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Development and Characterization of Human Primary Cholangiocarcinoma Cell Lines

      Cholangiocarcinoma (CCA) is the second most common primary liver tumor and has been associated with a late diagnosis, limited treatment options, and a 5-year survival rate of around 30%. CCA cell lines were first established in 1971, and since then, only 70 to 80 CCA cell lines have been established. These cell lines have been essential in basic and translational research to understand and identify novel mechanistic pathways, biomarkers, and disease-specific genes. Each CCA cell line has unique characteristics, reflecting a specific genotype, sex-related properties, and patient-related signatures, making them scientifically and commercially valuable. CCA cell lines are crucial in the use of novel technologies, such as three-dimensional organoid models, which help to model the tumor microenvironment and cell-to-cell crosstalk between tumor-neighboring cells. This review highlights crucial information on CCA cell lines, including: i) type of CCA (eg, intra- or extrahepatic), ii) isolation source (eg, primary tumor or xenograft), iii) chemical digestion method (eg, trypsin or collagenase), iv) cell-sorting method (colony isolation or removal of fibroblasts), v) maintenance-medium choice (eg, RPMI or Dulbecco's modified Eagle's medium), vi) cell morphology (eg, spindle or polygonal shape), and vii) doubling time of cells.
      Cholangiocarcinoma (CCA) is a heterogeneous group of malignancies originating from the biliary tree.
      • Sato K.
      • Glaser S.
      • Alvaro D.
      • Meng F.
      • Francis H.
      • Alpini G.
      Cholangiocarcinoma: novel therapeutic targets.
      CCA accounts for approximately 3% of all malignancies of the gastrointestinal system and is the second most common primary hepatic tumor, after hepatocellular carcinoma.
      • Khan S.A.
      • Tavolari S.
      • Brandi G.
      Cholangiocarcinoma: epidemiology and risk factors.
      ,
      • Fabris L.
      • Sato K.
      • Alpini G.
      • Strazzabosco M.
      The tumor microenvironment in cholangiocarcinoma progression.
      The prevalence of CCA varies broadly by region; while the prevalence is 1.6 per 100,000 people in the United States, it could be as high as 85 to 90 per 100,000 people in northeastern Thailand, where infection with Opisthorchis viverrini, a CCA risk factor, is endemic.
      • Kirstein M.M.
      • Vogel A.
      Epidemiology and risk factors of cholangiocarcinoma.
      Several additional risk factors for cholangiocarcinogenesis have been identified, such as Clonorchis sinensis infection, bile duct cyst, primary sclerosing cholangitis, hepatolithiasis, cholelithiasis, and inflammatory bowel disease.
      • Tyson G.L.
      • El-Serag H.B.
      Risk factors for cholangiocarcinoma.
      Despite the development of state-of-the-art therapies, the 5-year survival rate of CCA is still under 30%.
      • Loosen S.H.
      • Vucur M.
      • Trautwein C.
      • Roderburg C.
      • Luedde T.
      Circulating biomarkers for cholangiocarcinoma.
      ,
      • Ahn D.H.
      • Bekaii-Saab T.
      Biliary cancer: intrahepatic cholangiocarcinoma vs. extrahepatic cholangiocarcinoma vs. gallbladder cancers: classification and therapeutic implications.
      CCAs are commonly classified, according to anatomic location along the biliary tree, as extrahepatic or intrahepatic (EHCCA or IHCCA).
      • Kendall T.
      • Verheij J.
      • Gaudio E.
      • Evert M.
      • Guido M.
      • Goeppert B.
      • Carpino G.
      Anatomical, histomorphological and molecular classification of cholangiocarcinoma.
      EHCCA can be subcategorized as perihilar or distal.
      • Kendall T.
      • Verheij J.
      • Gaudio E.
      • Evert M.
      • Guido M.
      • Goeppert B.
      • Carpino G.
      Anatomical, histomorphological and molecular classification of cholangiocarcinoma.
      CCAs can also be subclassified by macroscopic growth pattern (mass forming, periductal infiltrating, intraductal, or mixed), cell of origin (cholangiocytes, goblet cells, hepatic stem cells, or biliary tree stem/progenitor cells), and microscopic features (adeno, squamous, adenosquamous, mucinous, undifferentiated, or sarcomatous).
      • Krasinskas A.M.
      Cholangiocarcinoma.
      ,
      • Cardinale V.
      • Carpino G.
      • Reid L.
      • Gaudio E.
      • Alvaro D.
      Multiple cells of origin in cholangiocarcinoma underlie biological, epidemiological and clinical heterogeneity.
      Although the importance of the organ-specific or cancer-specific microenvironment has been recently highlighted due to cell-to-cell crosstalk, two-dimensional cancer cell lines are one of the best sources for investigating the respective cancer type.
      • Masters J.R.
      Human cancer cell lines: fact and fantasy.
      ,
      • Mirabelli P.
      • Coppola L.
      • Salvatore M.
      Cancer cell lines are useful model systems for medical research.
      CCA cell lines have been used for almost 50 years to: i) better understand CCA properties, ii) investigate treatment options, iii) model the disease in vitro, and iv) generate in vivo xenograft models.
      • Buranrat B.
      • Prawan A.
      • Kukongviriyapan U.
      • Kongpetch S.
      • Kukongviriyapan V.
      Dicoumarol enhances gemcitabine-induced cytotoxicity in high NQO1-expressing cholangiocarcinoma cells.
      • Panrit L.
      • Plengsuriyakarn T.
      • Martviset P.
      • Na-Bangchang K.
      Inhibitory activities of plumbagin on cell migration and invasion and inducing activity on cholangiocarcinoma cell apoptosis.
      • Kotawong K.
      • Chaijaroenkul W.
      • Muhamad P.
      • Na-Bangchang K.
      Cytotoxic activities and effects of atractylodin and beta-eudesmol on the cell cycle arrest and apoptosis on cholangiocarcinoma cell line.
      • Wattanawongdon W.
      • Hahnvajanawong C.
      • Namwat N.
      • Kanchanawat S.
      • Boonmars T.
      • Jearanaikoon P.
      • Leelayuwat C.
      • Techasen A.
      • Seubwai W.
      Establishment and characterization of gemcitabine-resistant human cholangiocarcinoma cell lines with multidrug resistance and enhanced invasiveness.
      RPMI 7451 is the first known CCA cell line isolated by George Eugene Moore and his team.
      • Fogh J.
      • Fogh J.M.
      • Orfeo T.
      One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice.
      Dr. Moore, oncologist, surgeon, accomplished scientist, and director of the RPMI, pioneered many cancer studies, established several types of cancer cell lines, and formulated RPMI 1640, a widely used cell-growth medium.
      • Moore G.E.
      • Merrick S.B.
      • Woods L.K.
      • Arabasz N.M.
      A human squamous cell carcinoma cell line.
      • Morgan R.T.
      • Woods L.K.
      • Moore G.E.
      • McGavran L.
      • Quinn L.A.
      • Semple T.U.
      A human gallbladder adenocarcinoma cell line.
      • Moore G.E.
      Tumors.
      • Moore G.E.
      • Minowada J.
      Historical progress and the future of human cell culture research.
      • Pincock S.
      George Eugene Moore.
