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. Author manuscript; available in PMC: 2022 Jul 27.
Published in final edited form as: Methods Mol Biol. 2022;2455:117–129. doi: 10.1007/978-1-0716-2128-8_11

Culture of Mouse Liver Ductal Organoids

Junkai Yan 1, Yunling Tai 1, Huiping Zhou 1
PMCID: PMC9327439  NIHMSID: NIHMS1824221  PMID: 35212991

Abstract

3D organoid culture has become a powerful tool and model for various human diseases, including liver diseases, such as non-alcoholic steatohepatitis (NASH). Hepatic organoids have significant advantages over traditional primary cell cultures. The hepatic progenitor cells can be induced to form hepatic organoids. The established organoids can be passaged or cryopreserved for future use. The established hepatic organoids can be manipulated to study the disease progression of NASH-related fibrosis. Here, we describe a protocol to establish mouse liver ductal organoids.

Keywords: Organoids, Bile duct, Liver, Hepatocytes, Cholangiocytes

1. Introduction

The intrinsic ability of hepatic progenitor cells to self-renew and differentiate into different hepatic cells has allowed the development of 3D organoid cultures from mouse models and humans [1, 2]. The molecular pathways and growth factors involved in regulating progenitor proliferation, migration, and survival are highly conserved across all vertebrates. Even though the liver progenitor cells can be maintained in culture in the presence of growth factors they fail to exhibit long-term expansion. Recent advances in stem cell cultures have allowed the development of in vitro culture protocols for the expansion and differentiation of mouse and human primary liver cells by providing a gel-based extracellular matrix (ECM) environment together with the essential growth factors [3–5]. The established mouse hepatic organoids can be genetically manipulated, expanded, or cryopreserved for future use. Hepatocytes and cholangiocytes are two major cell types of epithelial cells in the liver with distinct physiological functions. The differentiation of hepatocytes and cholangiocytes is precisely controlled by multiple signaling pathways [6]. Bile ducts can form self-expanding organoids in a 3D culture system in the presence of growth factors and Wnt agonist. The ductal organoids can further differentiate into hepatocyte organoids by the withdrawal of Wnt activator and inhibiting TGF and notch signaling pathways [7–9]. Here, we described a detailed protocol to generate mouse liver ductal organoids from isolation and long-term expansion to genetic manipulation. These organoids can be used to study the disease progression of NASH-fibrosis.

2. Materials

2.1. Reagents

  1. HepatiCult™Organoid Growth Medium (basal medium) (Stemcell).

  2. HepatiCult™ Organoid Growth Medium (supplement) (Stemcell).

  3. DMEM/F-12 with 15 mM HEPES (Stemcell).

  4. Advanced DMEM/F-12 (Thermo Fisher).

  5. Anti-Adherence Rinsing Solution (Stemcell)

  6. Collagenase IV (Stemcell).

  7. Dispase (Stemcell).

  8. Basement Membrane Matrix (Corning).

  9. TrypLE Express (Thermo).

  10. Polyjet transfection reagent (SignaGen).

  11. DAPI solution (1 mg/mL)(ThermoFisher).

  12. Rat anti-cytokeratin 19 antibody (DSHB).

  13. Goat anti-rat IgG(H+L)-Alexa Fluor 594 (ThermoFisher).

  14. Normal rat IgG (ThermoFisher).

  15. Histowax (ThermFisher).

  16. Isofluorane, USP (Covetrus).

  17. Xylene.

  18. Ethanol, absolute.

2.2. Equipment

  1. Biological safety cabinet.

  2. Cell culture incubator with 5% CO2, 37 °C (Thermo Fisher).

  3. Bio-Rad ZOE Fluorescent Cell Imager.

  4. HM 355S Microtome (Microm).

  5. Temperature-controlled water bath with shaker (Thermo Fisher Scientific, MAXQ7000).

  6. Eppendorf centrifuge 5810R.

  7. Fine scissors and fine forceps.

  8. Pipette aid and calibrated pipettes (P10, P100, P200, P1000).

  9. Paraffin embedding equipment (Leica).

  10. Datex Ohmeda Tec Isotec 4 Isoflurane Anesthesia Vaporizer.

2.3. Plastic Supplies

  1. Conical centrifuge tubes (15 mL and 50 mL).

  2. Sterile filter tips (10 μL, 200 μL, 1000 μL).

  3. Serological pipettes (1, mL, 5 mL, 10 mL, and 25 mL).

  4. Cell culture dishes (100 mm).

  5. 24-well and 48-well suspension culture plates.

