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Jul 2021

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Generation, Maintenance and HBV Infection of Human Liver Organoids
人肝类器官的产生、维持和HBV感染   

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Abstract

Hepatitis B virus (HBV) infection represents a major public health problem infecting approximately 400 million people worldwide. Despite the availability of a preventive vaccine and anti-viral therapies, chronic HBV infection remains a major health issue because it increases the risk of developing liver cirrhosis and hepatocellular carcinoma (HCC). The lack of a relevant in vitro model for the study of the molecular mechanisms that drive HBV replication and latency, as well as HBV-related carcinogenesis, has been one of the major obstacles to the development of curative strategies. Here, we propose the use of human liver organoids as a platform for modeling HBV infection and related tumorigenesis. Human liver organoids can be seeded from both healthy and cirrhotic liver biopsies. They can be expanded in vitro when culturing in a medium containing a specific set of growth factors. When the culture medium is changed into a new medium containing growth factors that promote differentiation, organoids differentiate into functional hepatocytes, which makes them susceptible to infection with recombinant HBV. The novel in vitro primary model system described in this protocol can be utilized as a platform to study HBV pathogenesis and drug screening. Organoids generated from cirrhotic liver biopsies can be a potential tool for personalized medicine, and for modeling HCC and other liver diseases.


Graphic abstract:



Keywords: Healthy donor liver organoids (健康的供体肝脏类器官), Patient-derived liver organoids (患者来源的肝脏类器官), HBV-infection (HBV感染)

Background

Organoid culture technology involves the generation of adult stem cell-derived, genetically-stable, in vitro, 3D-organ models of human origin. A primary liver culture system based on isolation and expansion of primary cells, that allows for the long-term expansion of liver cells as organoids, has already been established previously (Huch et al., 2013, 2015). They are generated from in vitro expansion of Lgr5+ adult stem cells present in the liver, by culturing them in a specified culture medium. Establishment of the liver organoids culture was previously reported byBroutier et al. (2016). Liver organoids can be generated from both healthy and HBV-infected patient-derived liver tissue. In comparison with other in vitro cell hepatocytes models, liver organoids are expandable, biobankable and genetically stable. Recently, we have used liver organoids as a primary cell derived model to study HBV infection and associated tumorigenesis, as well as a personalized in vitro platform for anti-viral drug screening and drug induced toxicity (De Crignis et al., 2021). In our study, we used a modified version of the protocol byBroutier et al. (2016) for culturing and maintenance of liver organoids, where we introduced slight modifications to the isolation, as well as the expansion culture medium, to optimize liver organoid yield and viability. A detailed protocol for the generation, expansion, and differentiation of healthy and patient-derived liver organoids is described below. Differentiated liver organoids can be infected with recombinant HBV by spinoculation using concentrated virus.

Materials and Reagents

  1. Sterile syringe filter, 0.1 µm (Merck Millipore, catalog number: SLVV033RS )

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

  3. Sterile syringe (Omnifix, catalog numbers: 4616200V [20 mL]; 4616502F [50 mL])

  4. Cell strainer 70 µm nylon (Falcon, catalog number: 352350)

  5. 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])

  6. Pipette controller (Brand, catalog number: 26303)

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

  8. 15 mL Falcon tubes (Greiner, catalog number: 188285)

  9. 50 mL Falcon tubes (Greiner, catalog number: 227285)

  10. 1.5 mL microcentrifuge tubes (Biotix, catalog number: MT-0150-BC)

  11. 24-well suspension plates (Greiner, catalog number: 662102)

  12. 48-well suspension plates (Greiner, catalog number: 677102)

  13. 60 mm Petri dish (Thermo Fisher Scientific, catalog number: 150462)

  14. Disposable forceps (Servoprax, Mediware, catalog number: H7-301)

  15. CryoTubeTM vials (Thermo Scientific, Nunc, catalog number: 366656)

  16. Parafilm M (Sigma-Aldrich, catalog number: P7793)

  17. Ethanol absolute (Sigma-Aldrich, Merck Millipore, catalog number: 1009832500)

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

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

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

  21. Belzer UW Cold Storage Solution (transfer medium) (Bridge to Life, BTLBUW-2000)

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

  23. HEPES, 1 M Buffer Solution in 0.85% NaCl (Lonza, catalog number: BE17-737E)

  24. UltraGlutamine1 200 mM in 0.85% NaCl Solution (Lonza, catalog number: BE17- 605E/U1)

  25. EBSS (Earle's Balanced Salt Solution) (Thermo Fisher Scientific, catalog number: 24010-043)

  26. Collagenase D (Sigma-Aldrich, catalog number: 11088858001)

  27. Recovery cell culture freezing medium (Invitrogen, catalog number: 12648010)

  28. TrypLE Express (Thermo Fisher Scientific, Gibco, catalog number: 12604013)

  29. Cultrex Reduced Growth Factor Basement Membrane Extract (BME), Type 2, Pathclear (R&D Systems, catalog number: 3533-010-02)

  30. Bovine Serum Albumin (Sigma-Aldrich, catalog number: A7030)

  31. Phosphate Buffered Saline (PBS) (Thermo Fisher Scientific, Gibco, catalog number: 10010023)

  32. N2 Supplement (100×) (Thermo Fisher Scientific, Gibco, catalog number: 17502-048)

  33. B27 Supplement (without vit A) (Thermo Fisher Scientific, Gibco, catalog number: 12587010)

  34. N-acetyl Cysteine (Sigma-Aldrich, catalog number: A7250)

  35. Recombinant Human EGF (PeproTech, catalog number: AF-100-15)

  36. Recombinant [Leu15]-Gastrin I human (Sigma-Aldrich, catalog number: G9145)

  37. Recombinant Human HGF (PeproTech, catalog number: 100-39H)

  38. Primocin (Invivogen, catalog number: ant-pm-2)

  39. Recombinant human R-spondin 3 (conditioned medium, house made) (Broutier et al., 2016)

  40. Wnt 3A (conditioned medium, house made) (Broutier et al., 2016)

  41. Recombinant Human FGF-10 (PeproTech, catalog number: 100-26)

  42. Nicotinamide (Sigma-Aldrich, catalog number: N3376)

  43. TGF-β inhibitor (A83.01) (Tocris, catalog number: 2939)

  44. Forskolin (Sigma-Aldrich, catalog number: F3917)

  45. Rho-kinase inhibitor (ROCK Inhibitor, Y-27632) (Sigma-Aldrich, catalog number: SCM075)

  46. Recombinant Human Noggin (PeproTech, catalog number: 120-10C)

  47. Recombinant human BMP7 (PeproTech, catalog number: 120-03P)

  48. Recombinant human FGF19 (PeproTech, catalog number: 100-32)

  49. DAPT (Sigma-Aldrich, catalog number: D5942)

  50. Dexamethasone (Sigma-Aldrich, catalog number: D4902)

  51. PEG-6000 (Millipore, catalog number: 528877)

  52. 70% ethanol solution (100 mL) (see Recipes)

