协议文件描述了人类肝脏类器官的培养方法,以及它们对重组 HBV 的感染( Romal 等。 , 2022)。下面给出了该协议文件的非常简短的描述。
1.肝活检被分离成单细胞水平,与 BME/Ad+++ 混合,并培养成圆顶形液滴,使用能够生长 LGR5+ 成体肝干细胞和形成类器官的专用培养基。
2.类器官在允许人类肝脏类器官持续生长的膨胀培养基中培养。
3.将培养基转化为分化培养基,停止类器官的增殖并诱导其分化为肝细胞,并表达分化的肝细胞标志物和 HBV 受体 NTCP,这是感染类器官所必需的。
4.分化的类器官与适量的 HBV 混合并旋转感染。
5.旋转感染后,受感染的类器官在含有 BME/Ad+++ 的圆顶形液滴中的 24 孔细胞培养板中再次培养(D 部分, Romal中的步骤 31 等人, 2022)。


E.从上清液中分离和检测HBV DNA
上清液中的 HBV DNA 可以使用市售试剂盒或苯酚-氯仿分离方法进行分离。详细的协议描述如下:


# 使用商业分离试剂盒从上清液中分离 HBV DNA:
1.取 200 μL 的培养上清液(参见 D 部分,步骤 5 )。
2.QIAamp分离上清液中的 HBV DNA MinElute Virus Spin Kit,按照制造商的说明进行操作。


# 使用苯酚-氯仿分离法从上清液中分离 HBV DNA:
1.取 200 μL的上清液。
2.添加 300 µL的 1% SDS+ 0.1M NaHCO 3到MilliQ水中,辅以100 µg/mL蛋白酶 K。
3.将样品在 55 ° C下孵育1 小时。
4.短暂旋转以收集管底部的所有组件。
5.添加 500 μL 的苯酚-氯仿-异戊醇 (PCI),并彻底涡旋 10 秒。
6.以最大速度和室温离心 5 分钟。
7.将顶部水相转移到新的 1.5 mL 微量离心管中。
8.添加 500 μL 的24:1 氯仿-异戊醇 (CI),并彻底涡旋 10 秒。
9.以最大速度和室温离心 5 分钟。
10.将顶部水相转移到新的 1.5 mL 微量离心管中,加入 1 μL的糖原(20 mg/mL)和 30 μL 的3M NaOAc pH 5.2。涡旋 10 秒,加入 1 mL 的 100% 乙醇,再次彻底涡旋 10 秒。
11.将微量离心管在液氮中速冻或在 -80°C 下冷冻 30 分钟或过夜。
12.以最大速度和 4°C 旋转 DNA 30 分钟。
13.μL 的70% 乙醇清洗颗粒。
14.以最大速度和 4°C 降速 15 分钟。
15.小心去除上清液。
16.将 DNA 颗粒重新悬浮在 30 μL 的无核酸酶水中。
17.将 DNA 储存在 -20°C。


# 检测HBV DNA
零件最终浓度
2 × LightCycler480 Probes Master(罗氏)1 × (12.5 µL )
正向引物 (100 µM )0.5 µM
反向引物 (100 µM )0.5 µM
探针 (50 µM )0.1 µM
DNA模板4 µL
无核酸酶水x µL
总反应体积25 µL
* 引物和探针序列:正向 (5'-GCAACTTTTTCACCTCTGCCTA-3')
反向 (5'-AGTAACTCCACAGTAGCTCCAAATT-3')
探头 (FAM-TTCAAGCCTCCAAGCTGTGCCTTGGGTGGC-BHQ1)
1.准备反应混合物并短暂离心以收集 PCR 板底部的所有组分。
2.运行扩增,从 95°C 开始 10 分钟,然后是 95°C 10 秒、60°C 30 秒和 72°C 10 秒的 45 个循环。


