Cholangiocytes were obtained from the contents of gallbladders or from the connective tissues resected from the portal area of livers during the Kasai operation. Informed consent for the research use of resected tissues was obtained from the parents of the patients before the surgery. In our surgical strategy, cholangiography through the gallbladder is usually performed before the Kasai operation to confirm the diagnosis and classification of BA. Before cholangiography, the contents of the gallbladder are aspirated with a fine needle and a syringe and then centrifuged at 2,000–3,000×g for 3 min to precipitate the cells. The supernatant is then removed. If the amount of the gallbladder content is small, a few clusters of cells are obtained by washing the needle and syringe used for aspiration in the culture medium.

Connective tissues are surgically resected from the portal area of the liver with the remnants of the disturbed bile ducts in them. Small fragments of resected tissues are then dissected as much as possible using a fine blade and spread in culture medium.

Throughout this study, all cells or tissue fragments were cultured at 37 °C with 5% CO2. The contents of our conditioned medium are shown in Table 1. A cocktail of Y-27632 (Rock inhibitor), CHIR99021 (GSK-3 inhibitor), and A83-01 (ALK4/5/7 inhibitor) was added to the medium (Table 1) to maintain cell growth (Katsuda et al. 2017), but these compounds were not added to the medium when the cells were used for experiments to avoid the effects of the inhibitors. The medium was replaced every three to four days. After the cells attached to the bottom of dish and started to grow stably, the immortalizing genes SV40T and hTERT were introduced to the cells using a commercially available lentivirus system (Cat# G256, G200; Applied Biological Materials Inc., BC, Canada). Transdux (Cat# LV850A-1; System Bioscience, CA, USA) was added to the culture medium according to the manufacturer’s instructions, and 2–5 MOI (multiplicity of infection) of virus with each gene was introduced to the cells. First, SV40T-Lenti was added to the medium and incubated for one night, and then the medium was changed. After checking that the cells had not been obviously damaged, hTERT-Lenti was added to the medium and cultured overnight. After changing the medium, the culture was continued without adding any selection reagent.

For passage, cells were stripped from the dish using Accutase (Cat# 12679-54; Nacalai Tesque, Inc., Kyoto, Japan), incubated for 10 min at 37 °C, and then detached from the bottom by tapping the dish. The cells were washed once with the base medium DMEM/F12 or phosphate-buffered saline (PBS) and then seeded into the new culture medium. Stabilized cells were also cryopreserved for later experiments. Cells were stripped from the dish and washed with PBS once. The cells were then retrieved by centrifuging at 500 × g for 2 min, after which the supernatant was removed, and the cells were suspended in the cryopreservation reagent LaboBanker 2 (Cat# BLB-2; TOSC Japan Ltd., Tokyo, Japan) and transferred into cryo tubes. The tubes were placed in a − 80 °C freezer for at least 2 nights and then placed in nitrogen oxide for further preservation.

Thawing was performed according to the manufacturer’s instructions. In brief, the medium was warmed to 37 °C before thawing, and then the tube of frozen cells was thawed in a 37 °C water bath. Cells were transferred into a 10-fold volume of culture medium and centrifuged. The supernatant was removed, and the cells were suspended in new medium and then cultured at 37 °C with 5% CO2.

Cells were seeded in eight-well glass slides and incubated until they attached to the slide and spread. Attached cells were fixed with 4% paraformaldehyde and incubated for 15 min in room air. The slides were washed and activated in pH 6.0 citrate buffer, heated in a microwave for 10 min with intervals to prevent boiling, and then soaked in methanol with 0.3% H2O2 for 30 min to inactivate the internal peroxidase. After washing with PBS and incubation with blocking buffer (Blocking One Histo, Cat# 06349-64; Nacalai Tesque, Inc.) for 1 h in room air, the primary antibody (anti-CK19 antibody, 1:200; Cat# ab220193, RRID: AB_2814863; Abcam, Cambridge, UK) was reacted overnight at 4 °C. Slides were then washed, and a secondary antibody (Envision + System-HPR Labeled Polymer Anti-mouse, Cat# K4000; Agilent, CA, USA) reaction was performed for 1 h in room air. After washing, 3,3’Diaminobenzidine (DAB) was used for staining following the manufacturer’s protocol. Cell nuclei were counterstained with hematoxylin for 1 min.