  6. 2-mL Cryogenic vials.

  7. 70-μm Cell strainer and 35μm Reversible Cell strainer (STEMCELL).

3. Methods

3.1. Preparation of Reagents and Materials

  1. HepatiCult™ Organoid Growth Medium: Thaw the Supplement at 2–8 °C overnight. Add 5 mL of Supplement to 94 mL of Basal Medium. Add 1 mL antibiotics. Mix thoroughly. Warm to room temperature (15–25 °C) before use.

  2. Tissue Dissociation Cocktail: Use the sterile technique to prepare 60 mL of Tissue Dissociation Cocktail (sufficient for one mouse liver) by combining 7.5 mL Collagenase IV, 7.5 mL Dispase, 45 mL DMEM/F-12 with 15 mM HEPES. Warm to room temperature (15–25 °C) before use.

  3. Thaw the Basement Membrane Matrix on ice.

  4. Pre-wet Conical Tubes and Serological Pipettes: Conical tubes and serological pipettes that come in contact with hepatic ducts should be pre-wetted, as hepatic ducts frequently adhere to their surfaces, significantly reducing organoid yield. Pre-wet tubes and pipettes on the day of the experiment. For each liver being processed, pre-wet 1 × 15 mL conical tubes and 1 × 50 mL conical tube by adding 5 mL of Anti-Adherence Rinsing Solution to a 15 mL conical tube or 20 mL to a 50 mL conical tube. Swirl to coat the tube. Aspirate any remaining Rinsing Solution from the coated tubes. Add 5 mL or 20 mL of Advanced DMEM/F-12 for 15 mL and 50 mL tubes, respectively, and swirl to coat the tubes. Aspirate any remaining medium from coated conical tubes. Immediately before use in step 15, Subheading 3.2, pre-wet 1 × 10 mL serological pipette by pipetting the Anti-Adherence Rinsing Solution up and down for 1–2 min, followed by Advanced DMEM/F-12.

3.2. Organoid Initiation

  1. Euthanize mouse by isoflurane inhalation using an Isoflurane Anesthesia Vaporizer with 5% isoflurane.

  2. Dissect the mouse on ice.

  3. Harvest the liver and transfer it into a 100-mm petri dish containing 10 mL ice-cold DMEM/F-12 (Fig. 1).

  4. Swirl dish to wash liver. Use forceps to transfer liver to a new culture dish containing 10 mL ice-cold DMEM/F-12.

  5. Cut the liver into small pieces (3–5 mm) while the liver is submerged in DMEM/F-12 medium using fine scissors. Transfer liver pieces and DMEM/F-12 into a 50 mL centrifuge tube using a 25 mL serological pipette.

  6. Add 10 mL of ice-cold DMEM/F-12 and pipette up and down a few times with a 10 mL pipette to remove red blood cells and fat. Leave the tube on ice for 5 min to allow liver pieces to settle by gravity. Aspirate and discard the supernatant.

  7. Add 10 mL of pre-warmed Tissue Dissociation Cocktail (see Subheading 3.1) to liver pieces. Incubate tube in a 37 °C water bath for 20 min.

  8. Remove the tube from water bath. Using a 10 mL serological pipette, vigorously pipette liver pieces up and down seven times with medium-high force.

  9. Let liver pieces settle by gravity for 1 min.

  10. Using a 10 mL serological pipette, remove and discard the supernatant (including digested cells suspended in it).

  11. Repeat steps 7-10 (digestion cycle #2). Using a 10 mL serological pipette, transfer supernatant to a new pre-cold 50 mL tube. Keep on ice.