# 分析及预期结果:
lenti -HBV 构建体的质粒稀释制成的标准曲线,范围为 2 至 2 × 10 4个质粒拷贝。使用标准曲线计算 HBV DNA 拷贝数。图 3A 表示不同样品与 HBV 特异性引物和探针的 PCR 曲线。对于每个 PCR 反应,使用 4 µL 的 DNA 模板,每个样品条件的 DNA 制备起始体积为 30 µL。未感染的类器官和感染热灭活 HBV 的类器官的培养上清液用作阴性对照。 HepG2.2.15 细胞的培养上清液用作阳性对照。使用 2、20、200 和 2000 个拷贝的含有 1.3 mer HBV 基因组的lenti -HBV 质粒构建体生成标准曲线。在未感染的类器官或被热灭活的 HBV 感染的类器官中没有观察到扩增。受感染的类器官、HepG2.2.15 细胞和标准质粒稀释液的荧光信号曲线分别以紫色、红色和绿色表示。图 3B 表示不同样品条件下的单个和平均(循环阈值)Ct 值。标准曲线 (图 3C) 是通过在 y 轴上绘制不同标准质粒稀释度的 Ct 值和在 x 轴上标准质粒拷贝的相应对数来制备的。图 3C 中还提到了标准线的 r 2值和方程。使用受感染的类器官和 HepG2.2.15 细胞的平均 Ct 值和标准线方程,计算每毫升相应培养上清液中存在的 HBV DNA 拷贝数(图 3C)。图 3D 中的条形图描述了不同样品条件下的 HBV DNA 拷贝数。


 
图 3.从培养上清液中检测 HBV DNA 的预期结果。
A) 不同样品条件的 PCR 曲线。 B) 不同样品条件下观察到的 Ct 值和平均 Ct 值,C) 使用标准质粒的不同稀释度的 Ct 值生成标准曲线,并计算存在于受感染类器官和 HepG2 中的 HBV DNA 拷贝.2.15 细胞。 D) 不同样本中存在的 HBV DNA 拷贝数的条形图。 y 轴代表 HBV DNA 拷贝数,x 轴代表不同的样本条件。


F.细胞内HBV DNA和cccDNA的分离检测
cccDNA的可用技术都有其自身的局限性。因此,建议同时使用多种技术。在我们的研究中,我们用 T5 核酸外切酶处理全基因组 DNA 和碱裂解质粒 DNA 分离,然后进行质粒安全消化以区分cccDNA和rcDNA 。详细的协议描述如下:


1.准备一个含有 10 mL Ad+++ 的 15 mL Falcon 管。
2.的每个孔中取出培养基(参见D 部分,步骤 5)。
3.在每个有机体孔中加入 1 mL Ad+++ ,并在每个 15 mL Falcon 管中收集 4-8 个孔的 24 孔有机体板。
4.在冰上孵育 30 分钟。
5.200 × g和 4°C 离心 5 分钟。
6.去除上清液。
7.加入 60 – 100 µL TrypLE (用于解离贴壁细胞和原代人类细胞培养物的胰蛋白酶的替代品) ,并在室温下机械消化 30 – 60 秒。
8.用冰冷的 PBS 清洗有机体。
9.200 离心机 × g和 4°C 5 分钟。
10.去除上清液,留下消化的有机物颗粒。


# 全基因组 DNA 分离,然后进行 T5 核酸外切酶消化
T5 核酸外切酶降解所有线性和部分环状 dsDNA。然而,该酶不会完全降解环状超螺旋 dsDNA。 T5 核酸外切酶用于去除整个基因组 DNA 制剂中存在的rcDNA ,它是部分双链的,但不会降解完全环状和双链的cccDNA 。
1.沉淀(F 部分,第 10 步)用于使用DNeasy Blood and Tissue 试剂盒进行全基因组 DNA 分离。
2.按照制造商的说明继续隔离。
3.μL 的无核酸酶水中从柱中洗脱最终 DNA 。
4.使用 25 μL 进行 T5 外切酶消化,剩余的 25 μL 用于检测细胞内 HBV DNA。
5.按照制造商的说明,继续使用 T5 核酸外切酶进行消化。
6.通过添加 EDTA 使消化失活,最终浓度为 11 mM。 
7.QIAquick PCR Purification Kit 纯化完全灭活的反应体积。
8.按照制造商的说明继续纯化。
9.μL 的无核酸酶水中从柱中洗脱纯化的 DNA 。