      There is currently a growing interest in CCA-related basic and translational research, due to poor outcomes with the currently available treatment options. For recently developed novel experimental models [eg, three-dimensional (3D) organoids] and treatments, CCA cell lines have been used for the identification of novel mechanistic pathways, biomarkers, and disease-specific genes. Therefore, previously isolated human primary CCA cell lines, their isolation methods, and known essential characteristics are summarized herein to help researchers in their future studies.

      Human Primary CCA Cell Lines

      The first CCA cell line was established about a half-century ago, in 1971 in the United States
      • Fogh J.
      • Fogh J.M.
      • Orfeo T.
      One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice.
      ; more than half of currently available CCA cell lines were generated in Japan between 1975 and 1990. To date, researchers from Italy, Germany, China, South Korea, and Thailand have reported on the isolation of CCA cell lines and related experiments. In the past 2 decades, researchers have reported the isolation of multiple CCA cell lines in Thailand more than in any other country, possibly due to the high prevalence of the disease in that geographic region.
      • Kirstein M.M.
      • Vogel A.
      Epidemiology and risk factors of cholangiocarcinoma.
      Each CCA cell line is unique in reflecting a specific genotype, sex-related properties, and patient-related signatures, making them especially valuable for scientific and commercial applications. It would undoubtedly be helpful to have an international and collective cell bank that includes cell-identification analysis from each study to appropriately define the CCA properties and prevent any misidentification of the existing cell lines.
      • Dirks W.G.
      • Faehnrich S.
      • Estella I.A.
      • Drexler H.G.
      Short tandem repeat DNA typing provides an international reference standard for authentication of human cell lines.
      For instance, the ETK-1 CCA cell line was retracted in 2004 after a short tandem repeat polymorphism analysis revealed that the ETK-1 and SSP-25 cell lines were identical.
      • Yoshino K.
      • Iimura E.
      • Saijo K.
      • Iwase S.
      • Fukami K.
      • Ohno T.
      • Obata Y.
      • Nakamura Y.
      Essential role for gene profiling analysis in the authentication of human cell lines.
      ,
      • Huang C.K.
      • Iwagami Y.
      • Aihara A.
      • Chung W.
      • de la Monte S.
      • Thomas J.M.
      • Olsen M.
      • Carlson R.
      • Yu T.
      • Dong X.
      • Wands J.
      Anti-tumor effects of second generation β-hydroxylase inhibitors on cholangiocarcinoma development and progression.
      However, later, it was understood that SSP-25 should have been retracted instead of ETK-1 because the SSP-25 and RBE cell lines were isolated from the same patient, but their short tandem repeat profiling results did not match (https://cell.brc.riken.jp/en/rcb/rbessp-25_announce, last accessed May 5, 2022). As a result, the ETK-1 cells have been distributed under the name of SSP-25 since 2004, but it is unclear when that cell line was initially misidentified. It is also unclear what happened to the SSP-25 cell line. Similarly, the M156, M213, and M214 cell lines were regarded as having been isolated from three separate patients; however, results from recent short tandem repeat profiling showed that they were isolated from the same patient.
      • Sripa B.
      • Seubwai W.
      • Vaeteewoottacharn K.
      • Sawanyawisuth K.
      • Silsirivanit A.
      • Kaewkong W.
      • Muisuk K.
      • Dana P.
      • Phoomak C.
      • Lert-Itthiporn W.
      • Luvira V.
      • Pairojkul C.
      • Teh B.T.
      • Wongkham S.
      • Okada S.
      • Chamgramol Y.
      Functional and genetic characterization of three cell lines derived from a single tumor of an Opisthorchis viverrini-associated cholangiocarcinoma patient.
      Cross-contamination of cell lines is a long-standing problem that has resulted in scientific errors
      • Markovic O.
      • Markovic N.
      Cell cross-contamination in cell cultures: the silent and neglected danger.
      and might initiate inaccurate data chains. Various levels of the cell-culturing process have been blamed for cross-contamination, including: i) accidental inoculation, ii) mislabeling, iii) confusion in freeze-thawing, iv) work with more than one cell line in a biosafety cabinet at the same time, and v) contamination of the stock bottle of media with cells.
      • Rojas A.
      • Gonzalez I.
      Cell line cross-contamination: a detrimental issue in current biomedical research.
      ,
      • Nelson-Rees W.A.
      • Daniels D.W.
      • Flandermeyer R.R.
      Cross-contamination of cells in culture.
      Additionally, cancer cells may carry a greater risk for cross-contamination due to their greater capacity for proliferation. Even a minimal amount of inoculation of cancer cell lines could suppress other cell lines in the culture and take their place over time. Therefore, the preservation and maintenance of the cell line are as important and challenging as is the establishment of one.
      In a different case, CHGS was mentioned as a CCA cell line in 2015 by Zach et al
      • Zach S.
      • Birgin E.
      • Rückert F.
      Primary cholangiocellular carcinoma cell lines.
      and is listed in the Cellosaurus database (accession number CVCL_M272; https://web.expasy.org/cellosaurus/CVCL_M272, last accessed May 5, 2022). However, the first mention of CHGS, in 1988 by Katoh et al,
      • Katoh H.
      • Shinbo T.
      • Otagiri H.
      • Saitoh M.
      • Saitoh T.
      • Ishizawa S.
      • Shimizu T.
      • Satoh A.
      • Tazawa K.
      • Fujimaki M.
      Character of a human cholangiocarcinoma, CHGS, serially transplanted to nude mice.
      indicates that CHGS is a CCA tissue line passaged in mice, and that cancer cells have not been isolated from this tissue line. Similarly, the CC-CL-1 cell line was studied as a CCA cell line in a research study referred to in previously published studies; however, none of the references cited in the research study contained any information regarding the CC-CL-1 cell line, generating doubt about the credibility of the cell line.
      • Caca K.
      • Feisthammel J.
      • Klee K.
      • Tannapfel A.
      • Witzigmann H.
      • Wittekind C.
      • Mössner J.
      • Berr F.
      Inactivation of the INK4a/ARF locus and p53 in sporadic extrahepatic bile duct cancers and bile tract cancer cell lines.
      In addition to primary CCA cell lines, derivative CCA cell lines have been established for the study of drug resistance. QBC939/ADM is doxorubicin resistant; HuCCT1-G100, YSCCC-G100, RTFK-1, KKU-M139/GEM, KKU-213B/GEM, MT-CHC01R1.5, and SNU-1196/GR are gemcitabine resistant; and KKU-M055/46 and KKU-213B/246 are 5-fluorouracil–resistant CCA cell lines.
      • Wattanawongdon W.
      • Hahnvajanawong C.
      • Namwat N.
      • Kanchanawat S.
      • Boonmars T.
      • Jearanaikoon P.
      • Leelayuwat C.
      • Techasen A.
      • Seubwai W.
      Establishment and characterization of gemcitabine-resistant human cholangiocarcinoma cell lines with multidrug resistance and enhanced invasiveness.
      ,
      • Liu Z.H.
      • He Y.P.
      • Zhou Y.
      • Zhang P.
      • Qin H.
      Establishment and identification of the human multi-drug-resistant cholangiocarcinoma cell line QBC939/ADM.
      • Sato J.
      • Kimura T.
      • Saito T.
      • Anazawa T.
      • Kenjo A.
      • Sato Y.
      • Tsuchiya T.
      • Gotoh M.
