Human Liver Organoid Culture Protocol

This protocol is intended as a guide only, for full experimental details please read the reference provided.

Introduction

This protocol provides a procedure for subculturing normal human liver organoids. This protocol was modified from the submerged method described in Huch, M. et al. (2015) Long-term culture of genome-stable bipotent stem cells from adult human liver. Cell 160: 299.

The protocol provided below is intended to culture organoids from normal human liver tissues using Cultrex™ UltiMatrix Reduced Growth Factor (RGF) Basement Membrane Extract as a scaffold. The majority of reagents used in this protocol were sourced from the Bio-Techne brands of R&D Systems™ and Tocris Bioscience™.

Equipment

  1. Cell culture incubator (37 °C, 5% CO2)
  2. Cell culture hood with laminar flow
  3. Centrifuge with refrigeration and swinging bucket rotor
  4. 37 °C water bath
  5. Ice bucket
  6. Laboratory refrigerator
  7. Pipet aid and serological pipettes (5 mL)
  8. Micropipettes and tips (2–200 μL)
  9. Conical tubes, 15 mL and 50 mL, sterile
  10. 24-well plate, tissue-culture treated, sterile
  11. Vacuum pump
  12. Medium filtration unit, 0.1 μm, 500 mL, sterile
  13. Syringe, 50 mL, sterile
  14. Syringe filter, 0.2 μm, sterile
  15. Cell culture waste container

Other Required Reagents

  1. Distilled (DW) or deionized water (DI)
  2. Phosphate buffered saline (PBS)

Materials

TABLE 1. Materials Needed for Human Liver Organoid Culture

Product Name

Supplier

Catalog #
Cultrex Organoid Harvesting Solution R&D Systems 3700-100-01
Cultrex UltiMatrix Reduced Growth Factor Basement Membrane Extract  R&D Systems BME001-05
Advanced DMEM/F-12 Cell Culture Medium Thermo Fisher 12634010
GlutaminePlus R&D Systems B90210
HEPES  Tocris Bioscience 3173
N21-MAX Supplement  R&D Systems AR008
N-2 MAX Supplement R&D Systems AR009
N-Acetylcysteine  Tocris Bioscience 7874
Gastrin I (Human) Tocris Bioscience 3006
Nicotinamide Tocris Bioscience 4106
Y-27632 dihydrochloride (Rho Kinase inhibitor) Tocris Bioscience 1254
A 83-01 (ALK5 inhibitor) Tocris Bioscience 2939
Forskolin Tocris Bioscience 1099
DAPT Tocris Bioscience 2634
Dexamethasone Tocris Bioscience 1126
Recombinant Human EGF R&D Systems 236-EG
Recombinant Human R-Spondin 1  R&D Systems 4645-RS
Recombinant Human Noggin  R&D Systems 6057-NG
Recombinant Human FGF-10  R&D Systems 345-FG
Recombinant Human FGF-19 R&D Systems 969-FG
Recombinant Human BMP-7 R&D Systems 354-BP
Recombinant Human HGF R&D Systems 294-HG
Recombinant Human Wnt-3a R&D Systems 5036-WN

Reagent Preparation

Use aseptic technique at all times during this protocol. This protocol is optimized for human liver organoids. Organoids from other tissues may have different culture requirements.

1. Thaw Cultrex UltiMatrix RGF Basement Membrane Extract (BME) on ice for four hours or overnight at 2 - 8 °C (on ice in the refrigerator).

2. Prepare Liver Organoid Initiation Medium (TABLE 2), Liver Organoid Expansion Medium (TABLE 3), and Liver Organoid Differentiation Medium (TABLE 4).

3. Sterile filter the media

 

TABLE 2. Human Liver Organoid Initiation Medium

Reagent Name

[FINAL]
Advanced DMEM/F-12 Cell Culture Medium NA
N21-MAX Supplement 1X
GlutaminePlus 2 mM
HEPES 10 mM
Penicillin/Streptomycin 1X
N-2 MAX Supplement 1X
Nicotinamide 10 mM
A 83-01 5 µM
N-Acetylcysteine 1.25 mM
Recombinant Human FGF-10 100 ng/mL
Recombinant Human R-Spondin 1 0.5 µg/mL
Gastrin I Human 10 nM
Recombinant Human BMP-7 25 ng/mL
Recombinant Human EGF 50 ng/mL
Recombinant Human HGF 25 ng/mL
Recombinant Human Noggin 25 ng/mL
Recombinant Human Wnt-3a 100 ng/mL
Forskolin 10 µM
Y-27632 dihydrochloride 10 µM