  12. Repeat step 11, collecting supernatant into the 50 mL tube on ice after each digestion. Continue repeating these steps until liver pieces have been dissociated into hepatic ducts and no liver pieces remain. This usually requires up to 6 × 20 min digestion cycles. The pooled supernatant volume from digestion cycles 2–6 will be ~50 mL.

  13. Attach a 70 μm cell strainer to a new 50 mL conical tube. Using a 25 mL serological pipette, pass the pooled supernatant (from step 11) through the strainer. Discard the strainer.

  14. Attach a 37 μm Reversible Strainer to a new 50 mL conical tube. Using a 25 mL serological pipette, pass the flow-through from step 12 through the strainer. Discard the new flowthrough.

  15. Reverse the strainer onto a pre-wetted 50 mL conical tube (see Subheading 3.1). Using a 10 mL serological pipette, carefully add 10–12 mL of cold (2–8 °C) advanced DMEM/F-12 to the reversed strainer to wash hepatic ducts into the pre-wetted tube.

  16. Using a pre-wetted 10 mL serological pipette (see Subheading 3.1), pipette the hepatic ducts (collected in step 14) up and down 3–5 times to create an even suspension. Immediately transfer to a pre-wetted 15 mL conical tube (see Subheading 3.1).

  17. Centrifuge the tube at 290 × g for 5 min at 8 °C. Aspirate as much of the supernatant as possible without disturbing the pelleted ducts, leaving 10–20 μL in each tube (the pellet is often not visible) and place tubes on ice.

  18. Using a 200 μL pipette, add 200 μL of thawed Matrigel® on top of the pellet. Without generating bubbles, gently mix the duct-Matrigel® suspension by pipetting up and down 5–8 times, going to only the first stop of the pipettor.

  19. Add 50 μL suspension to the center of 1 well of the 24-well plate to form a dome. While dispensing, gradually move the pipette tip upwards so that the ducts are evenly distributed throughout the dome. Dispense only to the first stop of the pipettor to avoid generating bubbles on top of the dome.

  20. Repeat step 18 for the remaining pellets.

  21. Place the lid on the culture plate. Carefully place the plate in an incubator at 37 °C and 5% CO2 for 10 min to let the domes solidify.

  22. Remove the plate from the incubator and place it in the biosafety cabinet.

  23. Without disturbing the domes, carefully add 750 μL of room temperature (15–25 °C) HepatiCult™ Organoid Growth Medium against the side of each well containing a dome. Do not pipette directly onto the domes.

  24. Add sterile PBS to any unused wells. Place the lid on the culture plate. Incubate the plate at 37 °C and 5% CO2.

  25. Replace the medium every 2–3 days for up to 1 week by carefully aspirating the medium and adding 750 μL of fresh HepatiCult™ Organoid Growth Medium at room temperature (15–25 °C).

  26. Monitor organoids daily. They should be passaged before the lumen turns dark and collapses (Fig. 2). During early passages, this usually occurs at Day 4–6, and during later passages at Day 6 and 7. Proceed to Subheading 3.3 when organoids are ready for passaging.

Fig. 1.

Fig. 1

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Schematic flowchart of isolation and culture of mouse ductal organoids

Fig. 2.

Fig. 2

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Representative images of ductal organoids in culture

3.3. Passaging Organoids

Passage organoids about 6 days after seeding. For the initial passage, it usually requires combining four wells (P0 organoids) to split into six wells. After the initial passage, use a 1:6 to 1:12 split ratio in subsequent passages depending on the experimental design. Thaw Matrigel® on ice (50 μL/well to be passaged). Place Advanced DMEM/F-12 on ice.

  1. Prepare HepatiCult™ Organoid Growth Medium and bring to room temperature (15–25 °C).

  2. Without touching the dome, aspirate and discard the medium in each well to be passaged.

  3. Using a 1 mL pipettor, forcefully add 1 mL of cold Advanced DMEM/F-12 to the center of each dome and let sit for 1 min.