# 用于碱裂解质粒 DNA 分离,然后进行质粒安全消化
1.在 800 μL 的冰冷细胞裂解缓冲液中重新悬浮颗粒(F 部分,步骤 10)。
2.在冰上孵育 10 分钟。
3.加入等体积的碱裂解缓冲液。
4.将样品在 37°C 下孵育 30 分钟。
5.通过添加 3 M 醋酸钾 (CH 3 COOK) (pH 5.0) 中和 DNA,最终浓度为 0.6 M。
6.以最大速度和室温离心 5 分钟。
7.将上清液转移到两个新的 1.5 mL 微量离心管中,并加入 ±900 μL 的苯酚-氯仿-异戊醇 (PCI)。彻底涡旋 10 秒。
8.以最大速度和室温离心 5 分钟。
9.将顶部水相转移到新的 1.5 mL 微量离心管中。
10.重复步骤 7 - 9。
11.以最大速度和室温离心 5 分钟。
12.添加 500 μL 的丁醇:异丙醇(7:3),并彻底涡旋 10 秒。
13.以最大速度和室温离心 5 分钟。
14.将每个管的底层转移到两个新的 1.5 mL 微量离心管中。 (两个管子的底层将平均分配到四个管子中。)
15.添加 200 μL 的 7.5 M 乙酸铵 (CH 3 COONH 4 )、1 μL 的糖原储备 (20 mg/mL) 和 1 mL 的 100% 乙醇。
16.在液氮中快速冷冻微量离心管,或在 -80°C 冷冻 30 分钟或过夜。
17.以最大速度和 4°C 旋转 DNA 30 分钟。
18.用 1 mL的70% 乙醇清洗颗粒。
19.以最大速度和 4°C 降速 15 分钟。
20.小心去除上清液。
21.将 DNA 颗粒重新悬浮在 50 μL 的无核酸酶水中。
 暂停步骤: DNA 可在 -20°C 下保存一年,直至进行进一步消化。
22.取 25 μL 的 DNA。
23.按照制造商的协议,使用安全的质粒消化样品。
24.消化后,用 PBS 将体积加到 400 μL。 
25.添加 400 μL 的苯酚-氯仿-异戊醇 (PCI)。彻底涡旋 10 秒。
26.以最大速度和室温离心 5 分钟。
27.取顶部水相并转移到新的 1.5 mL 微量离心管中。
28.添加 400 μL 的24:1 氯仿-异戊醇 (CI)。彻底涡旋 10 秒。
29.以最大速度和室温离心 5 分钟。
30.将顶部水相转移到新的 1.5 mL 微量离心管中,从库存中添加 1 μL糖原(20 mg/mL)和 20 μL 3M NaOAc pH 5.2。涡旋 10 秒,加入 1 mL 的 100% 乙醇,并彻底涡旋 10 秒。
31.在液氮中快速冷冻微量离心管,或在 -80°C 冷冻 30 分钟或过夜。
32.以最大速度和 4°C 旋转 DNA 30 分钟。
33.μL 的70% 乙醇清洗颗粒。
34.以最大速度和 4°C 降速 15 分钟。
35.小心去除上清液。
36.将 DNA 颗粒重新悬浮在 30 μL 的无核酸酶水中。


# 检测细胞内HBV DNA
零件最终浓度
10 × PCR 缓冲液1 × (2.5 µL )
50 毫米氯化镁21.75 毫米
10 mM dNTP 混合物400微米
正向引物 (100 µM )0.5 µM
反向引物 (100 µM )0.5 µM
探针 (50 µM )0.15 µM
铂 Taq DNA 聚合酶0.04 单位/ µL
DNA模板7.5 µL
无核酸酶水x µL
总反应体积25 µL
* 引物和探针序列:正向 (5'-GCAACTTTTTCACCTCTGCCTA-3')
反向 (5'-AGTAACTCCACAGTAGCTCCAAATT-3')
探头 (FAM-TTCAAGCCTCCAAGCTGTGCCTTGGGTGGC-BHQ1)
3.准备反应混合物并短暂离心,以收集 PCR 板底部的所有组分。
4.从 95°C 开始扩增 10 分钟,然后进行 45 个循环,即 95°C 10 秒、60°C 30 秒和 72°C 10 秒。


# cccDNA的检测
零件最终浓度
2 × LightCycler480 Probes Master(罗氏)1 × (10 µL )
正向引物 (100 µM )1微米
反向引物 (100 µM )1微米
探针 (50 µM )0.2 µM
二甲基亚砜4%
cccDNA模板4.2 µL
无核酸酶水x µL
总反应体积20 µL
* 引物和探针序列:正向 (5'-GTCTGTGCCTTCTCATCTGC-3')
反向 (5'-AGTAACTCCACAGTAGCTCCAAATT-3')
探头 (FAM-TTCAAGCCTCCAAGCTGTGCCTTGGGTGGC-BHQ1)
1.准备反应混合物并短暂离心,以收集 PCR 板底部的所有组分。
2.et al. , 2017)所述,从 95°C 开始扩增 10 分钟,然后是 95°C 15 秒和 61°C 1 分钟的 50 个循环。