      Gene expression analysis for predicting gemcitabine resistance in human cholangiocarcinoma.
      • Saiki Y.
      • Yoshino Y.
      • Fujimura H.
      • Manabe T.
      • Kudo Y.
      • Shimada M.
      • Mano N.
      • Nakano T.
      • Lee Y.
      • Shimizu S.
      • Oba S.
      • Fujiwara S.
      • Shimizu H.
      • Chen N.
      • Nezhad Z.K.
      • Jin G.
      • Fukushige S.
      • Sunamura M.
      • Ishida M.
      • Motoi F.
      • Egawa S.
      • Unno M.
      • Horii A.
      DCK is frequently inactivated in acquired gemcitabine-resistant human cancer cells.
      • Varamo C.
      • Peraldo-Neia C.
      • Ostano P.
      • Basiricò M.
      • Raggi C.
      • Bernabei P.
      • Venesio T.
      • Berrino E.
      • Aglietta M.
      • Leone F.
      • Cavalloni G.
      Establishment and characterization of a new intrahepatic cholangiocarcinoma cell line resistant to gemcitabine.
      • Namwat N.
      • Amimanan P.
      • Loilome W.
      • Jearanaikoon P.
      • Sripa B.
      • Bhudhisawasdi V.
      • Tassaneeyakul W.
      Characterization of 5-fluorouracil-resistant cholangiocarcinoma cell lines.
      The KKU-213L5 cell line is a derivative of the KKU-213A cell line, with high metastatic activity.
      • Uthaisar K.
      • Vaeteewoottacharn K.
      • Seubwai W.
      • Talabnin C.
      • Sawanyawisuth K.
      • Obchoei S.
      • Kraiklang R.
      • Okada S.
      • Wongkham S.
      Establishment and characterization of a novel human cholangiocarcinoma cell line with high metastatic activity.
      Tables 1 and 2 summarize the CCA cell lines available between 1971 and 2000, and between 2001 from 2021, respectively, from the English and non-English published literature. Tables 1 and 2 include: i) the type of CCA (eg, intra- or extrahepatic), ii) isolation source (eg, primary tumor or xenograft), iii) tissue-digestion method (eg, trypsin or collagenase), iv) cell-sorting and -purification methods (colony isolation or removal of fibroblasts), v) maintenance-medium choice (eg, RPMI or DMEM), vi) cell morphology (eg, spindle or polygonal shape), and vii) doubling time of cells.
      Table 1Human Primary Cholangiocarcinoma Cell Lines Established between 1971 and 2000
      YearCountryCell nameDiagnosisIsolation sourceDigestion methodSorting methodMaintenance mediumCell morphologyDoubling timeReferences
      11971USARPMI 7451CCAEagle's MEM + 10% FBS + nonessential amino acids + 60 pg/mL gentamicinTightly adherent, monolayer, polygonal shaped cells
      • Fogh J.
      • Fogh J.M.
      • Orfeo T.
      One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice.
      ,
      • Fogh J.
      • Wright W.C.
      • Loveless J.D.
      Absence of HeLa cell contamination in 169 cell lines derived from human tumors.
      ,
      • Storto P.D.
      • Saidman S.L.
      • Demetris A.J.
      • Letessier E.
      • Whiteside T.L.
      • Gollin S.M.
      Chromosomal breakpoints in cholangiocarcinoma cell lines.
      21975JapanH-1 or H1CCA
      • Storto P.D.
      • Saidman S.L.
      • Demetris A.J.
      • Letessier E.
      • Whiteside T.L.
      • Gollin S.M.
      Chromosomal breakpoints in cholangiocarcinoma cell lines.
      ,
      • Enjoji M.
      • Nakashima M.
      • Honda M.
      • Sakai H.
      • Nawata H.
      Hepatocytic phenotypes induced in sarcomatous cholangiocarcinoma cells treated with 5-azacytidine.
      31981JapanOZIHCCAAscitesNo digestionColony isolation by trypsin and EDTA-soaked filter; other cells removed by enzymatic and mechanical treatmentsWilliams' E + 10% newborn calf serum + P/SLarge nucleus with 1 to 2 nucleoli; dark cytoplasm; high nucleus/cytoplasm ratio; pavement-like proliferation. Abundant production of gel-like substance48 hours
      • Homma S.
      • Nagamori S.
      • Fujise K.
      • Yamazaki K.
      • Hasumura S.
      • Sujino H.
      • Matsuura T.
      • Shimizu K.
      • Kameda H.
      • Takaki K.
      Human bile duct carcinoma cell line producing abundant mucin in vitro.
      41984GermanyEGI-1CCAPrimary tumorMEM + 10% FBS + 2× MEM amino acids (essential and nonessential) + 4 mmol/L l-glutamine + 1 mmol/L sodium pyruvateMonolayer, adherent, polymorphic cells45–50 hours
      • Okaro A.C.
      • Deery A.R.
      • Hutchins R.R.
      • Davidson B.R.
      The expression of antiapoptotic proteins Bcl-2, Bcl-X(L), and Mcl-1 in benign, dysplastic, and malignant biliary epithelium.
      51985JapanHChol-Y1CCAPrimary tumorNo digestionFibroblasts spontaneously disappeared in 2 monthsHam's F12Uniform, monolayer cells; including abundant granules; prominent round nucleus with multiple nucleoli52 hours
      • Yamaguchi N.
      • Morioka H.
      • Ohkura H.
      • Hirohashi S.
      • Kawai K.
      Establishment and characterization of the human cholangiocarcinoma cell line HChol-Y1 in a serum-free, chemically defined medium.
      61985GermanySK-ChA-1 or WITTEHCCAAscitesNo digestionLight trypsin treatmentCMRL + 15% + P/SAdherent, spindle- to polygonal shaped, polymorphic cells; proliferating as single adherent cells or small clusters48 hours
      • Knuth A.
      • Gabbert H.
      • Dippold W.
      • Klein O.
      • Sachsse W.
      • Bitter-Suermann D.
      • Prellwitz W.
      • Meyer zum Büschenfelde K.H.
      Biliary adenocarcinoma. Characterisation of three new human tumor cell lines.
      ,
      • Vickers S.M.
      • Jhala N.C.
      • Ahn E.Y.
      • McDonald J.M.
      • Pan G.
      • Bland K.I.
      Tamoxifen (TMX)/Fas induced growth inhibition of human cholangiocarcinoma (HCC) by gamma interferon (IFN-gamma).
      71987JapanKMCH-1IHCCA + HCCPrimary tumor0.5 U/mg type IV collagenase in PBS, 37°C, 30 minutesColony isolation by trypsinDMEM + 20% FBS + 35 μmol/L sodium bicarbonate + P/SLarge, round nucleus with multiple nucleoli; abundant clear cytoplasm; pavement-like proliferation; microvilli on the luminal surfaces39 hours
      • Murakami T.
      • Yano H.
      • Maruiwa M.
      • Sugihara S.
      • Kojiro M.
      Establishment and characterization of a human combined hepatocholangiocarcinoma cell line and its heterologous transplantation in nude mice.
      81988JapanHuH-28IHCCAPrimary tumor (frozen-thawed)500 U/mL trypsinRPMI 1640 + 20% FBS + %0.2 lactalbumin hydrolysateSpindle- to polygonal shaped cells80 hours
      • Kusaka Y.
      • Tokiwa T.