 

TABLE 3. Human Liver Organoid Expansion Medium

Reagent Name 

[FINAL]
Advanced DMEM/F-12 Cell Culture Medium NA
N21-MAX Supplement 1X
GlutaminePlus 2 mM
HEPES 10 mM
Penicillin/Streptomycin 1X
N-2 MAX Supplement 1X
Nicotinamide 10 mM
A 83-01 5 µM
N-Acetylcysteine 1.25 mM
Recombinant Human FGF-10 100 ng/mL
Recombinant Human R-Spondin 1 0.5 µg/mL
Gastrin I Human 10 nM
Recombinant Human BMP-7 25 ng/mL
Recombinant Human EGF 50 ng/mL
Recombinant Human HGF 25 ng/mL
Forskolin 10 µM

 

TABLE 4. Human Liver Organoid Differentiation Medium

Reagent Name 

[FINAL]
Advanced DMEM/F-12 Cell Culture Medium NA
N21-MAX Supplement 1X
GlutaminePlus 2 mM
HEPES 10 mM
Penicillin/Streptomycin 1X
N-2 MAX Supplement 1X
A 83-01 0.5 µM
Recombinant Human FGF-19 100 ng/mL
Gastrin I Human 10 nM
Recombinant Human BMP-7 25 ng/mL
Recombinant Human EGF 50 ng/mL
Recombinant Human HGF 25 ng/mL
DAPT 10 µM
Dexamethasone 30 µM

Methods for Expanding and Differentiating Human Liver Organoids

Expansion

1. Prepare a suspension of isolated and dissociated human liver tissues as detailed in Huch, M. et al. (2015) Long-term culture of genome-stable bipotent stem cells from adult human liver. Cell 160: 299.

2. Resuspend the cell pellet in Cultrex UltiMatrix RGF Basement Membrane Extract (BME) and aliquot into wells by dispensing 50 μL of the Cultrex UltiMatrix RGF BME liver cell mixture in the center of each well of a 24-well plate to create domes. Note: The Cultrex UltiMatrix RGF BME domes should not touch the sides of the well.

Placement of Cultrex UltiMatrix RGF BME Organoid Mixture in a 24-well and 6-well Plate.

FIGURE 1. Placement of Cultrex UltiMatrix RGF BME/Organoid Mixture in a 24-well or 6-well Plate. (A) Placement of Cultrex UltiMatrix RGF BME/organoid mixture in the center of a well in a 24-well plate or (B) placement of multiple domes within a well of a 6-well plate.

3. Incubate the plate in the cell culture incubator for 15 minutes to polymerize the Cultrex UltiMatrix RGF BME.

4. Add the appropriate volume (~500 μL/well) of Liver Organoid Initiation Medium (TABLE 2). Note: Medium should be gently pipetted into the corner of the well away from the Cultrex UltiMatrix RGF BME/ organoids to prevent their disruption.

5. Return the plate containing the organoid cultures to the cell culture incubator to promote growth.

6. After 3 days, aspirate the culture medium from each well and add fresh Liver Organoid Expansion Medium (TABLE 3) every other day (~500 μL/well). Note: Medium should be gently aspirated from and pipetted into the corner of the well away from the Cultrex UltiMatrix RGF BME/organoids to prevent their disruption.

7. Passage organoids (See Passage Liver Organoids section) or start differentiation after 7-10 days.

 

Differentiation

1. Following 7-10 days of expansion, aspirate the Liver Organoid Expansion Medium, wash once with PBS, then add an equal volume (~500 μL/well) of Liver Organoid Differentiation Medium (TABLE 4). Incubate for an additional 11-13 days.

2. The culture medium should be aspirated from each well and replaced with fresh Liver Organoid Differentiation Medium every other day. Note: Medium should be gently aspirated from and pipetted into the corner of the well away from the Cultrex UltiMatrix RGF BME/organoids to prevent their disruption.

Brightfield images of undifferentiated and differentiated human liver organoids

FIGURE 2. Undifferentiated and Differentiated Human Liver Organoids. Representative brightfield images of undifferentiated (left) and differentiated (right) human liver organoid that were cultured using Cultrex UltiMatrix RGF Basement Membrane Extract (R&D Systems, Catalog # BME001-05) and the other reagents listed in this protocol.