# 分析和预期结果:
用于检测细胞内 HBV DNA 和cccDNA的两种 PCR 均包括由含有1.3mer HBV 基因组的慢乙肝质粒构建体的质粒稀释制成的标准曲线,质粒拷贝数范围为 2 至 2 × 10 4 。使用标准曲线计算细胞内 HBV DNA 和cccDNA拷贝数。图 4A 表示不同样品的 PCR 曲线,其中包含cccDNA特异性引物和探针。每个 PCR 反应使用 4.2 µL 的 DNA 模板,每个样品条件的 DNA 制备起始体积为 30 µL。来自未感染的类器官和 HepG2.2.15 细胞培养上清液的 DNA 制剂(仅应包含rcDNA )用作阴性对照。为了生成标准曲线,使用了 2、20、200 和 2000 个拷贝的含有 1.3 mer HBV 基因组的慢病毒-HBV质粒构建体。在未感染的类器官(红色)或 HepG2.2.15 细胞培养上清液(海军蓝)中没有观察到扩增。受感染有机体和标准质粒稀释液的荧光信号曲线分别以紫色和绿色表示。图 4B 表示不同样品条件下的单个 Ct 值和平均 Ct 值。标准曲线 (图 4C) 是通过在 y 轴上绘制不同标准质粒稀释的 Ct 值和在 x 轴上标准质粒拷贝的相应对数来制备的。图 4C 中还提到了标准线的 r 2值和方程。使用受感染有机体的平均 Ct 值和标准线方程,计算受感染有机体样本中存在的cccDNA拷贝数(图 4C)。图 4D 中的条形图描述了不同样本中存在的cccDNA拷贝数。


 
图 4. cccDNA检测的预期结果。 
A) 不同样品条件的 PCR 曲线。 B) 不同样品条件下观察到的 Ct 值和平均 Ct 值,C) 使用标准质粒的不同稀释度的 Ct 值生成标准曲线,并计算受感染类器官样品中存在的cccDNA拷贝。 D)不同样品中cccDNA拷贝数的条形图。 y 轴代表cccDNA拷贝数,x 轴代表不同的样本。


图 5A 表示具有 HBV 特异性引物和探针的不同样品的 PCR 曲线。对于每个 PCR 反应,使用 7.5 µL 的 DNA 模板,每个样本的 DNA 制备起始体积为 30 µL。未感染的类器官用作阴性对照。 HepG2.2.15 细胞用作阳性对照。为了生成标准曲线,使用了 2、20、200 和 2000 个拷贝的含有 1.3 mer HBV 基因组的慢病毒-HBV质粒构建体。在未感染的类器官样本中没有观察到扩增。受感染的类器官、HepG2.2.15 细胞和标准质粒稀释液的荧光信号曲线分别以紫色、红色和绿色表示。图 5B 表示不同样品的单个 Ct 值和平均 Ct 值。标准曲线 (图 5C) 是通过在 y 轴上绘制不同标准质粒稀释的 Ct 值和在 x 轴上标准质粒拷贝的相应对数来制备的。图 5C 中还提到了标准线的 r 2值和方程。使用受感染的类器官和 HepG2.2.15 细胞的平均 Ct 值和标准线方程,计算存在的细胞内 HBV DNA 拷贝数(图 5C)。图 5D 中的条形图描述了不同样本中存在的细胞内 HBV DNA 拷贝数。


 
图 5.从培养上清液中检测细胞内 HBV DNA 的预期结果。 
A) 不同样品条件的 PCR 曲线。 B) 不同样品条件下观察到的 Ct 值和平均 Ct 值,C) 使用标准质粒的不同稀释度的 Ct 值生成标准曲线,并计算受感染类器官中存在的细胞内 HBV DNA 拷贝数和HepG2.2.15 细胞。 D) 不同样本中存在的细胞内 HBV DNA 拷贝数的条形图。 y 轴代表细胞内 HBV DNA 拷贝数,x 轴代表不同的样品条件。