  4. Vigorously pipette up and down 15 times, being careful not to generate bubbles.

  5. Combine the contents of all wells into one 15 mL conical tube.

  6. Centrifuge the tube at 290 × g for 5 min at 8 °C. Aspirate as much of the supernatant as possible without disturbing the pelleted organoids.

  7. Add appropriate volumes of thawed Matrigel® on top of the pellet. Without generating bubbles, gently mix the duct-Matrigel® suspension by pipetting up and down 5–8 times, going to only the first stop of the pipettor.

  8. Add 50 μL suspension to the center of 1 well of the 24-well plate to form a dome. While dispensing, gradually move the pipette tip upwards so that the ducts are evenly distributed throughout the dome. Dispense only to the first stop of the pipettor to avoid generating bubbles on top of the dome.

  9. Repeat step 9 for the remaining pellets.

  10. Place the lid on the culture plate. Carefully place the plate in an incubator at 37 °C and 5% CO2 for 10 min to let the domes solidify.

  11. Remove the plate from the incubator and place it in the biosafety cabinet.

  12. Without disturbing the domes, carefully add 750 μL of room temperature (15–25 °C) HepatiCult™ Organoid Growth Medium against the side of each well containing a dome. Do not pipette directly onto the domes.

  13. Add sterile PBS to any unused wells. Place the lid on the culture plate. Incubate the plate at 37 °C and 5% CO2.

3.4. Organoid Cryopreservation

  1. Culture the organoid for 5–7 days until they are 80–90% confluent. Remove all of the HepatiCult™ Organoid Growth Medium from each well.

  2. Put 1 mL cold DMEM/F-12 with 15 mM HEPES into the well and break up the Matrigel® containing the organoids by pipetting up and down with an Advanced DMEM/F-12 wetted 1000 μL pipette tip.

  3. Transfer suspension containing organoids, combining wells in a single 15 mL centrifuge tube.

  4. Centrifuge the tube at 290 × g for 5 min at 2–8 °C. Remove and discard the supernatant, being careful not to disturb the organoid pellet.

  5. Wash the organoid pellet by resuspending in 10 mL of cold Advanced DMEM/F-12, and gently pipette the contents to help break down the pellet. Centrifuge the suspension at 290 × g for 5 min at 2–8 °C and then remove and discard the supernatant carefully.

  6. Resuspend organoids with the freezing medium containing DMSO (HepatiCult™ Organoid Growth Medium:FBS:DMSO = 1:2:1) and transfer them into a cryovial. Transfer the tube to −80 °C. After 24 h at −80 °C, transfer to liquid nitrogen for long-term storage.

3.5. Organoid Recovery

  1. Warm HepatiCult™ Organoid Growth Medium to room temperature (15–25 °C) and prepare 25% FBS DMEM/F12 with 15 mM HEPES for wash medium.

  2. Place the 24-well tissue culture plate in a 37 °C incubator for 1 h.

  3. Incubate the cryovial in the 37 °C water bath and thaw completely until the freezing medium becomes liquid.

  4. Transfer freezing medium to a 15 mL centrifuge tube with 3 mL wash medium.

  5. Centrifuge 290 × g for 5 min at 2–8 °C. Aspirate the supernatant carefully without disturbing the pellet and place it on ice.

  6. Using a pipettor with a 200 μL pipette tip, add 50 μL of thawed Matrigel® on top of the pellet. Mix Matrigel® suspension by pipetting up and down 5–8 times.

  7. Add the 50 μL suspension to the center of 1 well of the 24-well plate to form a dome. Place the lid on the 24 well tissue culture plate, place the plate in an incubator at 37 °C and 5% CO2 for 10 min to let domes solidify.

  8. Without disturbing the domes, add 750 μL of room temperature (15–25 °C) HepatiCult™ Organoid Growth Medium into each well containing a dome carefully. Do not pipette directly onto the domes.