FIGURE 3. Characterization of Differentiated Human Liver Organoids. Adult stem cell-derived liver organoids were cultured using Cultrex UltiMatrix RGF Basement Membrane Extract (R&D Systems, Catalog # BME001-05) and the other reagents listed in this protocol.  Differentiated human liver organoids were stained for proteins characteristic of differentiated hepatocytes using a Sheep Anti-Human Cytokeratin 19 Polyclonal Antibody (R&D Systems, Catalog # AF3506), a Goat Anti-Human HNF3-beta Polyclonal Antibody (R&D Systems, Catalog # AF2400), and a Mouse Anti-Human Serum Albumin Monoclonal Antibody (R&D Systems, Catalog # MAB1455). Organoids were counterstained with DAPI (Tocris, Catalog # 5748).   

Staining of differentiated hepatocytes in human liver organoids using antibodies against Cytokeratin 19, HNF3-beta, and Albumin.

Passaging Liver Organoids

1. View liver organoids under the microscope. Each well should contain approximately 500 organoids for optimal growth. Organoid cultures exhibiting rapid growth may be split 1:4 during passaging, while slow growing cultures may benefit from a 1:1 split. Make this determination prior to harvesting to estimate reagent needs prior to starting. Note: Organoid density is important for optimal growth; too many organoids will strain culture resources, while too few organoids lack paracrine signaling necessary to sustain growth.

2. Transfer the 24-well plate containing liver organoids from the cell culture incubator to the cell culture hood.

3. Aspirate the medium without disturbing the Cultrex UltiMatrix RGF BME-contained organoids at the bottom of the well.

4. Gently wash each well with 10 volumes of cold (2 - 8 °C) PBS (TABLE 5). Be careful not to disrupt the basement membrane matrix-containing organoids.

 

TABLE 5. Suggested Volumes of PBS and Organoid Harvesting Solution

Plate Type

Volume of Basement Membrane Matrix

Volume of PBS and Organoid Harvesting Solution
96-well plate 5 µL 50 µL
48-well plate 25 µL 250 µL
24-well plate 50 µL 500 µL

5. Aspirate the PBS and add 10 volumes of cold (2 - 8 °C) Cultrex Organoid Harvesting Solution to each well (TABLE 5).

6. Incubate the plate at 2 - 8 °C or on ice for 30 - 90 minutes with moderate shaking. This incubation is complete when the basement membrane matrix dome is no longer visible at the bottom of the well and the organoids are seen floating at the bottom of the well. Note: Dislodging the dome with a cell scraper or pipet may accelerate this process.

7. Once the matrix depolymerizes, transfer the contents of the well into a tube on ice. Single wells may be transferred to a microtube while multiple domes may necessitate a 15 mL or 50 mL conical tube.

8. Centrifuge the tube at 500 x g for 5 minutes at 2 - 8 °C in a swinging bucket rotor to pellet the organoids. Aspirate the supernatant.

9. Wash the organoids with 10 volumes of cold (2 - 8 °C) PBS and repeat centrifugation at 500 x g for 5 minutes at 2 - 8 °C in a swinging bucket rotor to pellet the organoids. Aspirate the PBS. Add fresh ice-cold Liver Organoid Expansion Medium.

10. Pipet up and down three times with a serological pipette to mix the organoids.

11. Centrifuge the tube at 500 x g at room temperature for 3 minutes.

12. Aspirate the medium but be careful not to disturb the organoid pellet.

13. Resuspend the organoids in Cultrex UltiMatrix RGF Basement Membrane Extract and dispense 50 μL of the mixture in the center of each well of a 24-well plate to form domes. Follow the density/splitting ratios recommended in Passaging Protocol Step 1. Note: The Cultrex UltiMatrix RGF BME domes should not touch the sides of the well.

14. Incubate the plate in the cell culture incubator for 15 minutes to polymerize the Cultrex UltiMatrix RGF Basement Membrane Extract.

15. Add 500 μL of Liver Organoid Initiation Medium per well. Note: Medium should be gently pipetted into the corner of the well away from the Cultrex UltiMatrix RGF BME/organoids to prevent their disruption.

16. Return the plate containing organoid cultures to the cell culture incubator to promote organoid growth. Follow the Liver Organoid Expansion Protocol.