All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of The University of Michigan.

1. Preparation of Equipment and Materials for Mouse EHBD Isolation

1. Prepare seeding medium and washing buffer (Table of Materials) in 50 mL conical tubes and keep them at 4 °C or on ice until use.
2. Set up a surgical table (Figure 1B). Prepare sterilized surgical instruments (Figure 1C).
3. Place a sterile 24-well plate in the 37 °C tissue culture incubator to pre-warm it.
4. Place an aliquot of basement matrix on ice. Use basement matrix only when it is completely liquefied.

2. EHBD Isolation and Biliary Organoid Culture

1. Isolation and preparation of a single cell suspension of mouse EHBD
   1. Euthanize an adult mouse (older than 2 months) according to the institutional guidelines. Place the mouse in a supine position. Open the abdominal cavity using a midline approach and retract the liver to rest on the diaphragm.
   2. Identify the common bile duct located immediately below the liver hilum by gently pulling the proximal duodenum with a hemostat. Separate EHBD from the surrounding tissues using a scalpel blade. Holding the proximal end of the common bile duct with forceps, dissect it distally just above its juncture with the duodenum, then dissect the proximal end of the duct from the liver (Figure 1D). Immediately place isolated EHBD (Figure 1E) into cold washing buffer.
   3. Remove the EHBD from the washing buffer and mince into 0.5 mm sections using a sterile scalpel blade. Place the tissue on a glass plate on ice during the procedure (Figure 1B).
   4. Place the EHBD sections into a tube containing 500 µL of the dissociation buffer. Incubate for 20 min at 37 °C. Neutralize the dissociation buffer by adding 500 µL of ice-cold cell culture medium.
   5. Triturate the cell suspension up and down progressing through 18 G and 20 G needles, 20 times each. Filter the cell suspension through a 70 µm cell strainer and collect the flow-through in a 50 mL tube.
      NOTE: Pre-condition the strainer with 500 µL of sterile phosphate-buffered saline (PBS) prior to filtering to facilitate the passage of the cell suspension.
2. Establishing EHBD organoids
   1. Centrifuge the flow-through from step 2.1.5 at 300 x g for 5 min at 4 °C.
   2. Carefully remove the supernatant. Resuspend the cells in 1 mL of ice-cold sterile PBS. Transfer the resuspension into a new 1.5 mL tube. Repeat step 2.2.1.
   3. After centrifugation, carefully remove the supernatant from washed cells collected at the tube bottom. Resuspend the cell pellet in 120 µL of liquefied ice-cold basement matrix by pipetting up and down using P200 tips.
      NOTE: Cell pellet resuspension in basement matrix has to be performed on ice-bath.
   4. Plate 40 µL of the cell resuspension in basement matrix into the center of a well in a pre-warmed 24-well plate.
      NOTE: Avoid suctioning air while manipulating basement matrix to prevent bubble formation.
   5. Return the plate with cells resuspended in basement matrix to the 37 °C tissue culture incubator for 15 min or until basement matrix is solidified. Add 600 µL of the seeding medium warmed up to 37 °C to each well (Table of Materials). Return the plate to the 37 °C tissue culture incubator.
   6. Replace the seeding medium with 600 µL of the fresh organoid culture medium in 3 days and every 3 days thereafter. Monitor organoid growth with an inverted microscope. Use organoids for a downstream application or split every 7 to 9 days before accumulation of intraluminal debris and organoid collapse are observed (Figure 2A).

3. EHBD Organoid Passage and Storage

1. Passage of EHBD organoids 1:3 to 1:4 every 7 to 9 days
   1. Remove the medium from the well and add 400 µL of ice-cold PBS. Resuspend the organoids by gently pipetting the mixture up and down 10 times in the well. Transfer the mixture to a 1.5 mL tube.
   2. Passage the mixture through a 25 G needle 4 times to dissociate the organoids. Centrifuge the mixture at 400 x g for 4 min at 4 °C.
   3. Carefully remove the supernatant and resuspend the cells in basement matrix (1:3 to 1:4) for further culturing (step 2.2.4.) or wash the cells with ice cold PBS for further processing.
      NOTE: Typically, 250-300 cells are plated into the 24-well plate for downstream applications. Plating efficiency can be evaluated by bright field microscopy using an inverted microscope on day 3-5 after passaging by counting the number of organoids and calculating their percent from initial cell number. mRNA can be isolated from EHBDOs washed in PBS using the standard protocol using guanidinium thiocyanate-phenol-chloroform extraction.
2. Long-term storage of EHBD organoids
   1. Remove the medium from the well and wash the organoids with room temperature PBS. Remove PBS from the well without disturbing the basement matrix drop.
   2. Add 500 µL of ice-cold cell freezing medium to the well. Gently resuspend the organoids in liquefied basement matrix and cell freezing medium and transfer the mixture into cryogenic vials.
   3. Store the vials at -80 °C for 48 h. Transfer the vials to a nitrogen tank for long-term storage in a vapor phase.

4. EHBD Organoid Pprocessing for Paraffin Embedding

1. Resuspend the EHBDOs in 500 µL of ice-cold PBS (4 °C) by pipetting up and down 5 to 10 times. Collect resuspended EHBDO in liquefied basement matrix in a 1.5 mL tube.
   NOTE: To avoid breaking organoids, cut off the bottom 2-3 mm of a P1000 tip and remove the supernatant very carefully.
2. Centrifuge EHBD organoids at 350 x g for 5 min. Carefully remove the supernatant without disturbing the organoid pellet.
3. Add 1 mL of ice-cold 4% paraformaldehyde (PFA) to the organoids and incubate the organoids in 4% PFA overnight at 4°C. Remove 4% PFA from the organoids using a P1000 tip after overnight incubation.
4. Add 1000 µL of room temperature PBS to the tube with the organoids and incubate for 5 min at room temperature (RT). Centrifuge the tube with organoids in PBS at 350 x g for 5 min. Repeat this process two more times.
5. Remove PBS and add 1 mL of 30% ethanol to the organoids. Incubate for 5 min at RT.
6. Centrifuge the tube at 350 x g for 5 min at RT. Remove 30% ethanol. Add 1 mL of 70% ethanol and incubate for 5 min at RT.
7. Centrifugate at 350 x g for 5 min. Remove 70% ethanol. Add 1 mL of 100% ethanol and incubate for 5 min at RT.
   NOTE: Organoids can be kept in 100% ethanol at room temperature for up to 48 h before further processing.
8. Heat specimen processing gel in a microwave for 20 s or until liquefied. Add 50 µL of specimen processing gel into the tube with organoids. Place the tube on ice until the specimen processing gel is solidified.
9. Remove the drop of specimen processing gel with organoids from the tube and place between the blue sponge pads in a cassette for further processing in the tissue processor. Use 15 min for each step in the paraffin embedder during further processing..
10. Section paraffin-embedded organoids in specimen processing gel at 4 µm. Proceed with immunohistochemical staining as previously described¹⁶.