G.HBV RNA的分离和检测
HBV RNA 的分离可以使用商业试剂盒和Trizol RNA 分离程序进行。详细的协议描述如下:
1.从有机体中取出介质。
2.+++收集24 孔板的1 – 2 个孔(参见D 部分;步骤 5 ) ,通过移液器混合 4 – 5 次,然后转移到 1.5 mL 微量离心管中。
  关键步骤: 24 孔板的一个孔,密度为 90%,或者如果密度低,则使用 24 孔板的两个孔。样品可在-80 °C下保存六个月,也可直接分离。
3.在冰上孵育 10-20分钟。
4.将样品在 200 × g和 4°C 下离心 5 分钟。
5.去除上清液,留下有机体颗粒。


# 使用商业试剂盒分离 RNA
1.500 µL 细胞裂解缓冲液( RealiaPrep RNA Cell Miniprep System, Promega)重悬类器官颗粒(参见G 部分,步骤 5 )。
2.按照制造商的说明继续隔离。
3.μL 的无核酸酶水中洗脱 RNA 。
4.将 RNA 储存在 -80°C,直到进一步处理。


# 使用Trizol RNA 分离方案分离 RNA 
1.用 1 mL 的Trizol溶解有机体颗粒(参见G 部分,步骤 5 ) ,通过上下移液, 并剧烈涡旋20 – 40 秒。
2.在室温下孵育样品 5 分钟。
3.加入 200 μL氯仿,剧烈涡旋样品 20 秒,室温孵育2-3分钟。
4.将样品在 12,000 × g和 4°C 下离心 15 分钟。
5.小心地将上层水相转移到新的 1.5 mL 微量离心管中,不要干扰界面。
6.加入 500 沉淀 RNA μL异丙醇,颠倒试管 4 到 5 次,室温孵育 10 分钟。
7.将样品在 12,000 × g和 4°C 下离心 10 分钟。
8.去除上清液。
 关键步骤:为避免丢失 RNA 沉淀,尤其是当沉淀不可见或非常小时,请留下约 50 μL的上清液。
9.加入 1 mL 75% 乙醇,在 7,500 × g和 4°C 下离心 5 分钟。
 关键步骤:不要涡旋 RNA 沉淀。
10.重复第9步,完全去除洗涤缓冲液。
 关键步骤:为确保完全去除洗涤缓冲液,对样品进行短暂离心,以除去管边缘的剩余洗涤缓冲液。
11.将颗粒风干 10 分钟。
12.将颗粒重新悬浮在 40 μL的无核酸酶水中。
13.将 RNA 样品储存在 -80°C。


#DNase l治疗
1.使用 1 μL样品使用NanoDrop 2000/2000c 分光光度计测量 RNA 浓度。
2.使用 300 – 1,000 ng RNA 进行 DNase l 处理。
3.按照制造商的说明完成 DNase 处理。


# cDNA合成
1.使用 DNase l 处理的样品与 Superscript II Reverse Transcriptase 试剂盒进行 cDNA 合成。
2.按照制造商的说明并使用随机引物完成 cDNA 合成。


# 检测HBV RNA
零件最终浓度
10 × PCR 缓冲液1 × (2.5 µL )
50 毫米氯化镁21.75 毫米
10 mM dNTP 混合物400微米
正向引物 (100 µM )0.5 µM
反向引物 (100 µM )0.5 µM
探针 (50 µM )0.15 µM
铂 Taq DNA 聚合酶0.04 单位/ µL
cDNA 模板(在水中按 1:2.5 或 1:5 稀释,基于用于 cDNA 合成的 RNA 起始量)4 µL
无核酸酶水x µL
总反应体积25 µL
* 引物和探针序列 (HBV):正向 (5'-GCAACTTTTTCACCTCTGCCTA-3')
反向 (5'-AGTAACTCCACAGTAGCTCCAAATT-3')
探头 (FAM-TTCAAGCCTCCAAGCTGTGCCTTGGGTGGC-BHQ1)
* 引物和探针序列(Beta-2-微球蛋白):
前锋(5'- AGCGTACTCCAAAGATTCAGGTT-3')
反向(5'- ATGATGCTGCTTACATGTCTCGAT-3')
探头(FAM- TCCATCCGACATTGAAGTTGACTTACTG-BHQ1)