  53. DMEM +/+ (50 mL) (see Recipes)

  54. Digestion solution (10 mL) (see Recipes)

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

  56. 0.1% BSA/PBS (10 mL) (see Recipes)

  57. 2× Human liver organoid basal medium (2× LBM) (50 mL) (see Recipes)

  58. Human liver organoid Isolation medium (IM) (20 mL) (see Recipes)

  59. Human liver organoid Expansion medium (EM) (20 mL) (see Recipes)

  60. Human liver organoid Expansion medium without wnt3A supplemented with BMP7 (EMB) (20 mL) (see Recipes)

  61. Human liver organoid Differentiation medium (DM) (20 mL) (see Recipes)

Equipment

  1. Calibrated micropipette (VWR, Gilson, catalog numbers: 613-5945 [P10]; 613-5946 [P20]; 613-5948 [P200]; 613-5949 [P1000])

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

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

  4. Bright field microscope (Leica DMIL microscope and a DFC420C camera)

  5. Shaker (Thermo Scientific, model: 88881102)

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

  7. Eppendorf Centrifuge 5417 R for 1.5 mL tubes (Eppendorf, model: F45-30-11)

  8. Milli-Q® IQ 7000 Ultrapure Lab Water System

  9. Water bath 37°C (Grant, VFP)

  10. -80°C freezer (Panasonic, catalog number: MDF-794-PE)

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

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

  13. -20°C freezer (Bosch, GSD-3513)

  14. Liquid nitrogen storage

  15. Scalpels (Blade 10 or 11) (Swann Morton, catalog number: 233-5364 or 233-5363)

  16. Scalpels (Blade 12D, double edge) (Swann Morton, catalog number: 233-5516)

  17. Alcohol-resistant cryogenic scientific marker (LabTAG, catalog number: SM-1)

  18. Cell counting chamber (Marienfeld, Bürker-Türk, catalog number: 1 630-1545)

Procedure

  1. Dissociation of human liver hepatocytes and seeding of organoids

    Safety instructions for working with human tissue:

    1. While handling human tissue, work inside the biosafety cabinet.

    2. Take precautions while working with human tissue (i.e., wear lab coats, gloves, sterilize the working space, etc.)

      Critical Step:

    1. Warm up a 24/48-well suspension plate in the incubator at 37°C.

    2. Prepare all the buffers/solution required for the procedure prior to start.


    1. After surgical excision, collect the sample immediately in a 15 mL or 50 mL Falcon tube, depending on the size of the biopsies and preserve in 5–10 mL of Belzer UW cold storage solution (commercial transfer medium provided by surgeons to transfer the biopsies) at 4°C until processing.

      Optional step: Biopsies can be stored up to 48 h at 4°C. We recommend processing the tissue immediately after surgery.

    2. Remove the basal medium and wash the tissue twice with 10 mL of DMEM +/+ (see Recipes) using a 10 mL pipette.

    3. Place the tissue into a 60 mm Petri dish using sterile tweezers (Figure 1).



      Figure 1. Human liver biopsies from donor (left) and patient (right).


    4. Add 1-2 mL of Ad+++ medium on top of the tissue and mince it into pieces of 0.5-1 mm3, using two scalpels (mince the tissue until it can be easily triturated with a p1000 pipette) (Figures 2-3).

      Critical Step: Use a scalpel blade 12D and scalpel blade 10 or 11 to cut the tissue. Try to slice the tissue gently and vertically with the scalpels, instead of pressing or mashing the tissue at an angle with the side of the scalpels with force. Excessive force can cause cell rupture.



      Figure 2. Mincing of liver biopsy.



      Figure 3. Minced liver tissue from donor (left) and patient (right).


    5. Transfer the minced tissue with a P1000 pipette to a 15 mL Falcon tube and top up to 10 mL with ice-cold Ad+++. Gently pipette up and down with a 10 mL pipette, to wash the liver pieces.

    6. Centrifuge at 100 × g and 4°C for 10 min.

      Critical Step: For biobanking the tissue:

      1. Divide the minced tissue into two after centrifugation and place half in a cryoTube containing 1 mL of freezing media.

      2. Label with an alcohol-resistant marker and transfer the cryotube in the pre-cooled (4°C) freezing container.

      3. Transfer the freezing container to -80°C.

      4. Transfer the samples within 2-3 days from the -80°C freezer to liquid nitrogen, for long-term storage.

      5. Organoids can be generated from frozen tissue, albeit with lower efficiency. Frozen tissue can also be used for genomic, transcriptomic, and proteomics studies.

      6. To generate organoids from frozen minced tissue, take the sample out from the liquid nitrogen tank and transfer on dry ice. Take the vial and place it in a water bath (37°C) for 40-60 s (~90% of the sample is thawed) and immediately add 1 mL of Ad+++ dropwise to the vial. Transfer the volume to a 15 mL Falcon tube containing 10 mL of Ad+++. Continue further from step 6.

    7. Discard the supernatant and add a sufficient volume (4-6 mL based on the size of the tissue, 4 mL for smaller tissue, and 6 mL for larger tissue) of pre-warmed digestion solution.

    8. Homogenize the tissue gently with digestion solution by inverting the tube 5-8 times and pipetting up and down with a P1000 pipette.

    9. Place the tube containing the homogenized sample horizontally in a rack and transfer it to a shaker incubator at 100-120 rpm and 37°C for 45 min-1 h (Figure 4).

      Critical Step: Check every 15 min, to make sure not to over-digest the tissue. Whenever almost all the pieces of tissue are digested, start the next step immediately.



      Figure 4. Placement of minced tissue on the shaker inside the incubator.

    10. Resuspend the cells by pipetting up and down with a P1000 pipette (Figure 5).



      Figure 5. Digested liver biopsy.

    11. Neutralize the digestion by adding 10 mL of cold Ad+++.

    12. Pass the digested material through a 70 µm cell strainer (Figure 6) and collect the material in a 50 mL Falcon tube.

      Optional step: If any partially digested tissue remains on the strainer, collect it with a p1000 pipette and incubate further with 1 mL of TrypLE at 37°C and 5% CO2 for 10–15 min. After incubation, follow step 12 again.

      Optional step: If any debris or plastic pieces from the Petri dish are still present in the solution, repeat step 12 to remove these.



      Figure 6. Straining of digested liver biopsy.

    13. Add pre-warmed Ad+++ (37°C) to increase the volume to 25–30 mL.

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

    15. Remove the supernatant. Resuspend the cell pellet in 10 mL of cold Ad+++ with a 10 mL pipette and transfer the resuspended cells to a 15 mL Falcon tube.

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

    17. Remove the supernatant and resuspend the cell pellet in 2 mL of Ad+++. Count the cells and mix the appropriate number of cells with BME, which will polymerize and form a dome like structure (drop), to give the organoids mechanical support to grow in 3D.