      • Sato J.
      Establishment and characterization of a cell line from a human cholangiocellular carcinoma.
      91989JapanHuCC-T1IHCCAAscitesNo digestionIS-RPMI medium used to eliminate fibroblastsRPMI 1640 + 0.2% lactalbumin hydrolysatePolygonal to spindle-shaped, abundant and clear cytoplasm, proliferation in pavement arrangement74 hours
      • Miyagiwa M.
      • Ichida T.
      • Tokiwa T.
      • Sato J.
      • Sasaki H.
      A new human cholangiocellular carcinoma cell line (HuCC-T1) producing carbohydrate antigen 19/9 in serum-free medium.
      ,
      • Nakabayashi H.
      • Taketa K.
      • Yamane T.
      • Miyazaki M.
      • Miyano K.
      • Sato J.
      Phenotypical stability of a human hepatoma cell line, HuH-7, in long-term culture with chemically defined medium.
      101989JapanMECCCAPleural effusionNo digestionRPMI 1640 + 20% FBSPolymorphic, epithelial-like cells; high nucleus/cytoplasm ratio40 hours
      • Yoshida K.
      • Tomizawa H.
      • Ota T.
      • Nagashima T.
      • Kikuchi H.
      • Watanabe H.
      • Hashizaki K.
      • Yonaha A.
      [Establishment and characterization of human cholaginocarcinoma, MEC, producing carbohydrate antigen 19-9].
      111990USAPCI:SG231IHCCAPrimary tumorType II collagenase, 37°C, 3 hoursα MEM (Earle's salts) with nucleosides + 10% FBS + 60 pg/mL gentamicinTightly adherent, monolayer, polygonal shaped cells; nonadherent cell clusters that formed three-dimensional tubular structures resembling bile ducts
      • Storto P.D.
      • Saidman S.L.
      • Demetris A.J.
      • Letessier E.
      • Whiteside T.L.
      • Gollin S.M.
      Chromosomal breakpoints in cholangiocarcinoma cell lines.
      121991ThailandHuCCA-1IHCCAPrimary tumorNo digestionFibroblasts gradually decreased with each passage and disappeared in a monthHam's F12 + P/SMonolayer, adherent, polygonal shaped epithelial cells with occasionally multiple, round to oval nuclei; granule-filled cytoplasms; piling up of cells and occasional gland-like appearances55 hours
      • Sirisinha S.
      • Tengchaisri T.
      • Boonpucknavig S.
      • Prempracha N.
      • Ratanarapee S.
      • Pausawasdi A.
      Establishment and characterization of a cholangiocarcinoma cell line from a Thai patient with intrahepatic bile duct cancer.
      131991JapanKMBCEHCCAXenograftCollagenase 150–250 U/mg, 37°C, 90–110 minutesFibroblasts scraped away and completely disappeared after several passageDMEM + 5% FBS + 12 mmol/L sodium bicarbonate + P/SPolymorphic epithelial-like cells; one or more large, irregular, round to oval nuclei with a few prominent nucleoli; relatively poor, round to polygonal cytoplasm; pavement-like proliferation; tubular formation30 hours
      • Yano H.
      • Maruiwa M.
      • Murakami T.
      • Fukuda K.
      • Ito Y.
      • Sugihara S.
      • Kojiro M.
      A new human pleomorphic hepatocellular carcinoma cell line, KYN-2.
      ,
      • Yano H.
      • Maruiwa M.
      • Iemura A.
      • Mizoguchi A.
      • Kojiro M.
      Establishment and characterization of a new human extrahepatic bile duct carcinoma cell line (KMBC).
      141992USACC-LP-1IHCCAPrimary tumor0.05% (w/v) collagenase type IV, 0.002% (w/v) DNase type 1, 90 minutesCentrifugation on double-layer (75%/100% v/v) Ficoll-Hypaque gradients; fibroblasts removed by differential trypsinizationDMEM + 15% FBS + 2 mmol/L l-glutamine + antibioticsCobblestone-like proliferation of monolayers; stratification of cells in some areas180 hours
      • Shimizu Y.
      • Demetris A.J.
      • Gollin S.M.
      • Storto P.D.
      • Bedford H.M.
      • Altarac S.
      • Iwatsuki S.
      • Herberman R.B.
      • Whiteside T.L.
      Two new human cholangiocarcinoma cell lines and their cytogenetics and responses to growth factors, hormones, cytokines or immunologic effector cells.
      151992CC-SW-172 hours
      161992JapanKMC-1IHCCAXenograft0.5 mg/nL type IV collagenase in PBS, 37°C, 60–80 minutesTumor cells suppressed the fibroblasts by the timeDMEM + 10% or 5% FBSMonolayer, pavement-like proliferation; clear cytoplasm, oval-shaped nuclei; tubular formation; some cells with mucin in the cytoplasm54 hours
      • Iemura A.
      • Maruiwa M.
      • Yano H.
      • Kojiro M.
      A new human cholangiocellular carcinoma cell line (KMC-1).
      171993ChinaQBC939EHCCAPrimary tumorType I collagenase, DNase I, plasminaseFibroblasts gradually decreased by passagingRPMI 1640 + 10% FBSMonolayer, polymorphic, adherent cells; round or oval nuclei; high nucleus/cytoplasm ratio24 hours
      • Wang S.
      [Establishment of extrahepatic cholangiocarcinoma cell line].
      ,
      • Wang B.
      • Yang R.
      • Wu Y.
      • Li H.
      • Hu Z.
      • Chen Y.
      • Zou S.
      Sodium valproate inhibits the growth of human cholangiocarcinoma in vitro and in vivo.
      181994JapanTKEHCCAAscitesNo digestionColony isolation using 0.33% agarRPMI 1640 + 15% FBS + 2 mmol/L glutamine + 1 mmol/L sodium pyruvateMonolayer, adherent proliferation; forming gland-like structures; lobated or dark, large nuclei29 hours
      • Watanabe M.
      • Chigusa M.
      • Takahashi H.
      • Nakamura J.
      • Tanaka H.
      • Ohno T.
      High level of CA19-9, CA50, and CEA-producible human cholangiocarcinoma cell line changes in the secretion ratios in vitro or in vivo.
      191995JapanOCUCh-LM1EHCCAPrimary tumorNo digestionFibroblasts gradually decreased and disappeared in 1 monthDMEM + 10% FBS + 2 mmol/L l-glutamine + 0.5 mmol/L sodium pyruvate + P/SMonolayer, pavement-like proliferation; clear cytoplasm and oval nuclei31 hours
      • Yamada N.
      • Chung Y.S.
      • Arimoto Y.
      • Sawada T.
      • Seki S.
      • Sowa M.
      Establishment of a new human extrahepatic bile duct carcinoma cell line (OCUCh-LM1) and experimental liver metastatic model.
      201995JapanTFK-1EHCCAPrimary tumor1000 U/mL dispase, 37°C, 30 minutesFibroblasts removed by mechanical scraping and differential attachment selection with trypsinRPMI 1640 + 10% FBS + P/SPolygonal epithelial monolayers; pavement-like proliferation37 hours
      • Saijyo S.
      • Kudo T.
      • Suzuki M.
      • Katayose Y.
      • Shinoda M.
      • Muto T.
      • Fukuhara K.
      • Suzuki T.
      • Matsuno S.