  9. Add sterile PBS to any unused wells. Place the lid on the culture plate. Incubate the plate at 37 °C and 5% CO2.

3.6. Transfection in Hepatic Organoids

  1. Preparation of a single-cell mixture. Culture the organoids for 5–7 days, and then disrupt the basement matrix containing the organoids (drops in the center of the well) with cooled DMEM/F12 by scraping and pipetting up and down.

  2. Pool three wells of a 24-well plate of organoids in a 15-mL centrifuge tube. Centrifuge the tube at 200 × g for 5 min at room temperature.

  3. Aspirate the supernatant without disrupting the pellets. Add 10 mL cooled DMEM/F12 and repeat the centrifugation step to wash off the basement matrix.

  4. Remove the supernatant from the pellet generated above. Try to remove as much supernatant as possible without disturbing the pellet.

  5. Resuspend the organoids in 1 mL of pre-warmed TrypLE Express (#12605-028, Thermo) per tube and transfer to one well of a 24-well plate.

  6. Pre-wet a 1 mL- syringe with Anti-Adherence Rinsing Solution and DMEM/F12 as described above, and vigorously pipette the TrypLE mixture containing organoids up and down approximately ten times.

  7. Incubate the mixture at 37 °C for 5 min. Check the TrypLE digest solution every 2 min under a bright-field microscope. Stop the digestion when the majority (90–95%) of the material consists of single cells.

  8. Add 10 mL of cold Advanced DMEM/F12 to stop the digestion, and centrifuge the tube at 300 × g for 5 min at 4 °C.

  9. Remove the supernatant and resuspend the pellet in 450 μL of Advanced DMEM/F12. Keep on ice until the transfection.

  10. Preparation of transfection mixture (e.g. polyjet, SignaGen). Add 50 μL of transfection mixture containing 1 μg plasmids and 3 μL of transfection reagent to 450 μL of single-cell suspension.

  11. Seal the plate with Parafilm and centrifuge in a pre-warmed centrifuge at 32 °C at 600 × g for 1 h.

  12. Remove the Parafilm and incubate it in a tissue culture incubator at 37 °C for 2 h.

  13. Transfer the transfection mixture from the well to a microcentrifuge tube.

  14. Centrifuge the mixture at 400 × g for 5 min at room temperature.

  15. Seed the organoids as described above.

3.7. Organoid Staining

  1. Use two wells (24-well plates) of 80–90% confluent organoids (100–200 organoids) for staining.

  2. Pre-wet a 15-mL centrifuge tube with 5 mL PBSB (0.1% BSA in PBS).

  3. Pre-wet a 1000μL pipette tip with PBSB by pipetting up and down for 1–2 min.

  4. Remove the medium and add 1 mL of cold PBSB. Using the coated pipette, gently suspend the basement matrix in the cold PBSB and carefully transfer the organoid suspension to a pre-wetted 15-mL centrifuge tube.

  5. Gently add cold PBS to the top and allow the organoids to settle under gravity on ice (10 min).

  6. Remove the PBS with extra precaution.

  7. Add 13 mL of cold PBS and incubate the mixture on ice for 30 min. To wash the basement matrix off, invert the tube five to ten times or gently pipette up and down three to five times.

  8. Allow the organoids to settle under gravity. Carefully remove the PBS.

  9. Repeat the wash-step one to three times.

  10. Add 4 mL of freshly prepared fixative (e.g. 4% paraformaldehyde) to the organoids and incubate them for 30 min on ice.

  11. Gently remove the fixative and replace it with 10 mL of cold PBS. Let the tube stand on ice for 10 min to allow the organoids to settle under gravity. Carefully aspirate the PBS. There are two methods for staining, whole-mount staining and paraffin embedding. Continue to Subheading 3.8 for whole-mount staining, or to Subheading 3.9 for paraffin embedding.