    18. Count the cells with a hemocytometer to seed approximately 10,000-15,000 cells/25 µL drop in a 48-well plate.

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

    20. Resuspend the cells with the appropriate volume of Ad+++ and BME to make the drops (each drop contains 1/3 Ad+++ and 2/3 BME, i.e., 8.3 µL of Ad+++ plus 16.6 µL of BME per 25 µL-drop in a 48-well plate).

      Critical Step: When homogenizing the BME/Ad+++ mixture and making the drops, it is important to avoid making any bubbles.

      Critical Step: While handling the BME, always keep the BME on ice, and be quick when making the drops. The BME will start to solidify if removed from ice for longer than 1 min.

    21. Take the pre-warmed plate from the incubator and start making the drops (Figure 7).

      Note: The volume of a drop for each well of a 48-well plate is 25 µL. For a 24-well plate, both 3× 25 µL drops or 1× 50 µL drop can be used.



      Figure 7. BME drops containing hepatocytes.


    22. Incubate at 37°C and 5% CO2 for 30–45 min, and let the BME polymerize.

    23. Add 250 µL (48-well plate) or 500 µL (24-well plate) of warm human liver organoid isolation medium (IM) in each well (Figure 8).



    Figure 8. Adding the organoids culture medium.


Critical step:

  1. Add the medium gently on the side of the well. Avoid adding the medium directly on top of the drop.

  2. During the first seven days, change the medium every 3–4 days and use IM.

  3. After 1 week of seeding the organoids, change IM to human liver expansion medium.

  4. Organoids will be visible within 2–14 days post seeding. Daily monitoring using a bright field microscope is required to identify growing organoids (Figure 9).



    Figure 9. Generation of organoids from liver biopsy.


  1. Expansion and maintenance of organoids in culture

    Split the organoids every 7–10 days according to organoid density and size (>500–1,000 µm). Here, the term splitting means digesting the organoids enzymatically and mechanically, and distributing the digested organoids into the required number of wells by means of mixing the digested organoids with BME/Ad+++ and making the drops. The general split ratio is 1:4–1:8, depending on growth rate of the donor organoid lines.

    Critical step: Pre-warm the 24-well suspension plate

    1. Prepare 1× 15 mL Falcon tube containing 10 mL of ice-cold Ad+++.

    2. Discard the medium from each well.

    3. To collect the drops, take 4 mL of ice-cold Ad+++ from the 15 mL Falcon tube and add 1 mL into each well containing organoids that need to be split.

    4. Disrupt the drop containing organoids by scraping with P1000 pipette.

    5. Collect the disrupted organoids and transfer to one 15 mL Falcon tube. A maximum of 4 wells can be collected in one 15 mL Falcon tube (e.g., if 8 wells are split, the organoids can be collected in two 15 mL Falcon tubes).

      Note: Follow step 3–5 for each 4 wells of organoids.

    6. Triturate 3–5 times with a 10 mL pipette.

    7. Keep the Falcon tubes on ice for 20–45 min. Invert the Falcon tubes every 10 min, this can help the BME to dissolve efficiently.

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

    9. Discard the supernatant.

    10. Add 60–100 µL of TrypLE to the pellet of organoids. Digest the organoids mechanically at room temperature for 1–5 min, by pipetting with a P200 pipette.

      Note: Check the size of the digested organoids under the microscope. The desired size depends on the experimental requirements. Make sure that the digested organoids are uniform in size.

    11. Add 10 mL of ice-cold Ad+++ to stop the digestion and centrifuge at 200 × g and 4°C for 5 min.

    12. Discard the supernatant completely.

    13. Resuspend the digested organoid pellet with the appropriate amount of 1/3 Ad+++ and 2/3 BME (e.g., if splitting two wells 1:4, then 8 drops need to be made; if 6 drops are split 1:6, then 36 drops need to be made).

    14. Make drops of appropriate volume based on the plate that is used.

    15. Allow the drops to polymerize at 37°C and 5% CO2 for 30–45 min.

    16. Add 250 µL (48-well plate) or 500 µL (24-well plate) of warm EM.

    17. Change the medium every 2–3 days (Figure 10).



    Figure 10. Liver organoids in expansion medium.


  2. Hepatic differentiation of liver organoids

    1. Culture liver organoids with EM-wnt3A+BMP7 for 3–5 days prior to differentiation, to allow the organoids to recover and completely reform after splitting.

      Critical step: If the organoids are big, this might result in breaking of organoids during spin infection. Therefore, start differentiating the liver organoids when they are small (50–350 µm).

    2. Wash the drops gently two times with 500 µL of warm Ad+++.

    3. Add 250 µL (48-well plate) or 500 µL (24-well plate) of warm human liver organoid differentiation medium (DM).

    4. Change the medium every 2–3 days. Differentiate the organoids for 7 days (Figure 11).



    Figure 11. Liver organoids in differentiation medium.


  3. HBV infection

    The infection of human liver organoids can be done either without the addition of PEG or with addition of 4% PEG, which plays a role in increasing the infection efficacy.


    Safety instructions for working with recombinant HBV:

    1. Handling HBV should only be done by HBV vaccinated personnel.

    2. While handling HBV, work inside the biosafety cabinet

    3. Take proper precautions while working with HBV (i.e., wear lab coat, gloves, sterilize the working space, etc.)


    1. Remove the medium from each well.

    2. Collect the organoids by disrupting the drops gently with a 10 mL pipette.

    3. Pull together 3–4 wells of organoids into a 1× 15 mL Falcon tubes containing 10 mL ice cold Ad+++

    4. Pipette up and down gently 3–5 times with a 10 mL pipette.

    5. Incubate on ice for 50–60 min.

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

    6. Centrifuge the differentiated organoids at 100 × g and 4°C for 10 min.

    7. Discard the supernatant.

    8. Thaw the pre-titered recombinant HBV stock on ice.

    9. Centrifuge the virus at max speed and 4°C for 10 min.

    10. Prepare 500 μL of Ad+++ containing 1–10 × 107 copies of HBV (or heat inactivated HBV as control) with or without 4% PEG. Resuspend a pellet of washed differentiated organoids corresponding to 3–4 wells (of 24-well plate) in the 500 μL virus mix.

    11. Divide the volume over a 48 (250 μL/well) or 24-well plate (500 μL/well)

    12. Seal the plate with parafilm (Figure 12).



      Figure 12. Parafilm sealed plate containing differentiated liver organoids and HBV.


    13. Place the sealed plates in the pre-warmed (32°C) Eppendorf centrifuge (5810 R) and spin infect at 600 × g and 32°C for 60 min.

    14. Remove the parafilm.

    15. Triturate gently the organoids with a P1000 pipette.

      Critical step: If the organoids are too big, snip the tip of the P1000 before triturating.

    16. Incubate at 37°C and 5% CO2 for 5 h.

    17. Collect the organoids in a 15 mL Falcon tube with a 10 mL pipette.

    18. Centrifuge at 100 × g and 4°C for 10 min.

    19. Discard the supernatant containing the virus.

      Critical step: Virus can be recycled 2–3 times. It is recommended to check the virus titer before each subsequent infection.