1.准备反应混合物,并短暂离心以收集 PCR 板底部的所有组分。
2.从 95°C 开始扩增 10 分钟,然后进行 45 个循环,即 95°C 10 秒、60°C 30 秒和 72°C 10 秒。


# 分析和预期结果:
Beta-2-microglobulin (B2M) 用作分析 cDNA 样品表达的管家控制。使用 2 -ΔΔCt方法计算倍数增加。图 6A 表示不同样品的 PCR 曲线,其中 HBV 特异性引物和探针,以及 B2M 特异性引物和探针。对于每个 PCR 反应,使用 4 µl cDNA 模板。未感染的类器官和感染热灭活HBV的类器官用作阴性对照。 HepG2.2.15 细胞用作阳性对照。未感染的类器官、感染的类器官、HepG2.2.15 细胞和感染热灭活 HBV 的类器官的荧光信号曲线分别用蓝色、紫色、红色和绿色表示。图 6B 表示不同样本的单个 Ct 值和平均 Ct 值,以及与热灭活 HBV 感染的类器官相比,确定受感染类器官和 HepG2.2.15 细胞中 HBV RNA 表达倍数变化的计算。在未感染的类器官中没有 HBV RNA 的表达。因此,未感染的类器官样本被排除在倍数变化计算之外。图 6C 中的条形图描述了不同样本中 HBV RNA 表达的倍数变化。
 
图 6. HBV RNA 检测的预期结果。 
A) 使用 HBV 特异性(左)和 B2M 特异性(右)引物和探针的不同样品条件的 PCR 曲线。 B) 观察到的 Ct 值和平均 Ct 值,以及与感染热灭活 HBV 的类器官相比,不同样品的 HBV RNA 倍数变化计算。 C) 不同样本中存在的 HBV RNA 倍数变化的条形图。 y 轴代表倍数变化,x 轴代表不同的样本条件。