      Establishment of a new extrahepatic bile duct carcinoma cell line, TFK-1.
      211996JapanKMCH-2IHCCA + HCCPrimary tumor150–250 U/mg collagenase in PBS, 37°C, 90–110 minutesFibroblasts removed by mechanical scrapingDMEM + 5% FBS + 12 mmol/L sodium bicarbonate + P/SMonomorphic polygonal cells; large round to oval nuclei with a few prominent nucleoli; pavement-like proliferation44 hours at 20th passage; 32 hours at 55th passage
      • Yano H.
      • Iemura A.
      • Haramaki M.
      • Momosaki S.
      • Ogasawara S.
      • Higaki K.
      • Kojiro M.
      A human combined hepatocellular and cholangiocarcinoma cell line (KMCH-2) that shows the features of hepatocellular carcinoma or cholangiocarcinoma under different growth conditions.
      221997JapanETK-1IHCCAAscitesNo digestionLimiting dilutionRPMI 1640 + 10% FBS + 10 mmol/L HEPES + 2 mmol/L l-glutamine + 0.1 mmol/L nonessential amino acids + 1 mmol/L sodium pyruvate + 0.005 mmol/L β-mercaptoethanolSmall polygonal cells; round to oval nuclei and prominent nucleoli; uniform, monolayer with a pavement-like proliferation71 hours
      • Enjoji M.
      • Nakashima M.
      • Honda M.
      • Sakai H.
      • Nawata H.
      Hepatocytic phenotypes induced in sarcomatous cholangiocarcinoma cells treated with 5-azacytidine.
      231997JapanICBD-1EHCCAPrimary tumorNo digestionFibroblasts gradually decreased and disappeared in 1 monthRPMI 1640 + 10% FBS + 0.5 mmol/L sodium bicarbonate + 2 mmol/L l-glutamine + P/SLarge round to oval nuclei with a few nucleoli; relatively poor, polygonal to oval cytoplasm; pavement-like proliferation31.5 hours
      • Takiyama I.
      • Terashima M.
      • Ikeda K.
      • Kawamura H.
      • Kashiwaba M.
      • Tamura G.
      • Suto T.
      • Nakashima F.
      • Sasaki R.
      • Saito K.
      Establishment and characterization of a new human extrahepatic bile duct carcinoma cell line (ICBD-1).
      241997JapanSSP-25∗IHCCAPrimary tumorNo digestionFibroblasts gradually disappeared, limiting dilutionRPMI 1640 + 10% FBSSpindle-shaped cells64 hours
      • Enjoji M.
      • Sakai H.
      • Nawata H.
      • Kajiyama K.
      • Tsuneyoshi M.
      Sarcomatous and adenocarcinoma cell lines from the same nodule of cholangiocarcinoma.
      25RBE∗Monolayer of polygonal cells, pavement-like proliferation45 hours
      261998JapanTBCN-1EHCCAPrimary tumorRPMI 1640 + 10% FBS54 hours
      • Koike N.
      • Todoroki T.
      • Kawamoto T.
      • Yoshida S.
      • Kashiwagi H.
      • Fukao K.
      • Ohno T.
      • Watanabe T.
      The invasion potentials of human biliary tract carcinoma cell lines: correlation between invasiveness and morphologic characteristics.
      271999ThailandKKU-213A∗IHCCAPrimary tumor0.25% trypsin-EDTA, 37°C, 1 hourDMEM + 10% FBS + A/ASmall, spindle-shaped cells23 hours
      • Sripa B.
      • Seubwai W.
      • Vaeteewoottacharn K.
      • Sawanyawisuth K.
      • Silsirivanit A.
      • Kaewkong W.
      • Muisuk K.
      • Dana P.
      • Phoomak C.
      • Lert-Itthiporn W.
      • Luvira V.
      • Pairojkul C.
      • Teh B.T.
      • Wongkham S.
      • Okada S.
      • Chamgramol Y.
      Functional and genetic characterization of three cell lines derived from a single tumor of an Opisthorchis viverrini-associated cholangiocarcinoma patient.
      28KKU-213B∗Irregular polygonal cells; high nucleus to cytoplasmic ratio; patch-like structure24.5 hours
      29KKU-213C∗Irregular polygonal cells; high nucleus to cytoplasmic ratio25.6 hours
      301999JapanYSCCCCCARPMI 1640 + 10% FBSLymphocyte-like
      • Sugiyama H.
      • Onuki K.
      • Ishige K.
      • Baba N.
      • Ueda T.
      • Matsuda S.
      • Takeuchi K.
      • Onodera M.
      • Nakanuma Y.
      • Yamato M.
      • Yamamoto M.
      • Hyodo I.
      • Shoda J.
      Potent in vitro and in vivo antitumor activity of sorafenib against human intrahepatic cholangiocarcinoma cells.
      , https://cellbank.brc.riken.jp/cell_bank/CellInfo/?cellNo=RCB1549&lang=, last accessed May 5, 2022
      312000JapanHBDCEHCCAAscitesNo digestionCentrifugation on triple-layer (75%/100%/25) Ficoll-Hypaque gradients; colony isolation using porcelain cloning ringsWilliams' E + 10% FBS + 2 ng/mL HGF + 2 mmol/L l-glutamine + 6 mmol/L glucose + 0.5 mmol/L sodium bicarbonate + 100 ng/mL kanamycin + 10 ng/mL fungizonePolygonal or spindle-shaped polymorphic cells; occasional large vacuoles in the cytoplasm; forming small clusters or clumps; one or more large, irregular, round or oval nuclei with a few prominent nucleoli; pavement-like proliferation32 hours
      • Jiao W.
      • Yakushiji H.
      • Kitajima Y.
      • Ogawa A.
      • Miyazaki K.
      Establishment and characterization of human hilar bile duct carcinoma cell line and cell strain.
      CCA, cholangiocarcinoma; DMEM, Dulbecco's modified Eagle's medium; EHCCA, extrahepatic cholangiocarcinoma; FBS, fetal bovine serum; HCC, hepatocellular carcinoma; IHCCA, intrahepatic cholangiocarcinoma; MEM, minimal essential medium.
      Table 2Human Primary Cholangiocarcinoma Cell Lines Established between 2001 and 2021
      YearCountryCell nameDiagnosisIsolation sourceDigestion methodSorting methodMaintenance mediumCell morphologyDoubling timeReferences
      12001Republic of KoreaChoi-CKIHCCAPrimary tumorNo digestionDifferential trypsinization, scraping, or G418 treatment (100 μg/mL)Opti-MEM + 10% FBS + 30 mmol/L sodium bicarbonate + antibioticsPolygonal and compact cells with indistinct cell boundaries
      • Kim D.G.
      • Park S.Y.
      • You K.R.
      • Lee G.B.
      • Kim H.
      • Moon W.S.
      • Chun Y.H.
      • Park S.H.
      Establishment and characterization of chromosomal aberrations in human cholangiocarcinoma cell lines by cross-species color banding.
      2Cho-CKPolygonal and compact cells with indistinct cell boundaries
      3JCKPolygonal morphology and some spindle-shaped cells
      4SCKMonolayer, epithelioid or spindle-shaped cells; fewer polygonal shaped cells
      52002ThailandHubCCA-1IHCCAIntrahepatic biliary fluidHam's F12 + 20% FBS + P/SAdherent, monolayer, polygonal to spindle-shaped cells
      • Panichakul T.