    20. Resuspend the organoids with 10 mL of ice-cold Ad+++.

    21. Centrifuge at 100 × g and 4°C for 10 min.

    22. Repeat steps 20–21 4–5 times.

    23. Remove the supernatant completely.

    24. Resuspend the pellet with an appropriate volume of 1/3 Ad+++ and 2/3 BME.

    25. Make the drops in pre-warmed plate. Keep the number of wells the same as the number of wells that were collected for infection (i.e., if 12 wells of differentiated organoids are used for infection, the organoids can be seeded back into 12 wells post spin infection.)

    26. Incubate the plate at 37°C and 5% CO2 for 30–45 min.

    27. Add 250 µL (48-well plate) or 500 µL (24-well plate) of warm EM supplemented with 10 µM Rho-kinase inhibitor.



      Figure 13. Hepatitis B virus (HBV)-infected liver organoids.


    28. The next day, remove the medium of organoids.

    29. Keep the medium to titer.

    30. Gently wash the drops with 500 µL of pre-warmed Ad+++ four times.

    31. Add 250 µL (48-well plate) or 500 µL (24-well plate) of pre-warmed DM.

    32. Change the medium every 2–3 days depending on experiment.

Recipes

  1. 70% ethanol solution (100 mL)

    70 mL of ethanol absolute

    30 mL of Milli-Q treated Water

  2. DMEM +/+ (50 mL)

    49.45 mL of DMEM

    500 µL of FBS

    50 µL of Penicillin-Streptomycin

    Filter with 0.2 µm filter

  3. Digestion solution (10 mL)

    2.5 mL of 10 mg/mL Collagenase D

    7.49 mL of EBSS

    10 µL of Rho-kinase inhibitor

    Filter with 0.2 µm filter

    Incubate at 37°C for 15–20 min

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

    485 mL of DMEM

    5 mL of HEPES

    5 mL of Ultraglutamine

    5 mL of Penicillin-Streptomycin

  5. 0.1% BSA/PBS (10 mL)

    10 mg of BSA

    Top up to 10 mL with PBS

    Filter with 0.2 µm filter

  6. 2× Human liver organoid basal medium (2× LBM) (50 mL)

    1 mL of N2 supplement

    2 mL of B27 supplement

    250 µL of 500 mM N-acetyl Cysteine (NAC in water)

    10 µL of 500 µg/mL hEGF in 0.1% BSA/PBS

    10 µL of 100 µM Gastrin in PBS

    50 µL of 50 µg/mL HGF in 0.1% BSA/PBS

    200 µL of Primocin

    46.48 mL of Ad+++

  7. Human liver organoids isolation medium (IM) (20 mL)

    10 mL of 2× LBM

    4 mL of R-spondin 3 conditioned medium

    2 mL of Wnt3A conditioned medium

    20 µL of 100 µg/mL FGF10 in 0.1% BSA/PBS

    200 µL of 1 M Nicotinamide in PBS

    5 µL of 20 mM TGF-β inhibitor (A83.01) in DMSO

    10 µL of 20 mM Forskolin in DMSO

    20 µL of 10 mM Rho-kinase inhibitor in DMSO

    20 µL 25 µg/mL Noggin in 0.1% BSA/PBS

    3.725 mL of Ad+++

    Filter with 0.1 µm filter

  8. Human liver organoids Expansion medium (EM) (20 mL)

    10 mL of 2× LBM

    4 mL of R-spondin 3 conditioned medium

    250 µL of Wnt3A conditioned medium

    20 µL of 100 µg/mL FGF10 in 0.1% BSA/PBS

    200 µL of 1 M Nicotinamide in PBS

    5 µL of 20 mM TGF-β inhibitor (A83.01) in DMSO

    10 µL of 20 mM Forskolin in DMSO

    5.515 mL of Ad+++

    Filter with 0.1 µm filter

  9. Human liver organoids Expansion medium without wnt3A supplemented with BMP7 (EMB) (20 mL)

    10 mL of 2× LBM

    4 mL of R-spondin 3 conditioned medium

    20 µL of 100 µg/mL FGF10 in 0.1% BSA/PBS

    200 µL of 1M Nicotinamide in PBS

    5 µL of 20mM TGF-β inhibitor (A83.01) in DMSO

    10 µL of 20mM Forskolin in DMSO

    20 µL of 25 µg/ml BMP7 in 0.1% BSA/PBS

    5.745 mL of Ad+++

    Filter with 0.1 µm filter

  10. Human liver organoids Differentiation medium (DM) (20 mL)

    10 mL of 2× LBM

    20 µL of 25 µg/mL BMP7 in 0.1% BSA/PBS

    20 µL of 100 µg/mL FGF19 in 0.1% BSA/PBS

    2 µL of 5 mM TGF-β inhibitor (A83.01) in DMSO

    20 µL of 10 mM DAPT in DMSO

    2 µL of 30 mM Dexamethasone in DMSO

    9.936 mL of Ad+++

Acknowledgments

The generation, maintenance and differentiation of human liver organoids part of the protocol has been adapted from previously published work (Broutier et al., 2016).

Competing interests

The authors declare no conflict of interest.

References

  1. Huch, M., Dorrell, C., Boj, S. F., van Es, J. H., Li, V. S., van de Wetering, M., Sato, T., Hamer, K., Sasaki, N., Finegold, M. J., et al. (2013). In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration. Nature 494(7436): 247-250.
  2. Huch, M., Gehart, H., van Boxtel, R., Hamer, K., Blokzijl, F., Verstegen, M. M., Ellis, E., van Wenum, M., Fuchs, S. A., de Ligt, J., et al. (2015). Long-term culture of genome-stable bipotent stem cells from adult human liver. Cell 160(1-2): 299-312.
  3. Broutier, L., Andersson-Rolf, A., Hindley, C. J., Boj, S. F., Clevers, H., Koo, B. K. and Huch, M. (2016). Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation. Nat Protoc 11(9): 1724-1743.
  4. De Crignis, E., Hossain, T., Romal, S., Carofiglio, F., Moulos, P., Khalid, M. M., Rao, S., Bazrafshan, A., Verstegen, M. M., Pourfarzad, F., et al. (2021). Application of human liver organoids as a patient-derived primary model for HBV infection and related hepatocellular carcinoma. Elife 10: e60747.