H.免疫荧光
 关键步骤:使用超低保留移液器吸头减少研磨时的样品损失。
首先,用洗涤介质或缓冲溶液预润湿移液器,然后重悬样品。
1.用 10 mL 冰冷的 Ad+++准备一个15 mL Falcon 管,并将其放在冰上。
2.所有孔中取出培养基(参见 D 部分,步骤 5 ) 。
3.在每口井中加入约 1 mL 的冰冷 Ad+++。
4.用 10 mL 移液器收集含有有机物的液滴,轻轻刮擦。
 关键步骤:打破有机体的空隙。
5.用 10 mL 移液器研磨混合三到四次。 
6.冰上孵育30 分钟至 1 小时。
 关键步骤:每 5 分钟将 Falcon 管倒置四到五次,混合有机体。这将有助于 BME有效地溶解。 
7.在 4°C 下以 100 × g离心5 分钟来沉淀类器官。
8.用10 mL 冰冷的 Ad+++洗涤 1 × ,用10 mL 移液器上下吹打四到五次混合。
9.C在 100下离心 × g和 4°C 5 分钟。
10.取出介质并用 10 mL 的冰冷 PBS 清洗一次。
11.取出培养基,用 200 μL 新鲜制备的固定液轻轻重悬颗粒 [ HBcAg使用 4% PFA 、肝细胞核因子 4 α (HNF4α)、牛磺胆酸钠协同转运多肽 (NTCP) 和白蛋白,或者HBsAg 使用 100% 丙酮] 。
 关键步骤:固定试剂和浓度可能因抗体而异。
12.在冰上孵育 30 分钟。
13.去除固定溶液并加入 10 mL 的冰冷 PBS。
14.100 × g和 4°C 离心 5 分钟。
15.取出 PBS。
16.在 PBS 中加入 1 mL 新鲜制成的 0.1 M 甘氨酸。
17.在室温下孵育 15-30 分钟。
 关键步骤:固定后用甘氨酸处理类器官将起到淬灭剂的作用。这可以帮助阻塞未反应的醛,这可能会导致背景荧光增加。 
18.加入 10 mL PBS,100 g 离心 × g 5 分钟。
 暂停步骤:固定类器官可在 4 °C下储存3-4 周。
19.取出 PBS。
20.HBcAg 、HNF4α 和 NTCP)中添加 1 mL 的 0.3% Triton、在 PBS(白蛋白)中添加 1% Triton X-100 或 100% 丙酮(HBsAg)来渗透有机体。
21.在室温下孵育 30 分钟。
22.100 度离心机 × g 5 min,除去透化液。
23.用 1 mL 的 0.5% FBS 在PBS中清洗一次。
24.100 度离心机 × 克 和室温5分钟。
25.添加 1 mL 的封闭缓冲液(PBSTD;PBS + 0.5% 血清 + 0.3% triton+ 1% DMSO,用于HBcAg 、HNF4α、白蛋白和 NTCP, 或 10% BSA+ 0.5% 血清在 PBS中用于HBsAg ),并在室温下孵育 2 小时。
 可选:使用山羊血清进行封闭步骤,以防所有二抗均来自山羊。
26.100 度离心机 × g和室温 5 分钟。
27.移除阻塞缓冲液。
28.PBS + 10%封闭缓冲液中稀释的一抗孵育有机体(检查制造商推荐的稀释度)。
29.在 4°C 的轨道摇床上孵育过夜。
30.100 度离心机 × g和 4°C 5 分钟。
31.去除一抗混合物。
32.在 PBS 中加入 10 mL 的 0.5% FBS。在室温下在轨道摇床上孵育 10 分钟。取出洗涤缓冲液并重复此步骤至少 3 次。
33.抗的 PBS 中加入 200 μL的 0.5% FBS ,并在室温下在黑暗的轨道摇床上孵育 2 小时。
34.去除二抗。
35.在 PBS 中加入 10 mL 的 0.5% FBS 进行清洗。在室温下在轨道摇床上孵育 10 分钟。
36.在 100 × g和室温下离心 5 分钟。
37.重复步骤 3 5 –3 6至少四到五次。
38.含有 5的 PBS 中加入 200 μL的 0.5% FBS μg /mL赫斯特。
39.在室温下在黑暗的轨道摇床上孵育15-20 分钟。
40.取出 Hoechst 并在 PBS 中用 1 mL 的 0.5% FBS 洗涤。
41.在室温下在黑暗中在轨道摇床上孵育 10 分钟。
42.100 度离心机 × g和室温 5 分钟。
43.使用塑料巴斯德吸管去除上清液,并将有机体转移到载玻片上。
44.加入一滴防褪色溶液,盖上盖玻片。
45.将载玻片存放在 4°C 的黑暗中。
46.使用 Leica LAS AF Lite 软件 (Leica SP5 confocal) 拍摄和处理图像(图 7)。


 
图 7. 免疫荧光染色显示 HBV 核心抗原 ( HBcAg )(洋红色)和肌动蛋白(绿色)的表达,在 HBV 感染 6 天后在健康供体肝脏类器官中进行。


I.从上清液中检测 HBsAg 和HBeAg
1.在感染后 3-6 天收集类器官上清液(参见 D 部分,步骤 5 )。
2.将样品在 200 × g和 4°C 下离心 5 分钟。
3.使用 50 μL上清液检测 HBsAg 或HBeAg 。
4.使用HBeAg (人类肝炎 Be 抗原 ELISA 试剂盒, Cusabio )或 HBsAg( Monolisa HBs Ag ULTRA,Bio-Rad)特异性试剂盒分别检测HBeAg和 HBsAg。


# 分析和预期结果:
市售的 ELISA 试剂盒用于检测培养上清液中的 HBsAg 和HBeAg 。图 8A 表示用于检测 HBsAg 的吸光度,测量为不同样品在 450 nm 波长处的光密度 (OD),使用试剂盒制造商提供的阴性样品的吸光度计算校正截止值,以及不同样品的信号截止值,以及描绘不同样品中 HBsAg 表达的条形图。图 8B 表示不同样品的检测HBeAg的吸光度为 OD 450 ,使用试剂盒制造商提供的阴性样品的吸光度计算校正的截止值,以及不同样品的截止值信号,以及描绘不同样品中HBeAg表达的条形图。
 
图 8.检测 HBsAg 和HBeAg的预期结果。 
A) 检测 HBsAg 的吸光度,测量不同样品的 OD 450 ,使用试剂盒制造商提供的阴性样品的吸光度计算校正的截止值,以及不同样品的截止值信号,如以及描绘不同样品中 HBsAg 表达的条形图。 y 轴代表 450 nm S/CO 处的 OD,x 轴代表不同的样品。 B) 检测HBeAg的吸光度,测量不同样品的 OD 450 ,使用试剂盒制造商提供的阴性样品的吸光度计算校正的截止值,以及不同样品的截止值的信号,以及作为描述不同样品中HBeAg表达的条形图。 y 轴代表 450 nm S/CO 处的 OD,x 轴代表不同样品。




