C57BL6 mice were purchased from Sankyo Labo Service Corporation, Inc. (Tokyo, Japan). CAG-Cre:ROSA-LSL-tdTomato mice were obtained by crossing B6.Cg-Tg(CAG-Cre)CZ-MO2Osb (CAG-Cre) mice from Riken BRC (30) with B6.Cg-Gt(ROSA)26Sortm(CAG-tdTOmato)Hze/J (ROSA-LSL-tdTomato) mice (The Jackson Laboratory, Bar Harbor, ME). Eight to twelve weeks old mice were used for cell isolation. All animal experiments were approved by the Sapporo Medical University Institutional Animal Care and Use Committee and were conducted according to institutional guidelines for ethical animal use.
High concentration type I collagen, and growth factor reduced Matrigel (MG) were purchased from Corning (Corning, NY). Epidermal growth factor (EGF) and hepatocyte growth factor (HGF) were purchased from Corning. Oncostatin M (OSM) was purchased from R&D systems (Minneapolis, MN). Tissue culture plates (24 well) were purchased from Greiner Bio-One (Kremsmünster, Austria). DMEM/F-12 medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10% FBS (MP Biomedicals, Santa Ana, CA), 10 mM nicotinamide (Sigma-Aldrich), 1 × 10-7 M dexamethasone (Dex, Sigma-Aldrich), and 1× ITS (Gibco, Grand Island, NY) was used as the basic medium. The growth medium was prepared by adding 5 ng/ml EGF and 5 ng/ml HGF to the basic medium. The differentiation medium was prepared by adding 1% DMSO (Sigma-Aldrich) to the basic medium.
To isolate hepatocytes and cholangiocytes from adult mouse liver, two-step collagenase perfusion was performed, as previously reported (9, 10). MHs and SHs were obtained from digested tissue, whereas undigested tissue was kept for isolating cholangiocytes. After collagenase perfusion, the cell suspension was centrifuged at 50 × g for 1 min. The pellet was suspended in Hanks’ balanced salt solution, mixed with Percoll and centrifuged at 50 × g for 15 min to eliminate dead cells, yielding MHs. The supernatant collected after centrifugation at 50 × g was further centrifuged at 115 × g for 3 min. The pellet was suspended in Hanks’ balanced salt solution, mixed with Percoll and centrifuged at 180 × g for 15 min to eliminate dead cells, yielding SHs. The residual tissue after collagenase perfusion was further digested with collagenase/hyaluronidase solution. Liberated cells were used for isolating cholangiocytes, based on the expression of EpCAM, by a magnetic cell sorter (MACS) (Miltenyi Biotec, Bergisch Gladbach, Germany).
Induction of the HBTOs
Cholangiocytes were resuspended in the growth medium and plated in 24-well plates coated with 200 ml of 4 mg/ml type I collagen gel prepared from high concentration type I collagen (Corning) at a density of 50,000 cells/well. Five days after plating, SHs were added to each well at a density of 50,000 cells/well. Following a further two days of incubation, the medium was replaced with differentiation medium supplemented with 10 ng/ml oncostatin M (OSM), and then overlaid with collagen gel containing 20% MG (Col-MG), which was prepared by mixing 2 mg/ml type I collagen gel and MG (v/v=4:1) on ice. The plate was incubated at 37°C for 3–4 h to form a gel, and then the differentiation medium was added. Culture medium was replaced with fresh medium every four days.
Immunostaining and confocal imaging
A sandwich culture was fixed in PBS containing 4% paraformaldehyde at 4°C for 30 min with gentle shaking. After washing with PBS, the samples were permeabilized in PBS containing 1% Triton X-100 at room temperature for 30 min. After blocking in Block ACE containing 0.1% Triton X-100, the samples were incubated with primary antibodies and then dye-conjugated secondary antibodies were applied. Nuclei were counterstained with Hoechst33342. The primary and secondary antibodies used for immunostaining are listed in Tables S2 and S3. Images were acquired using Zeiss LSM780 confocal laser scanning microscopes (Carl Zeiss, Jena, Germany) and Olympus FV3000 microscopes (Olympus, Tokyo, Japan).
Identification and quantification of the hepatobiliary connections in HBTOs
HBTOs were stained with phalloidin, anti-CK19, and anti-HNF4a antibodies, and then the phalloidin (+) luminal structures connecting CK19(+) ducts with HNF4a(+) hepatocyte clusters were counted. The same images were used to measure the boundary between hepatocyte clusters and the biliary tissue using Olympus cellSens software. Three to four different areas in four independent experiments were examined using the same method.
Uptake of chloromethyl fluorescein diacetate (CMFDA) and cholyl-lysine fluorescein (CLF)
Three to four weeks after Col-MG overlay, the medium was replaced with a differentiation medium containing 1 mg/ml CMFDA (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) or 1 mg/ml CLF (Corning). The wells were washed with the differentiation medium five times, after 10 min incubation with CMFDA and 30 min incubation with CLF. Images were taken with an Olympus fluorescence microscope. Three to four areas per well were selected, and the transport of CMFDA and CLF was examined by taking fluorescence and phase-contrast images at different time points. Experiments were repeated three times. Representative images are shown in Figure 3.