简介

[摘要]乙型肝炎病毒(HBV)感染是一个严重的公共卫生问题,全球约有4亿人受到感染。尽管有预防性疫苗和抗病毒疗法,慢性HBV感染仍然是一个主要的健康问题,因为它增加了发展为肝硬化和肝细胞癌(HCC)的风险。缺乏一个相关的体外模型来研究驱动HBV复制和潜伏期的分子机制,以及HBV相关的致癌作用,一直是治疗策略发展的主要障碍之一。在这里,我们建议使用人类肝脏类器官作为模拟HBV感染和相关肿瘤发生的平台。人类肝脏类器官可以从健康和肝硬化肝活检中提取。当在含有一组特定生长因子的培养基中培养时,它们可以在体外扩增。当培养基转变为含有促进分化的生长因子的新培养基时,类器官分化为功能性肝细胞,这使它们容易感染重组HBV。本方案中描述的新型体外原代模型系统可作为研究HBV发病机制和药物筛选的平台。肝硬化肝活检产生的类器官可能是个性化药物的潜在工具,并用于模拟肝癌和其他肝脏疾病。

图形摘要:

[背景]类器官培养技术涉及生成成人干细胞衍生的、遗传稳定的体外人体3D器官模型。先前已经建立了一个基于原代细胞分离和扩增的原代肝培养系统,该系统允许肝细胞作为类器官长期扩增(Huch et al.,2013,2015)。它们是通过在特定培养基中培养存在于肝脏中的Lgr5+成体干细胞的体外扩增产生的。Broutier等人(2016年)曾报道过肝类器官培养的建立。肝类器官可以从健康和HBV感染患者的肝组织中产生。与其他体外细胞肝细胞模型相比,肝类器官具有可扩展性、生物银行性和遗传稳定性。最近,我们使用肝类器官作为原代细胞衍生模型来研究HBV感染和相关的肿瘤发生,以及用于抗病毒药物筛选和药物诱导毒性的个性化体外平台(De Crignis et al.,2021)。在我们的研究中,我们使用了Broutier et al.(2016)提出的改进方案来培养和维持肝脏类器官,我们对分离和扩展培养基进行了轻微修改,以优化肝脏类器官的产量和生存能力。下文描述了健康和患者来源的肝类器官的产生、扩展和分化的详细方案。分化的肝类器官可通过使用浓缩病毒的自旋接种感染重组HBV。