      • Intachote P.
      • Wongkajorsilp A.
      • Sripa B.
      • Sirisinha S.
      Triptolide sensitizes resistant cholangiocarcinoma cells to TRAIL-induced apoptosis.
      ,
      • Panichakul T.
      • Wanun T.
      • Reutrakul V.
      • Sirisinha S.
      Synergistic cytotoxicity and apoptosis induced in human cholangiocarcinoma cell lines by a combined treatment with tumor necrosis factor-alpha (TNF-alpha) and triptolide.
      62002ThailandKKU-100IHCCAPrimary tumor0.025% trypsin-EDTA, 37°C, 1 hourFibroblasts removed with a cell scraper and by differential trypsinizationHam's F12 + 20% FBS + P/SCompact, polygonal to spindle-shaped cells; floating or clumping in a confluent monolayer; large nucleus containing two to five nucleoli and a clear cytoplasm72 hours
      • Panichakul T.
      • Wanun T.
      • Reutrakul V.
      • Sirisinha S.
      Synergistic cytotoxicity and apoptosis induced in human cholangiocarcinoma cell lines by a combined treatment with tumor necrosis factor-alpha (TNF-alpha) and triptolide.
      ,
      • Sripa B.
      • Leungwattanawanit S.
      • Nitta T.
      • Wongkham C.
      • Bhudhisawasdi V.
      • Puapairoj A.
      • Sripa C.
      • Miwa M.
      Establishment and characterization of an opisthorchiasis-associated cholangiocarcinoma cell line (KKU-100).
      72002Republic of KoreaSNU-245EHCCAPrimary tumorNo digestionDifferential trypsinization used to remove fibroblastsRPMI 1640 + 10% FBSMonolayer, adherent cells; trabecular arrangement with acinar formation54 hours
      • Ku J.L.
      • Yoon K.A.
      • Kim I.J.
      • Kim W.H.
      • Jang J.Y.
      • Suh K.S.
      • Kim S.W.
      • Park Y.H.
      • Hwang J.H.
      • Yoon Y.B.
      • Park J.G.
      Establishment and characterisation of six human biliary tract cancer cell lines.
      8SNU-1079IHCCAMonolayer, adherent cells; pleomorphic appearance with multiple cytoplasmic processes; numerous cytoplasmic vacuoles in some cells; some multinucleated cells72 hours
      9SNU-1196EHCCAMonolayer, adherent cells; trabecular pattern consisted of spindle to polygonal shaped cells having vesicular nuclei and multiple small nucleoli48 hours
      102004JapanTKKKIHCCADMEM (low glucose) + 10% FBSAdherent, monolayer, epithelial-like cells; pavement-like proliferation
      • Oka T.
      • Yamamoto H.
      • Sasaki S.
      • Ii M.
      • Hizaki K.
      • Taniguchi H.
      • Adachi Y.
      • Imai K.
      • Shinomura Y.
      Overexpression of beta3/gamma2 chains of laminin-5 and MMP7 in biliary cancer.
      112005ThailandKKU-M055IHCCAHam's F-12 + 10% FBS + P/S
      • Tepsiri N.
      • Chaturat L.
      • Sripa B.
      • Namwat W.
      • Wongkham S.
      • Bhudhisawasdi V.
      • Tassaneeyakul W.
      Drug sensitivity and drug resistance profiles of human intrahepatic cholangiocarcinoma cell lines.
      12KKU-OCA17
      132005JapanTBCN6EHCCAXenograft0.25% trypsin in PBS, 37°C, 10 minutesDMEM + 10% FBSAdherent, monolayer, polygonal cells38 hours
      • Ghosh M.
      • Koike N.
      • Tsunoda S.
      • Hirano T.
      • Kaul S.
      • Kashiwagi H.
      • Kawamoto T.
      • Ohkohchi N.
      • Saijo K.
      • Ohno T.
      • Ohno T.
      • Miwa M.
      • Todoroki T.
      Characterization and genetic analysis in the newly established human bile duct cancer cell lines.
      14TGBC-47
      152006ThailandKKU-M139CCAPrimary tumorHam's F12 or RPMI 1640 + 10% FBS + P/SAdherent, monolayer, polygonal cells; pavement-like proliferation17 hours
      • Wattanawongdon W.
      • Hahnvajanawong C.
      • Namwat N.
      • Kanchanawat S.
      • Boonmars T.
      • Jearanaikoon P.
      • Leelayuwat C.
      • Techasen A.
      • Seubwai W.
      Establishment and characterization of gemcitabine-resistant human cholangiocarcinoma cell lines with multidrug resistance and enhanced invasiveness.
      ,
      • Panichakul T.
      • Intachote P.
      • Wongkajorsilp A.
      • Sripa B.
      • Sirisinha S.
      Triptolide sensitizes resistant cholangiocarcinoma cells to TRAIL-induced apoptosis.
      162006ThailandRMCCA-1IHCCAPrimary tumorNo digestionDifferential trypsinization used to remove fibroblastsHam's F12 + 20% FBS + EGF + 250 μg/mL amphotericin + P/SCircular to spindle shape with many processes and ornamental fringes; granulated nucleus and cytoplasm48 hours
      • Rattanasinganchan P.
      • Leelawat K.
      • Treepongkaruna S.A.
      • Tocharoentanaphol C.
      • Subwongcharoen S.
      • Suthiphongchai T.
      • Tohtong R.
      Establishment and characterization of a cholangiocarcinoma cell line (RMCCA-1) from a Thai patient.
      172007ChinaHKGZ-CCIHCCAPrimary tumor1200–2000 U/mL collagenase and DNase in 10 mL/g DPBSDMEMAdherent, monolayer, epithelial-like cells48 hours
      • Ma S.
      • Hu L.
      • Huang X.H.
      • Cao L.Q.
      • Chan K.W.
      • Wang Q.
      • Guan X.Y.
      Establishment and characterization of a human cholangiocarcinoma cell line.
      182007JapanIHGGKIHCCARPMI 1640 + 10% FBS + P/SAdherent, monolayer, epithelial-like cells
      • Kokuryo T.
      • Senga T.
      • Yokoyama Y.
      • Nagino M.
      • Nimura Y.
      • Hamaguchi M.
      Nek2 as an effective target for inhibition of tumorigenic growth and peritoneal dissemination of cholangiocarcinoma.
      , http://www2.idac.tohoku.ac.jp/dep/ccr/TKGdate/TKGvo106/0623.html, last accessed May 5, 2022
      192009ThailandCL-2CCADMEM + 15% FBS + P/S
      • Akarasereenont P.
      • Aiamsa-ard T.
      • Chotewuttakorn S.
      • Thaworn A.
      Cholangiocarcinoma cell induced platelet aggregation via activation of thrombin and cyclooxygenase.
      ,
      • Chaijaroenkul W.
      • Viyanant V.
      • Mahavorasirikul W.
      • Na-Bangchang K.
      Cytotoxic activity of artemisinin derivatives against cholangiocarcinoma (CL-6) and hepatocarcinoma (Hep-G2) cell lines.
      20CL-6
      21CL-19
      222010JapanNCC-BD1EHCCAXenograftNo digestionFibroblasts removed by mechanical scrapingRPMI 1640 + 10% FBS + 2 mmol/L l-glutamine + P/SAdherent, monolayer, epithelial-like cells; pavement-like proliferation
      • Ojima H.