Cells were cultured in a 24-well dish and fixed with 4% paraformaldehyde for 15 min at room temperature. The plate was then washed, and the cells were permeabilized with PBS with 0.3% Triton X, rocking for 10 min at room temperature. The buffer was replaced with blocking buffer (Blocking One Histo, Cat# 06349-64; Nacalai Tesque, Inc.) and incubated for 1 h at room temperature. The blocking buffer was then removed, and double-primary antibody solution was added to the cells. The antibodies used were 1:200 for CK19 (Cat# ab220193, RRID:AB_2814863; Abcam) and 1:400 for SOX9 (Cat# AB5535, RRID:AB_2239761; Millipore, MA, USA)The plate was placed at 4 °C overnight, and then the samples were washed and incubated with fluorescent secondary antibody solution (both 1:500, Cat# A-11060, RRID:AB_2534107; Cat# A31566, RRID:AB_10374301; Thermo Fisher Scientific, MA, USA) in room air for 1 h. The samples were washed with 0.05% PBS-Tween20 and observed under a fluorescence microscope (BZ9000; Keyence, Osaka, Japan).

We tested two 3D culture methods: a Matrigel (Cat# 356234; Corning, NY, USA) and a Transwell system (Cat# 353492; Corning) (Fig. 1). The first method involved spheroid culture, while the second promoted vertical growth from 2D culture (2 + 1D culture).

Two 3D culture methods of cholangiocytes. A Sphere culture method: cells were seeded on a layer of Matrigel in a 24-well insert. B 2D + 1D culture method: cells were first cultured on the membrane of the Transwell insert until the cells covered approximately 50% of the surface, and then Matrigel was placed on top of the cell sheet. Conditioned medium is filled outside and inside the inserts

For the first method (spheroid culture), 100–200 µl of Matrigel was placed in a Falcon Transwell insert for a 24-well culture plate and then incubated at 37 °C. A total of 500 µl of conditioned culture medium was placed in the well to soak the bottom of the insert, and then 200 µl of cell suspension was added to the insert. After cells had formed small spheres in the gel, the culture medium, both in the insert and in the well, was exchanged every three to four days.

For the second method (2 + 1D culture), 2D culture was performed first in the Transwell. A total of 200 µl of cell suspension was placed in the Transwell insert for a 24-well plate with the bottom soaked in 500 µl of conditioned culture medium in the well. Cells were cultured at 37 °C with 5% CO2, and the culture medium was replaced every three to four days until the cells grew to cover more than 50% of the membrane. For vertical growth, the medium in the insert was completely removed, and 100 µl of Matrigel was added to the cells and then incubated at 37 °C with the bottom of the insert soaked in medium. After the Matrigel was added, 200 µl of fresh medium was further added to the insert, and the culture was continued until the cells had established 3D structures in the Matrigel.

Cholangiocytes reportedly take up and excrete rhodamine-123 into bile duct lumens, as has been observed using fluorescence microscopy. Rhodamine-123 (100 µM, Cat# R8004; Sigma-Aldrich, MO, USA) was added to the medium of the 3D culture well in which the 3D culture Transwell had been inserted and then incubated for 30 min at 37 °C until the chemical solution had entered the gel. The well was then washed with PBS three times. The cells were cultured in fresh medium for a few days and observed under a microscope with a 488-nm filter (BZ9000; Keyence). As a negative control, 100 µM verapamil (Cat# 36232-31; Nacalai Tesque, Inc.) was added to the culture medium to inhibit the uptake of rhodamine-123, and the medium was replaced with fresh medium without verapamil after overnight incubation.