关键字:健康的供体肝脏类器官, 患者来源的肝脏类器官, HBV感染

材料和试剂 1. 1.无菌注射器过滤器,0.1µm(默克密理博,目录号:SLVV033RS) 2. 2.无菌注射器过滤器,0.2μm(GE Healthcare,Whatman,目录号:6900-2502) 3. 3.无菌注射器(Omnifix,目录号:4616200V[20毫升];4616502F[50毫升]) 4. 4.单元过滤器70µm尼龙(Falcon,目录号:352350) 5. 5.带过滤器的低保留率无菌移液管头(Biotix,目录号:M-0010-9FC[P10];M-00209FC[P20];M-0200-9FC[P200];M-1000-9FC[P1000]) 6. 6.移液管控制器(品牌,目录号:26303) 7. 7.带过滤器的一次性无菌血清学移液管(VWR,目录号:89130-910[10毫升];89130890[25毫升]) 8. 8.15 mL Falcon管(Greiner,目录号:188285) 9. 9.50 mL Falcon管(Greiner,目录号:227285) 10. 10.1.5 毫升微量离心管(Biotix,目录号:MT-0150-BC) 11. 11.悬挂板(编号:2102,编号:6624) 12. 12.48井悬挂板(Greiner,目录号:677102) 13. 13.60 mm皮氏培养皿(赛默飞世尔科学公司,目录号:150462) 14. 14.一次性镊子(Servoprax,Mediware,目录号:H7-301) 15. 15.CryoTube TM小瓶(赛默科学公司,Nunc,目录号:366656)   16. 16.帕拉菲林M(西格玛·奥尔德里奇,目录号:P7793) 17. 17.无水乙醇(Sigma-Aldrich,默克密理博,目录号:1009832500) 18. 18.DMEM(Dulbecco改良的Eagle培养基)(赛默飞世尔科学公司,Gibco,目录号:41966029) 19. 19.胎牛血清(FBS)(摩羯座科学,目录号:FBS-12A) 20. 20.青霉素-链霉素(10000 U/mL)(赛默飞世尔科学公司,吉布科,目录号:15140122) 21. 21.Belzer UW冷藏解决方案(传输介质)(生命之桥,BTLBUW-2000) 22. 22.高级DMEM/F-12(赛默飞世尔科学公司,Gibco,目录号:12634028) 23. 23.HEPES,0.85%NaCl中的1M缓冲溶液(Lonza,目录号:BE17-737E) 24. 24.在0.85%NaCl溶液中加入200mM超谷氨酰胺(Lonza,目录号:BE17605E/U1) 25. 25.EBSS(厄尔平衡盐溶液)(赛默飞世尔科学公司,目录号:24010-043) 26. 26.胶原酶D(SigmaAldrich,目录号:11088858001) 27. 27.回收细胞培养冷冻培养基(Invitrogen,目录号:12648010) 28. 28.TripleExpress(赛默飞世尔科技有限公司,Gibco,目录号:12604013) 29. 29.Cultrex减少生长因子基膜提取物(BME),2型,Pathclear(研发系统,目录号3533-010-02) 30. 30.牛血清白蛋白(Sigma-Aldrich,目录号:A7300) 31. 31.磷酸盐缓冲盐水(PBS)(赛默飞世尔科学公司,Gibco,目录号:10010023) 32. 32.N2补充件(100×)(赛默飞世尔科技有限公司,Gibco,目录号:17502-048) 33. 33.B27补遗(不含维生素A)(赛默飞世尔科技有限公司,Gibco,目录号:12587010) 34. 34.N-乙酰半胱氨酸(Sigma-Aldrich,目录号:A7250) 35. 35.重组人EGF(PeproTech,目录号:AF-100-15) 36. 36.重组[Leu15]-胃泌素I人类(Sigma-Aldrich,目录号:G9145) 37. 37.重组人HGF(PeproTech,目录号:100-39H) 38. 38.Primocin(Invivogen,目录号:ant-pm-2) 39. 39.重组人R-海绵蛋白3(条件培养基,自制)(Broutier等人,2016年) 40. 40.Wnt 3A(条件培养基,自制)(Broutier等人,2016年) 41. 41.重组人FGF-10(PeproTech,目录号:100-26) 42. 42.烟酰胺(Sigma-Aldrich,目录号:N3376) 43. 43.TGF-β抑制剂(A83.01)(Tocris,目录号:2939) 44. 44.Forskolin(SigmaAldrich,目录号:F3917) 45. 45.Rho激酶抑制剂(岩石抑制剂,Y-27632)(Sigma-Aldrich,目录号:SCM075) 46. 46.重组人诺金(PeproTech,目录号:120-10C) 47. 47.重组人BMP7(PeproTech,目录号:120-03P) 48. 48.重组人FGF19(PeproTech,目录号:100-32) 49. 49.DAPT(Sigma-Aldrich,目录号:D5942) 50. 50.地塞米松(Sigma-Aldrich,目录号:D4902) 51. 51.PEG-6000(密理博,目录号:528877) 52. 52.70%乙醇溶液(100毫升)(见配方)   53. 53.DMEM+/+(50毫升)(见配方) 54. 54.消化液(10毫升)(见配方) 55. 55.Ad+++培养基(500毫升)(见配方) 56. 56.0.1%BSA/PBS(10毫升)(见配方) 57. 57.2×人肝类器官基础培养基(2×LBM)(50毫升)(见配方) 58. 58.人肝类器官分离培养基(IM)(20毫升)(见配方) 59. 59.人肝类器官扩张培养基(EM)(20毫升)(见配方) 60. 60.不含wnt3A、补充BMP7(EMB)的人肝类器官扩张培养基(20mL)(见配方) 61. 61.人肝类器官分化培养基(DM)(20毫升)(见配方) 设备 1. 1.校准微量移液管(VWR,Gilson,目录号:613-5945[P10];613-5946[P20];613-5948[P200];613-5949[P1000]) 2. 2.生物安全柜(贝克清洁空气,型号:BioVanguard II级生物安全柜) 3. 3.含5%二氧化碳、37°C的细胞培养箱(松下,目录号:MCO-170AICUVH-PE) 4. 4.明场显微镜(徕卡DMIL显微镜和DFC420C相机) 5. 5.振动筛(赛默科技,型号:88881102) 6. 6.Eppendorf离心机5810 R,用于15毫升Falcon试管和细胞培养板(Eppendorf,型号:A4-62) 7. 7.Eppendorf离心机5417 R,用于1.5毫升试管(Eppendorf,型号:F45-30-11) 8. 8.Milli-Q®IQ 7000超纯实验室用水系统 9. 9.37°C水浴(格兰特,VFP) 10. 10.-松下-794°C冷冻柜 11. 11.冷冻容器(NalgeneTM Cryo,目录号:5100-0001) 12. 12.冰箱(利勃海尔,目录号:CP3523-22) 13. 13.-20°C冷冻柜(博世GSD-3513) 14. 14.液氮储存 15. 15.手术刀(刀片10或11)(斯旺·莫顿,目录号:233-5364或233-5363) 16. 16.手术刀(刀片12D,双面)(斯旺·莫顿,目录号:233-5516) 17. 17.耐酒精低温科学标记(LabTAG,目录号:SM-1) 18. 18.细胞计数室(Marienfeld,Bürker-Türk,目录号:1630-1545) 程序 A. A.人体肝细胞的分离和类有机物的接种人体组织工作的安全说明: • •处理人体组织时,在生物安全柜内工作。   • •在使用人体组织时采取预防措施(例如,穿实验室工作服、戴手套、对工作空间进行消毒等) 关键步骤: a. a、 在37°C的培养箱中预热24/48孔悬浮板。 b. b、 开始前,准备好程序所需的所有缓冲液/溶液。 1. 1.手术切除后,根据活检组织的大小,立即在15毫升或50毫升的Falcon管中收集样本,并在4°C下保存在5-10毫升Belzer UW冷藏溶液(外科医生提供用于转移活检组织的商业转移介质)中,直至处理。  可选步骤:活组织检查可在4°C下保存48小时。我们建议在手术后立即处理组织。 2. 2.取下基本培养基,用10mL移液管用10mL DMEM+/+(见配方)清洗组织两次。 3. 3.使用无菌镊子将组织放入60mm培养皿中(图1)。 