      • Yoshikawa D.
      • Ino Y.
      • Shimizu H.
      • Miyamoto M.
      • Kokubu A.
      • Hiraoka N.
      • Morofuji N.
      • Kondo T.
      • Onaya H.
      • Okusaka T.
      • Shimada K.
      • Sakamoto Y.
      • Esaki M.
      • Nara S.
      • Kosuge T.
      • Hirohashi S.
      • Kanai Y.
      • Shibata T.
      Establishment of six new human biliary tract carcinoma cell lines and identification of MAGEH1 as a candidate biomarker for predicting the efficacy of gemcitabine treatment.
      23NCC-BD2EHCCA
      24NCC-CC1IHCCA
      25NCC-CC3-1∗IHCCA
      26NCC-CC3-2∗IHCCA
      27NCC-CC4-1IHCCA
      282013ChinaHCCC-9810IHCCARPMI 1640 + 10% FBS + P/S20 hours
      • Liu J.
      • Han G.
      • Liu H.
      • Qin C.
      Suppression of cholangiocarcinoma cell growth by human umbilical cord mesenchymal stem cells: a possible role of Wnt and Akt signaling.
      292015ItalyMT-CHC01IHCCAXenograft200 U/mL collagenase, 3 hours, 37°CKnockout/DMEM/F-12 + 10% FBS + P/SMonolayer, adherent cells40 hours
      • Cavalloni G.
      • Peraldo-Neia C.
      • Varamo C.
      • Casorzo L.
      • Dell'Aglio C.
      • Bernabei P.
      • Chiorino G.
      • Aglietta M.
      • Leone F.
      Establishment and characterization of a human intrahepatic cholangiocarcinoma cell line derived from an Italian patient.
      302016USAICC1IHCCATrypsin, 30 minutes, 37°CRPMI or DMEM/F12 + 5% FBS + P/S
      • Saha S.K.
      • Gordan J.D.
      • Kleinstiver B.P.
      • Vu P.
      • Najem M.S.
      • Yeo J.C.
      • Shi L.
      • Kato Y.
      • Levin R.S.
      • Webber J.T.
      • Damon L.J.
      • Egan R.K.
      • Greninger P.
      • McDermott U.
      • Garnett M.J.
      • Jenkins R.L.
      • Rieger-Christ K.M.
      • Sullivan T.B.
      • Hezel A.F.
      • Liss A.S.
      • Mizukami Y.
      • Goyal L.
      • Ferrone C.R.
      • Zhu A.X.
      • Joung J.K.
      • Shokat K.M.
      • Benes C.H.
      • Bardeesy N.
      Isocitrate dehydrogenase mutations confer dasatinib hypersensitivity and SRC dependence in intrahepatic cholangiocarcinoma.
      31ICC2
      32ICC5
      332016ChinaZJU-0826EHCCAPrimary tumorFibroblasts eliminated by differential trypsinization and differential attachmentRPMI 1640 + 10% FBS + antibioticsMonolayer, adherent, homogeneous cells with characteristic loose pleomorphic cells and rare multinucleated cells63 hours
      • Zhang Y.
      • Luo J.
      • Dong X.
      • Yang F.
      • Zhang M.
      • Zhao J.
      • Wang Q.
      • Zhou F.
      • Sun J.
      • Yang X.
      Establishment and characterization of two novel cholangiocarcinoma cell lines.
      34ZJU-1125IHCCAMonolayer, epithelial-like, adherent cells; polygonal, occasionally multinucleated44 hours
      352017GermanyCCC-5EHCCAPleural effusionNo digestionDMEM + 20% FBS: KFSM - 2:1Monolayer, spindle- to polygonal shaped cells, pavement-like proliferation; anaplastic, multinucleated giant cells60 hours
      • Zach S.
      • Grun J.
      • Bauer A.T.
      • Pilarsky C.
      • Grutzmann R.
      • Weng H.
      • Dooley S.
      • Wilhelm T.J.
      • Gaiser T.
      • Ruckert F.
      CCC-5, a new primary cholangiocellular cell line.
      362017ThailandKKU-023IHCCAPrimary tumor1 mg/mL collagenase, 37°C, 30–45 minutesFibroblasts aseptically removed with a cell scraperHam's F12 + 10% FBS + P/S + nonessential amino acids + 12.5 mmol/L HEPES + 50 μg/mL cefazolin + 10 μg/mL ciprofloxacin + 2.5 μg/mL amphotericin B + 5 μmol/L ROCK inhibitorOvoid to cuboid shape polygonal cells; seldom multinucleated; forming compact monolayer with occasional multinucleated cells34 hours
      • Saensa-Ard S.
      • Leuangwattanawanit S.
      • Senggunprai L.
      • Namwat N.
      • Kongpetch S.
      • Chamgramol Y.
      • Loilome W.
      • Khansaard W.
      • Jusakul A.
      • Prawan A.
      • Pairojkul C.
      • Khantikeo N.
      • Yongvanit P.
      • Kukongviriyapan V.
      Establishment of cholangiocarcinoma cell lines from patients in the endemic area of liver fluke infection in Thailand.
      37KKU-452EHCCASpindle-shaped cells; seldom multinucleated; segregated and spread surround17 hours
      382018ChinaICC-1IHCCAPrimary tumorNo digestion
      • Yu G.Y.
      • Wang X.
      • Zheng S.S.
      • Gao X.M.
      • Jia Q.A.
      • Zhu W.W.
      • Lu L.
      • Jia H.L.
      • Chen J.H.
      • Dong Q.Z.
      • Lu M.
      • Qin L.X.
      RA190, a proteasome subunit ADRM1 inhibitor, suppresses intrahepatic cholangiocarcinoma by inducing NF-KB-mediated cell apoptosis.
      39ICC-2
      402019ThailandKKK-D049IHCCAXenograft1000 U/mL collagenase + 0.1 mg/mL DNase I, 3 hoursStromal cells sequentially removed by partial trypsinization and mechanical removalDMEM + 10% FBS + P/SEpithelial-like cells; high nuclear to cytoplasmic ratio; tight clustering
      • Vaeteewoottacharn K.
      • Pairojkul C.
      • Kariya R.
      • Muisuk K.
      • Imtawil K.
      • Chamgramol Y.
      • Bhudhisawasdi V.
      • Khuntikeo N.
      • Pugkhem A.
      • Saeseow O.T.
      • Silsirivanit A.
      • Wongkham C.
      • Wongkham S.
      • Okada S.
      Establishment of highly transplantable cholangiocarcinoma cell lines from a patient-derived xenograft mouse model.
      41KKK-D068Epithelial-like, polygonal and spindle-shaped cells; high nuclear to cytoplasmic ratio
      42KKK-D131Epithelial-like, polygonal and spindle-shaped cells; high nuclear to cytoplasmic ratio
      43KKK-D138Epithelial-like, polygonal and spindle-shaped cells; high nucleus-to-cytoplasm ratio
      442021Italy82.3IHCCAXenograftCollagenase 200 U/mL, 3 hours, 37°C10% FBS + P/S + gemcitabineMonolayer, adherent, epithelial-like cells53 hours
      • Peraldo-Neia C.
      • Massa A.
      • Vita F.
      • Basiricò M.
      • Raggi C.