Detection of bilirubin in the organoids
Ten mg of bilirubin (Kanto Chemicals, Tokyo, Japan) was dissolved in 0.1 ml of DMSO. Then 200 ml of 0.1 M Na2CO3, 500 ml of FBS, 20 ml of 0.1 N HCl, and 180 ml of distilled water were added. The solution was diluted ten-fold with DMSO. The bilirubin solution was further diluted 100-fold with culture medium, to produce a 10 mg/ml bilirubin solution, which was then filtered using a pore size of 0.22 mm (31). HBTOs were incubated with a medium containing 10 mg/ml bilirubin for five days. Hall’s method (32) was used for the histochemical staining of bilirubin. The organoids were washed with PBS and fixed with 10% buffered neutral formalin for 5 min at room temperature, and then washed twice with distilled water. Fouchet’s reagent, containing 22.5% trichloroacetic acid and 1% ferric chloride, was added and the mixture was incubated at room temperature for 5–15 min, until green biliverdin was detected in the luminal networks of the organoids. Bilirubin detection was repeated three times at three or four weeks after Col-MG overlay. A representative image is shown in Figure 3.
Sandwich ELISA using goat anti-mouse ALB (Bethyl Laboratories, Montgomery, TX) and HRP-conjugated anti-mouse ALB antibodies (Bethyl Laboratories) was performed to measure ALB in the culture medium. Signal was detected with o-Phenylenediamine (OPD) (Sigma-Aldrich) and measured on an 800TS absorbance reader (BioTek, Winooski, VT). The HBTOs were kept for two to three months, and the ALB concentration in the culture medium was measured using ELISA every two weeks after Col-MG overlay. The long term culture for 8 weeks after Col-MG overlay was repeated three times. One out of three cultures were extended for additional 4 weeks. As a control, SHs were plated onto collagen gel in 24-well plates at a density of 5×104 cells/well and overlaid with Col-MG.
CYP activity was measured using Glo-CYP3A4-Assay and Glo-CYP1B1-Assay (Promega, Madison, WI). The substrate was added to the culture medium at the concentration shown in the protocol, and incubated for 30 min for CYP3A4 and 3 hours for CYP1B1. Assays using four wells per experiment were repeated twice for CYP1B1 and three times for CYP3A4. The average values of representative cultures are shown in Figure 4. As a control, SHs were cultured in a sandwich culture as HBTOs, and the CYP activity was examined.
Uptake of low density lipoprotein (LDL)
Four weeks after Col-MG overlay, HBTOs were incubated with the growth medium containing 2 mg/ml DiI-acetylated LDL (Alpha Acer, Havehill, MA) for 1 hour. Wells were washed with the growth medium twice, and images were taken with an Olympus fluorescence microscope. Uptake of LDL was examined twice and the representative images are shown in Figure 4.
MHs and SHs were isolated as described above in “Cell Isolation”. SHs were further purified by isolating CD31-CD45−EpCAM-ICAM-1+ cells using a FACSAriaII (9). Total RNA was extracted and used for synthesizing cDNA using PrimeScript 1st strand cDNA synthesis kits (Takara Bio Inc., Shiga, Japan). Quantitative PCR was performed using an ABI PRISM 7500 (Thermo Fisher, Scientific) with the primers 5′-atcctcgccctgctgatt-3′ and 5′-accaccgttctcctccgta-3′ for Cdh1, and 5′-tgtgaatgaactgaaggaaagc-3′ and 5′- atcctgcacccagctgtatt-3′ for Cldn2. Isolation of MHs and SHs was repeated four times and purified MHs and SHs were used for 1st strand cDNA synthesis and for quantitative PCR.
Separation of ECAD(+) MHs
MHs were incubated with PE-conjugated anti-ECAD antibody (BioLegend). ECAD(−) and (+) MHs were isolated using a FACSAria II. Five days before cell isolation, cholangiocytes were plated on type I collagen gel. Isolated MHs were plated onto collagen gel containing cholangiocyte colonies. Cell isolation and co-culture was repeated three times. The number of hepatobiliary connections was examined after staining with antibodies against HNF4a & CK19, phalloidin, and Hoechst 33342.
Culture of human reprogramming hepatocytes (hiCLiP)
hCLiPs were generated from primary human hepatocytes, as previously reported (22). For inducing hybrid HBTO, mouse cholangiocytes isolated from Tomato mice were plated on type I collagen gel. On the same day, a frozen stock of hCLiP was thawed and the hCLiPs were kept on dishes coated with type I collagen. On day 5, hCLiPs were treated with trypsin-EDTA and, after centrifugation, the cells were plated onto collagen gel, where cholangiocytes formed colonies. Two days after the hCLiPs were plated, 10 ng/ml human OSM was added to the culture, followed by an overlay of collagen gel containing 40% MG. The co-culture was repeated four times. Representative data are shown in Figure 6.
Unpaired two-tailed student t-tests were performed for the data pertaining to CYP1B1&CYP3A4 activity, ALB secretion, quantification of the number of hepatobiliary connections, and qPCR data using Microsoft Excel.