图1。供者(左)和患者(右)的人类肝脏活检。 4. 4.加上 1–2 毫升 属于 Ad+++媒体 在…上 顶部 属于 这个 组织 和 用绞肉机绞 把它变成 件 0.5–1 嗯3, 使用 二 手术刀 (切碎 这个 组织 直到 信息技术 可以 是 容易地 磨练 具有 A. p1000 移液管) (数字) 2 –3). 关键步骤:使用手术刀刀片12D和手术刀刀片10或11切割组织。试着用手术刀轻轻地垂直切割组织,而不是用力按压或捣碎与手术刀侧面成一定角度的组织。用力过大会导致细胞破裂。     图2。肝活检切碎。 图3。捐赠者(左)和患者(右)切碎的肝组织。 5. 5.用P1000移液管将切碎的组织移到15毫升的Falcon管中,用冰冷的Ad++加满10毫升。用10毫升移液管轻轻上下移液,清洗肝片。 6. 6.离心机 在 100 × G 和 4摄氏度 对于 10 敏。 关键步骤:生物银行组织: a. a、 离心后将切碎的组织一分为二,将一半放入含有1毫升冷冻介质的冷冻管中。 b. b、 用耐酒精标记标记,并将冷冻管转移到预冷(4°C)冷冻容器中。 c. c、 将冷冻容器转移至-80°c。 d. d、 在2–3天内将样品从-80°C冷冻柜转移到液氮中,以便长期储存。 e. e、 类有机物可以从冷冻组织中生成,尽管效率较低。冷冻组织也可用于基因组学、转录组学和蛋白质组学研究。 f. f、 要从冷冻切碎的组织中生成类有机物,请从液体中取出样本   氮气罐并在干冰上转移。取小瓶并将其置于水浴(37°C)中40–60 s(约90%的样品解冻),然后立即向小瓶中逐滴添加1 mL Ad++。将体积转移到含有10毫升Ad++的15毫升Falcon试管中。从第6步继续。 7. 7.丢弃上清液,并添加足量的预热消化液(根据组织大小4-6毫升,较小组织4毫升,较大组织6毫升)。 8. 8.将试管倒置5-8次,用P1000移液管上下移液,用消化液轻轻均匀组织。 9. 9.放置包含 均质样品 水平的 在架子上 转移 这是一个很好的例子 摇壶 恒温箱 在 100–120 转速 和 37摄氏度 对于 45 闵–1 H (图 4). 关键步骤:每15分钟检查一次,确保不会过度消化组织。当几乎所有的组织都被消化时,立即开始下一步。 图4。将切碎的纸巾放在培养箱内的摇壶上。 10. 10.用P1000移液管上下移液,重新培养细胞(图5)。 图5。消化性肝活检。   11. 11.加入10毫升冷Ad++,中和消化。 12. 12.将消化后的材料通过70µm的滤池过滤器(图6),并将材料收集在50 mL Falcon管中。  可选步骤:如果任何部分消化的组织残留在过滤器上,用p1000移液管收集,并在37°C和5%CO2下进一步与1毫升胰蛋白酶孵育10–15分钟。孵育后,再次执行步骤12。  可选步骤:如果溶液中仍然存在培养皿中的任何碎屑或塑料片,重复步骤12以去除这些碎屑或塑料片。 图6。消化肝活检的应变。 13. 13.加入预热的Ad++(37°C),将体积增加至25–30毫升。 14. 14.在200×g和4°C条件下离心5分钟。 15. 15.去除上清液。用10毫升移液管将细胞颗粒重新悬浮在10毫升冷Ad+++中,并将重新悬浮的细胞转移到15毫升Falcon管中。 16. 16.在200×g和4°C条件下离心5分钟。 17. 17.去除上清液,并将细胞颗粒重新悬浮在2毫升Ad++。对细胞进行计数,并将适当数量的细胞与BME混合,BME将聚合并形成穹顶状结构(滴),为类有机物提供3D生长的机械支持。 18. 18.用血液细胞仪计数细胞,在48孔培养皿中每25µL滴培养约10000–15000个细胞。 19. 19.在200×g和4°C条件下离心5分钟。 20. 20.用适当体积的Ad+++和BME重新放置细胞,以形成滴液(每个滴) drop包含1/3 Ad+++和2/3 BME,即每25µL添加8.3µL Ad+++和16.6µL BME- 滴 在里面 A. 48井 盘子)。 关键步骤:在均匀化BME/Ad+++混合物并进行滴注时,重要的是避免产生任何气泡。   关键步骤:在处理BME时,始终将BME保持在冰上,并且在滴下时要迅速。如果将BME从冰中取出超过1分钟,BME将开始固化。 21. 21.拿 这个 预热的 盘子 从…起 这个 培养箱 和 开始 制作 这个 滴 (图 7). 注:48孔板的每个孔的液滴体积为25µL。对于24孔板,可以使用3×25µL液滴或1×50µL液滴。 图7。含有肝细胞的BME滴剂。 22. 22.在37°C和5%CO2下培养30-45分钟,让BME聚合。 23. 23.在每个孔中加入250µL(48孔板)或500µL(24孔板)温热的人肝类器官分离培养基(IM)(图8)。 图8。加入类有机物培养基。 关键步骤: a. a、 将培养基轻轻地放在井的一侧。避免将培养基直接添加到培养基上   滴水。 b. b、 在前七天,每3-4天更换一次培养基,并使用IM。 c. c、 接种类器官1周后,将IM改为人肝扩张培养基。 d. d、 类有机物将在播种后2-14天内可见。需要使用亮场显微镜进行日常监测,以识别正在生长的类器官(图9)。 图9。从肝活检中生成类器官。 B. B.培养基中类有机物的扩展和维持 根据类有机物密度和大小(>500–1000µm),每7–10天拆分一次类有机物。在这里,术语拆分是指以酶和机械方式消化类有机物,并通过将消化后的类有机物与BME/Ad+++混合并制备液滴,将消化后的类有机物分配到所需数量的孔中。一般分割比为1:4–1:8,取决于供体类器官系的生长速率。 关键步骤:预热24井悬挂板 1. 1.制备1×15 mL Falcon管,其中含有10 mL冰冷的Ad++。 2. 2.从每口井中丢弃培养基。 3. 3.为了收集滴落物,从15毫升Falcon试管中取出4毫升冰凉的Ad++,然后向每个含有需要拆分的类有机物的孔中加入1毫升。 4. 4.用P1000移液管刮除含有类有机物的液滴。 5. 5.收集被破坏的类有机物并转移到一个15毫升的Falcon管中。在一个15ml Falcon管中最多可以收集4个孔(例如,如果8个孔被拆分,则类有机物可以收集在两个15ml Falcon管中)。   注:对于每4个类有机物孔,遵循步骤3-5。 6. 6.用10毫升移液管研磨3-5次。 7. 7.将猎鹰管置于冰上20-45分钟。每10分钟翻转一次猎鹰管,这有助于BME有效溶解。 8. 8.在200×g和4°C条件下离心5分钟。 9. 9.丢弃上清液。 10. 10.添加 60–100 微升 属于 锥虫 到 这个 弹丸 属于 类器官。 消化 这个 类器官 机械地 在房间里 温度 对于 1–5 敏, 通过 移液 具有 A. P200 移液管。 注意:在显微镜下检查消化的类有机物的大小。所需尺寸取决于实验要求。确保消化后的类有机物大小一致。 11. 11.加入10毫升冰冷Ad++,停止消化,并在200×g和4°C条件下离心5分钟。 12. 12.完全丢弃上清液。 13. 13.用适当量的1/3 Ad+++和2/3 BME(例如,如果将两个孔以1:4的比例分开,则需要制备8滴;如果将6滴以1:6的比例分开,则需要制备36滴)重新悬浮消化的类有机物颗粒。 14. 14.根据所用的盘子滴适量的液。 15. 15.让液滴在37°C和5%CO2下聚合30–45分钟。 16. 16.添加250µL(48孔板)或500µL(24孔板)热EM。 17. 17.每2-3天更换一次培养基(图10)。 图10。扩张培养基中的肝类器官。 C. C.肝类器官的肝分化 1. 1.文化 肝脏 类器官 具有 EM-wnt3A+BMP7 对于 3–5 天 先前的 到 区别 到 允许 这个 类器官 到 恢复 和 彻底地 改革 之后 分裂。 关键步骤:如果类有机物很大,这可能会导致旋转感染期间类有机物破裂。因此,当肝脏类器官较小(50–350µm)时,开始区分它们。 2. 2.用500µL温热的Ad++将滴剂轻轻清洗两次。   3. 3.添加250µL(48孔板)或500µL(24孔板)温热人肝类器官分化培养基(DM)。 4. 4.每2-3天更换一次培养基。将类有机物区分7天(图11)。 图11。肝类器官在分化培养基中。 D. 乙型肝炎病毒感染 人类肝脏类器官的感染既可以不添加PEG,也可以添加4%的PEG,这对提高感染效果起到了作用。 使用重组HBV的安全说明: • •只有接种乙肝疫苗的人员才能处理乙肝病毒。 • •处理HBV时,在生物安全柜内工作 • •在处理HBV时采取适当的预防措施(即,穿实验室工作服、戴手套、对工作空间进行消毒等) 1. 1.从每口井中取出培养基。 2. 2.用10毫升移液管轻轻搅动液滴,收集类有机物。 3. 3.将3-4孔类有机物收集到1×15 mL Falcon试管中,试管中含有10 mL冰镇Ad+++ 4. 