食谱


1.DMEM ++ 培养基(500 毫升)
445 毫升 DMEM
50 毫升胎牛血清
5毫升青霉素-链霉素


2.Ad+++ 培养基(500 毫升)
485 毫升 DMEM
5 毫升 HEPES
5 毫升超谷氨酰胺
5毫升青霉素-链霉素


3.70% 乙醇溶液 (100 mL)
70 毫升无水乙醇
30 毫升 Milli-Q 处理过的水


4.洗涤液
500 mL 1 × PBS,pH 7.4


5.洗脱缓冲液 (100 mL)
25 mL 10 × PBS,pH 7.4
75 mL Milli-Q 处理水


6.柱清洗缓冲液 (100 mL)
40 毫升 5M 氯化钠
60 mL Milli-Q 处理水


7.肝素柱储存缓冲液 (100 mL)
13 mL 10 × PBS,pH 7.4
20 毫升无水乙醇
67 mL Milli-Q 处理水


8.细胞裂解缓冲液 (50 mL)
100 μL 0.5M EDTA (pH 8.0)
250 μL 1 M Tris HCl (pH 7.5)
250 μL 10% Nonidet P-40
49.4 mL Milli-Q 处理水


9.碱裂解缓冲液 (50 mL)
5 毫升 1 M NaOH
30 毫升 10% SDS
15 mL Milli-Q 处理水


10.PBSTD (50 毫升)
250 μL FBS
150 μL Triton X-100
500 μL DMSO
49.1 毫升 PBS


11.10 × PBS(500 毫升)
8.9 克 Na 2 HPO 4 · 2H 2 O
1.2 克 KH 2 PO 4
40 克氯化钠
1克氯化钾
用 Milli-Q 处理的水将最终体积调整到 500 mL。




致谢


用于产生高滴度 HBV 库存的方案已改编自Wettengel最近发布的方案 等。 (2021) 并添加了必要的修改,以使病毒储备适合感染人类肝脏类器官。重组 HBV 感染人类肝脏类器官后检测不同 HBV 特异性病毒产物的方案改编自作者先前发表的工作 (De Crignis 等人, 2021)。




利益争夺


作者宣称没有利益冲突。




参考


De Crignis, E.、Hossain, T.、Romal, S.、Carofiglio, F.、Moulos, P.、Khalid, MM、Rao, S.、Bazrafshan, A.、Verstegen, MM 和 Pourfarzad, F. (2021 )。人肝类器官作为HBV感染和相关肝细胞癌患者来源的主要模型的应用。生命 10:e60747 。
Wettengel, JM, Linden, B., Esser, K., Laue, M., Burwitz, BJ 和 Protzer, U. (2021)。用于体外和体内应用的高效价乙型肝炎病毒的快速和稳健的连续纯化。病毒13(8):1503。
Winer,BY,Huang,TS,Pludwinski,E.,Heller,B.,Wojcik,F.,Lipkowitz,GE,Parekh,A.,Cho,C.,Shrirao,A.,Muir,TW等。 (2017)。可扩展肝脏共培养系统中的长期乙型肝炎感染。 国家通讯8(1): 125。
Roman, S.、Hossain, T. 和 Mahmoudi, T. (2022)。人类肝脏类器官的产生、维持和 HBV 感染。 生物协议12(6):e4358。

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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright Hossain et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Hossain, T., Romal, S. and Mahmoudi, T. (2022). Production of Recombinant Hepatitis B virus (HBV) and Detection of HBV in Infected Human Liver Organoids. Bio-protocol 12(8): e4392. DOI: 10.21769/BioProtoc.4392.
  2. De Crignis, E., Hossain, T., Romal, S., Carofiglio, F., Moulos, P., Khalid, M. M., Rao, S., Bazrafshan, A., Verstegen, M. M. and Pourfarzad, F. (2021). Application of human liver organoids as a patient-derived primary model for HBV infection and related hepatocellular carcinoma. Elife 10: e60747.
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