Huh28

Twenty human biliary tract cancer cell lines were collected from different sources. Huh-28 (ICC), HuCCT1 (ICC), OZ (CPHBD), NOZ (CGB) and OCUG-1 (CGB) cell lines were obtained from the Japanese Collection of Research Bioresources Cell Bank. TKKK (ICC), TFK-1 (CPHBD), TGBC1TKB (CGB), TGBC2 (CGB), TGBC14TKB (CGB), TGBC24TKB (CGB) and G-415 (CGB) cell lines were obtained from the RIKEN BioResource Center. SNU-1079 (ICC), SNU-245 (CPHBD), SNU-1196 (CPHBD) and SNU-308 (CGB) cell lines were obtained from the Korean Cell Line Bank. The Egi-1 (CPHBD) cell line was obtained from the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures. All cell lines were cultured as recommended by their respective cell banks. SK-ChA-1 (CPHBD), Mz-ChA-1 (CGB), Mz-ChA-2 (CGB) were obtained from Prof. Alexander Knuth (University Hospital of Zürich, Zürich, Switzerland) (32 (link)) and cultured in RPMI 1640 with 10 mM HEPES, 2 mM L-Glutamine, 1X MEM non-essential amino acids (Thermo Fisher Scientific), 100 U/ml penicillin, 100 µg/ml of streptomycin, and 10% fetal bovine serum (FBS). Human BMSCs (196hT) immortalized with the hTERT/GFP system (33 (link)) were cultured as described previously (34 (link)). The control cell line 293/hTNW that stably expresses human tenascin-W (27 (link)) was cultured in DMEM with 0.25 µg/ml of G418, 1.5 µg/ml of puromycin, and 10% FBS.

d,l-sulforaphane (1-isothiocyanate-4-methylsulphinylbutane, purity ≥ 98%) was purchased from Toronto Research Chemicals Inc. (Toronto, ON, Canada), and gemcitabine (2′-deoxy-2′,2′-difluorocytidine, purity ≥ 98%) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Two human iCCA cell lines, HuCCT-1 (cat: JCRB0425) and HuH28 (cat: JCRB0426) were obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). These cells were cultured in RPMI-1640 (Nacalai Tesque, Inc., Kyoto, Japan) supplemented with 10% fetal bovine serum (FBS) and 1% ampicillin/streptomycin. The primary human biliary epithelial cell line (HIBEpiC, cat: #5100) was purchased from ScienCell Research Laboratories, Inc. (Carlsbad, CA, USA). HIBEpiC cells were cultured in Epithelial Cell Medium (ScienCell Research Laboratories) supplemented with 2% FBS and 1% epithelial cell growth supplement (ScienCell Research Laboratories), and 1% ampicillin/streptomycin. The cells were grown at 37 °C in a 5% CO₂ atmosphere.

The non-malignant human cholangiocyte cell line MMNK-1 were obtained from the JCRB Cell Bank (Osaka, Japan) and cultured as recommended by the supplier. The CCA cell lines HuCCT1, HuH28 and OZ were obtained from the JCRB Cell Bank, and TFK-1 and RBE cells were provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT (Tsukuba, Japan) [37 (link)]. HuCCT1, HuH28, TFK-1 and RBE cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Thermo Fisher Scientific). OZ cells were cultured in Williams’ medium (Thermo Fisher Scientific) with 10% FBS and 1% penicillin-streptomycin. MMNK-1 cells were cultured in DMEM high-glucose medium (Thermo Fisher Scientific) with 5% FBS and 1% penicillin-streptomycin. All cells were cultured at 37° C in a 5% CO₂ incubator. For all EV studies, EV-depleted medium was prepared using Exosome-Depleted FBS (Thermo Fisher Scientific). TGF-β was obtained from EMD Millipore Corporation (Billerica, MA, USA). Cells were treated with 10 ng/ml TGF-β for 72 h to induce EMT.

Human BTC cell lines HuCC-T1 (JCRB0425 [29 ]), HuH-28 (JCRB0426 [30 ]), OCUG-1 (JCRB0191 [31 (link)]), OZ (JCRB1032 [32 (link)]), NOZ (JCRB1033 [33 (link)]), KKU-055 (JCRB1551), KKU-100 (JCRB1568 [34 (link)]), KKU-213 (JCRB1557), and immortalized human cholangiocyte cell line MMNK-1 (JCRB1554 [35 (link)]) were purchased from the Japanese Collection of Research Bioresources Cell Bank (JCRB). Human BTC cell lines EGI-1 (ACC385) and TFK-1 (ACC344 [36 (link)]) were purchased from the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ). All cell lines were cultured in L-glutamine-free Dulbecco’s Modified Eagle Medium (DMEM) containing 4.5 g/l glucose (Catalogue #: 11,960,044, Gibco, Thermo Fisher Scientific, Vienna, Austria). Medium was supplemented with 10% foetal bovine serum (FBS; Catalogue #: S-FBS-SA-015, Serana Europe GmbH, Pessin, Germany) and 1% Penicillin-Streptomycin (Catalogue #: P4333, Sigma-Aldrich Handels GmbH, Vienna, Austria). Cells were cultured under standard conditions (37°C, 21% O₂, 5% CO₂, and 98% humidity).

TFK1, RBE, SSP25, HuCCT1, HuCCA1, HuH28, KKU213A, KKU213B, KKU213C, KKU068, KKU131, KKU138, KKU055, KKU100, and YSCCC cell lines were obtained from the Japanese Collection of Research Bioresources (JCRB; Osaka, Japan). TFK1, RBE, SSP25, and YSCCC cells were maintained in Roswell Park Memorial Institute 1640 medium (RPMI 1640) (Gibco; Thermo Fisher Scientific, Waltham, MA, USA). HuCCA1 and KKU100 cells were maintained in Ham’s F-12 medium (Gibco; Thermo Fisher Scientific). HuCCT1, HuH28, KKU213A, KKU213B, KKU213C, KKU068, KKU131, KKU138, and KKU055 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco; Thermo Fisher Scientific). All cell lines were routinely tested for mycoplasma. For the construction of resistant cells, parental cells (KKU213C, TFK1, KKU068, and SSP25) were seeded in 12-well plates at 5 x 10⁴ cells/well in 1 milliliter (ml) of growth medium. Cells were treated with GEM/CIS using a stepwise dose-induction protocol (16 (link)).

HepG2, Hep3B, HLE, HLF, HuCCT1, and HuH28 cells were obtained from the Japanese Collection of Research Bioresources (Osaka, Japan). Plat-E cells were obtained from Cell Biolabs Inc. (San Diego, CA). HeLa, NIH/3T3, TALDO1 KO, HepG2, Hep3B, HCE, and HCF cells were cultured in Dulbecco’s modified Eagle’s Medium (DMEM; Sigma-Aldrich, St. Louis, MO) supplemented with 10% foetal bovine serum (FBS). Plat-E cells were cultured in DMEM containing 10% FBS, puromycin (1 μg/mL), and blasticidin S (10 μg/mL). HuCCT1 and HuH28 were cultured in RPMI-1640 (Sigma-Aldrich) supplemented with 10% FBS. The cells were maintained in a 37 °C incubator with 10% CO₂-humidified air.