4.用10毫升移液管轻轻上下移液3-5次。 5. 5.孵化 在…上 冰 50–60 敏。 关键步骤:每5分钟翻转一次flacon管,混合有机物。这将有助于BME有效溶解。 6. 6.将分化的类有机物在100×g和4℃下离心10分钟。 7. 7.丢弃上清液。 8. 8.在冰上解冻预先滴度的重组HBV储备。 9. 9.以最大速度和4°C离心病毒10分钟。 10. 10.制备500μL含有1-10×107份HBV(或作为对照的热灭活HBV)的含或不含4%聚乙二醇的Ad++。重新使用一粒洗涤过的分化类有机物   对应于500μL病毒混合物中的3-4个孔(24孔板)。 11. 11.将体积除以48(250μL/孔)或24孔板(500μL/孔) 12. 12.用parafilm密封板(图12)。 图12。含有分化肝类器官和HBV的旁膜密封板。 13. 13.将密封板放入预热(32°C)的Eppendorf离心机(5810 R)中,在600×g和32°C下旋转感染60分钟。 14. 14.取下薄膜。 15. 15.研磨 轻轻地 这个 类器官 具有 A. P1000 移液管。 关键步骤:如果类有机物太大,在研磨前剪断P1000的尖端。 16. 16.在37°C和5%CO2下培养5小时。 17. 17.用10毫升移液管在15毫升Falcon管中收集类有机物。 18. 18.在100×g和4°C条件下离心10分钟。 19. 19.丢弃 这个 上清液 包含 这个 病毒 关键步骤:病毒可以回收2-3次。建议在每次后续感染前检查病毒滴度。 20. 20.用10毫升冰冷的Ad++重新注入有机物。 21. 21.在100×g和4°C条件下离心10分钟。 22. 22.重复步骤20-21 4-5次。 23. 23.完全去除上清液。 24. 24.用1/3 Ad+++和2/3 BME的适当体积重新悬浮颗粒。 25. 25.在预热的盘子里滴一滴。保持孔的数量与收集用于感染的孔的数量相同(即,如果12个分化类器官的孔用于感染,则类器官可以在旋转感染后重新播种到12个孔中。) 26. 26.在37°C和5%CO2下培养培养皿。持续30-45分钟。 27. 27.添加250µL(48孔板)或500µL(24孔板)热EM,并添加10µM Rhokinase抑制剂。     图13。乙型肝炎病毒(HBV)感染的肝类器官。 28. 28.第二天,去除类有机物培养基。 29. 29.保持培养基的滴度。 30. 30.用500µL预热的Ad+++轻轻清洗滴剂四次。 31. 31.添加250µL(48孔板)或500µL(24孔板)预热DM。 32. 32.根据实验情况,每2-3天更换一次培养基。 食谱 1. 70%乙醇溶液(100毫升)70毫升无水乙醇 30 毫升Milli-Q处理水2。DMEM+/+(50毫升) 49.45 1毫升DMEM 500微升FBS 50 µL青霉素链霉素过滤器和0.2µm过滤器 3. 3.消化液(10毫升) 2.5 10毫克/毫升胶原酶D 7.49 1毫升EBSS 10µL Rho激酶抑制剂过滤器和0.2µm过滤器在37°C下培养15–20分钟 4. 4.Ad+++培养基(500毫升)485毫升DMEM 5 毫升HEPES 5 毫升超谷氨酰胺 5 青霉素链霉素5毫升。0.1%BSA/PBS(10毫升)   10 牛血清白蛋白毫克 用PBS过滤器和0.2µm过滤器加满10毫升 6. 6.2×人肝类器官基础培养基(2×LBM)(50毫升)1毫升N2补充剂 2 B27补充剂毫升 250µL 500 mM N-乙酰半胱氨酸(NAC在水中)10µL 500µg/mL hEGF在0.1%BSA/PBS中 10 PBS中的100µM胃泌素微升 50 0.1%牛血清白蛋白/PBS中50微克/毫升HGF的微升,200微升初霉素 46.48 广告的毫升数+++ 7. 7.人肝类器官分离培养基(IM)(20mL)10mL 2×LBM 4 1毫升R-spondin 3条件培养基2毫升Wnt3A条件培养基 20 0.1%BSA/PBS中的100µg/mL FGF10微升PBS中的1 M烟酰胺200µL 5 在DMSO中加入µL 20 mM TGF-β抑制剂(A83.01)在DMSO中加入10µL 20 mM Forskolin 20 10 mM Rho激酶抑制剂0.1%二甲基亚砜20µL 25µg/mL诺金0.1%牛血清白蛋白/PBS 3.725 mL Ad+++ 带0.1µm过滤器的过滤器 8. 8.人肝类器官扩张培养基(EM)(20mL)10mL 2×LBM 4 R-spondin 3条件培养基mL 250µL Wnt3A条件培养基 20 0.1%BSA/PBS中的100µg/mL FGF10微升PBS中的1 M烟酰胺200µL 5 在DMSO中加入µL 20 mM TGF-β抑制剂(A83.01)在DMSO中加入10µL 20 mM Forskolin 5.515毫升Ad+++过滤器和0.1µm过滤器 9. 9.不含wnt3A、补充BMP7(EMB)(20mL)10mL 2×LBM的人肝类器官扩张培养基 4 R-spondin 3条件培养基毫升 20 0.1%BSA/PBS中的100µg/mL FGF10微升PBS中的1M烟酰胺200µL   5 20mM TGF-β抑制剂(A83.01)在二甲基亚砜中的用量为10μL在二甲基亚砜中的20mM Forskolin 20 在0.1%BSA/PBS 5.745毫升Ad中加入25微克/毫升BMP7微升+++ 带0.1µm过滤器的过滤器 10. 10.人肝类器官分化培养基(DM)(20mL)10mL 2×LBM 20 0.1%BSA/PBS中的25微克/毫升BMP7微升 20 0.1%BSA/PBS中的100微克/毫升FGF19微升 2 5μL 5mM TGF-β抑制剂(A83.01)在DMSO中,20μL 10mM DAPT在DMSO中 2 在DMSO 9.936 mL Ad中加入µL 30 mM地塞米松+++ 致谢 该方案中人类肝脏类器官的生成、维持和分化部分改编自之前发表的工作(Broutier et al.,2016)。 竞争利益 作者声明没有利益冲突。 工具书类 1. 1.胡奇,M.,多雷尔,C.,日本银行,S.F.,范恩斯,J.H.,李,V.S.,范德韦特林,M.,佐藤,T.,哈默尔,K.,佐佐木,N.,Finegold,M.J.,等(2013)。体外培养诱导单个Lgr5+肝干细胞扩增 通过 Wnt驱动 再生 自然494(7436):247-250。 2. 2.赫赫,M.,盖哈特,H.,范博克塞尔,R.,哈默,K.,布洛克齐尔,F.,维斯特根,M.M.,埃利斯,E.,范文努姆,M.,富克斯,S.A.,德利格特,J.等人(2015年)。基因组稳定的双能干细胞的长期培养 从…起 成人 人类 肝脏 160号牢房(1-2):299-312。 3. 3.布鲁蒂埃,L.,安德森·罗尔夫,A.,辛德利,C.J.,日本银行,S.F.,克利夫斯,H.,古,B.K.和哈奇,M.(2016)。人和小鼠成体肝脏和胰腺3D的培养和建立 类器官 和 他们的 遗传的 操纵 Nat Protoc 11(9):1724-1743。 4. 4.E.德克里尼斯、T.侯赛因、S.罗马尔、F.卡罗菲利奥、P.莫洛斯、哈立德、M.M.拉奥、S.巴兹拉夫尚、A.维斯特根、M.M.波尔法扎德等(2021年)。应用 属于 人类 肝脏 类器官 像 A. 病人- 衍生 主要的,重要的 模型 对于 乙肝病毒 传染 和 相关的 肝细胞 癌症。 以利腓书10:e60747。

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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Romal, S., Hossain, T. and Mahmoudi, T. (2022). Generation, Maintenance and HBV Infection of Human Liver Organoids. Bio-protocol 12(6): e4358. DOI: 10.21769/BioProtoc.4358.
  2. De Crignis, E., Hossain, T., Romal, S., Carofiglio, F., Moulos, P., Khalid, M. M., Rao, S., Bazrafshan, A., Verstegen, M. M., Pourfarzad, F., et al. (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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