3.8. Whole-Mount Staining

  1. After fixing the organoids, remove the PBS and replace it with 4 mL of PBSDT blocking solution. Incubate organoids with gentle agitation for 1–3 h at room temperature. Let the tube stand for 10 min afterward to settle the organoids under gravity. Gently remove the blocking solution and add primary antibody diluted in 3 mL PBSDT blocking solution. Determination of the best diluent requires optimization based on the antibodies to be used (not suitable here). Incubate the mixture for 48 h with gentle agitation at 4 °C.

  2. Let the organoids settle under gravity for up to 5 min and remove the supernatant. Then wash the organoids by adding 4 mL of PBSB and incubate them at room temperature for 10 min.

  3. Repeat the wash-step three times.

  4. Incubate the organoids with secondary antibodies diluted 1: 250 in PBSB for 2 h at room temperature with gentle agitation.

  5. Let the organoids settle under gravity for up to 5 min and remove the supernatant. Then wash the organoids by adding 4 mL of PBSB, and incubate them at room temperature for 10 min.

  6. Repeat the wash-step three times.

  7. If nuclear staining is required, incubate the organoids with a nuclear dye (e.g., Hoechst or DAPI) diluted 1:2000 in PBS for 15 min.

  8. Wash the organoids by adding PBS, incubating with gentle agitation at room temperature for 10 min, letting the tube stand for 10 min, and gently removing the supernatant.

  9. At this point, the organoids can be imaged directly in suspension in PBS (Fig. 3). However, it is recommended to immediately mount the organoids on a glass slide using an agent such as Vectashield in order to preserve the fluorescence of the secondary antibody conjugates.

Fig. 3.

Fig. 3

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Representative images of immunofluorescent staining of cholangiocyte marker cytokeratin 19 (CK-19) (red). Nuclei were stained with DAPI (blue)

3.9. Paraffin Embedding

  1. Prepare 3% agarose solution with sterile PBS. Boil agarose using a microwave oven until it is thoroughly melted.

  2. Before agarose is solidified, resuspend organoids in 100 μL agarose using a 200μL pipette tip. Try pipetting up and down 1–2 times without generating bubbles.

  3. Immediately put the tube on ice that contains embedded organoids for 1 h. Take it out when the agarose is thoroughly solidified.

  4. Dehydrate the organoids in gradient ethanol (70–100%) according to the standard procedure for immunohistochemistry staining.

  5. Clear organoids using Xylene (two washes of ~20 min each).

  6. Impregnate the organoids with Histowax at 60 °C for 2 h.

  7. Transfer the organoids gently into the middle of a metal mold, add enough hot Histowax paraffin for embedding.

  8. Harden the paraffin on a cold plate, and remove the mold.

  9. Cut sections using a standard microtome and stain the slides according to the standard protocol for immunohistochemistry staining (Fig. 3).

Acknowledgments

We thank Mrs. Elaine Kennedy for proofreading and English editing. This work was supported by VA Merit Awards I01BX004033; Research Career Scientist Award IK6BX004477; National Institutes of Health Grants (R01 DK104893, R01DK-057543, and R21 AA026629-01).

4 Notes

1.

If the mouse has advanced liver firbrosis, the liver tissue may not be completely dissociated. Do not allow the organoid fragments to dissociate for too long, as this may result in decreased viability.

2.

In Subheading 3.2, step 15, make sure the tip of the serological pipette touches the surface of the strainer while the entire bottom surface is thoroughly rinsed. If fragments remain on the surface, use a serological pipette to gently scrape them off and transfer them to the tube gently. PBDT solution is PBS supplemented with 0.1–1% Triton X-100 (depending on the protein localization, membrane, or nucleus), 1% DMSO, and 1% BSA.

3.

Do not overgrow the organoids, as it is easier to conserve the shape of the organoids by using small organoids for this protocol.

4.

Wash steps in Subheading 3.7, step 7 are important for the removal of the basement matrix, which can interfere with staining.

5.

The immunostained organoids can be resuspended in PBS and stored at 4 °C for 1–2 weeks with light protection.

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