      • Bernabei P.
      • Ostano P.
      • Casorzo L.
      • Panero M.
      • Leone F.
      • Cavalloni G.
      • Aglietta M.
      A novel multidrug-resistant cell line from an Italian intrahepatic cholangiocarcinoma patient.
      CCA, cholangiocarcinoma; DMEM, Dulbecco's modified Eagle's medium; EGF, epidermal growth factor; EHCCA, extrahepatic cholangiocarcinoma; FBS, fetal bovine serum; IHCCA, intrahepatic cholangiocarcinoma; MEM, minimal essential medium; PBS, phosphate-buffered saline.

      Overview on Isolation Methods

      The general principles of the CCA cell–isolation protocol are summarized in Figure 1. CCA cell isolation requires either a solid sample (eg, primary tumor or xenograft) or a liquid sample (eg, ascites or pleural effusion). Samples can come from autopsy, surgery, paracentesis, or animal harvesting. Samples could be transferred into Ham's F12 culture medium at 4°C.
      • Sirisinha S.
      • Tengchaisri T.
      • Boonpucknavig S.
      • Prempracha N.
      • Ratanarapee S.
      • Pausawasdi A.
      Establishment and characterization of a cholangiocarcinoma cell line from a Thai patient with intrahepatic bile duct cancer.
      It is better to process the samples as soon as possible to avoid ischemia-related problems in cells. If the CCA sample needs to be frozen, freeze-thawing the tissue samples in 10% dimethyl sulfoxide in culture medium is a viable option for tissue preservation.
      • Kusaka Y.
      • Tokiwa T.
      • Sato J.
      Establishment and characterization of a cell line from a human cholangiocellular carcinoma.
      Our practice preserves samples mainly in histidine-tryptophan-ketoglutarate organ-preservation solution at 4°C until tissue processing.
      • Mangus R.S.
      • Tector A.J.
      • Agarwal A.
      • Vianna R.
      • Murdock P.
      • Fridell J.A.
      Comparison of histidine-tryptophan-ketoglutarate solution (HTK) and University of Wisconsin solution (UW) in adult liver transplantation.
      Figure thumbnail gr1
      Figure 1CCA cell isolation method. CCA cell lines can be isolated from either fresh samples (eg, HuCCA-1 cell line) or frozen-thawed samples (eg, huh-28 cell line) from primary tumors or from xenografts, which can be kept in different mediums. After manual dissociation, plated with or without digestion.
      CCA cell lines can be isolated using several methods.
      • Yamaguchi N.
      • Morioka H.
      • Ohkura H.
      • Hirohashi S.
      • Kawai K.
      Establishment and characterization of the human cholangiocarcinoma cell line HChol-Y1 in a serum-free, chemically defined medium.
      ,
      • Murakami T.
      • Yano H.
      • Maruiwa M.
      • Sugihara S.
      • Kojiro M.
      Establishment and characterization of a human combined hepatocholangiocarcinoma cell line and its heterologous transplantation in nude mice.
      Herein, the general perspective of isolation protocols is briefly summarized. All tissue processing should be performed under aseptic conditions in a biological safety cabinet with sterile or disposable surgical instruments. The first and the most essential step of CCA cell isolation from solid samples is cutting the tissue into small pieces (>1 mm in diameter).
      • Yamaguchi N.
      • Morioka H.
      • Ohkura H.
      • Hirohashi S.
      • Kawai K.
      Establishment and characterization of the human cholangiocarcinoma cell line HChol-Y1 in a serum-free, chemically defined medium.
      • Knuth A.
      • Gabbert H.
      • Dippold W.
      • Klein O.
      • Sachsse W.
      • Bitter-Suermann D.
      • Prellwitz W.
      • Meyer zum Büschenfelde K.H.
      Biliary adenocarcinoma. Characterisation of three new human tumor cell lines.
      • Vickers S.M.
      • Jhala N.C.
      • Ahn E.Y.
      • McDonald J.M.
      • Pan G.
      • Bland K.I.
      Tamoxifen (TMX)/Fas induced growth inhibition of human cholangiocarcinoma (HCC) by gamma interferon (IFN-gamma).
      • Murakami T.
      • Yano H.
      • Maruiwa M.
      • Sugihara S.
      • Kojiro M.
      Establishment and characterization of a human combined hepatocholangiocarcinoma cell line and its heterologous transplantation in nude mice.
      After that, the protocol could be continued either with or without enzymatic digestion. For the protocols without enzymatic digestion, a stainless-steel mesh is helpful for releasing CCA cells.
      • Yamaguchi N.
      • Morioka H.
      • Ohkura H.
      • Hirohashi S.
      • Kawai K.
      Establishment and characterization of the human cholangiocarcinoma cell line HChol-Y1 in a serum-free, chemically defined medium.
      Chemical digestion usually proceeds at 37°C, and the digestion time is dependent on the concentration and type of the enzyme used (Table 1). Several types of collagenases (types I, II, and IV) and trypsin have successfully been used for chemical digestion (Table 1). Plasminase to digest fibrin clumps and DNase I to lyse DNA leaked into cell suspension could be included in the dissociation protocol (Figure 1).
      • Wang S.
      [Establishment of extrahepatic cholangiocarcinoma cell line].
      After chemical dissociation, the cell suspension should be filtered with 70 μm of mesh and centrifuged
      • Murakami T.
      • Yano H.
      • Maruiwa M.
      • Sugihara S.
      • Kojiro M.
      Establishment and characterization of a human combined hepatocholangiocarcinoma cell line and its heterologous transplantation in nude mice.
      ; the pellet can then be resuspended in a variety of media conditions [RPMI, Dulbecco's modified Eagle medium, minimum essential medium, William's, and Ham's F12].
      • Homma S.
      • Nagamori S.
      • Fujise K.
      • Yamazaki K.
      • Hasumura S.
      • Sujino H.
      • Matsuura T.
      • Shimizu K.
      • Kameda H.
      • Takaki K.
      Human bile duct carcinoma cell line producing abundant mucin in vitro.
      • Okaro A.C.
      • Deery A.R.
      • Hutchins R.R.
      • Davidson B.R.
      The expression of antiapoptotic proteins Bcl-2, Bcl-X(L), and Mcl-1 in benign, dysplastic, and malignant biliary epithelium.
      • Yamaguchi N.
      • Morioka H.
      • Ohkura H.
      • Hirohashi S.
      • Kawai K.
      Establishment and characterization of the human cholangiocarcinoma cell line HChol-Y1 in a serum-free, chemically defined medium.
      ,
      • Murakami T.
      • Yano H.
      • Maruiwa M.
      • Sugihara S.
      • Kojiro M.
      Establishment and characterization of a human combined hepatocholangiocarcinoma cell line and its heterologous transplantation in nude mice.
      ,
      • Miyagiwa M.
      • Ichida T.
      • Tokiwa T.
      • Sato J.
      • Sasaki H.
      A new human cholangiocellular carcinoma cell line (HuCC-T1) producing carbohydrate antigen 19/9 in serum-free medium.
      Stromal fibroblasts are the primary contaminating cells in CCA cell cultures.
      • Vaquero J.
      • Aoudjehane L.
      • Fouassier L.
      Cancer-associated fibroblasts in cholangiocarcinoma.
      After cell attachment, CCA cells can be purified using scraping of the contaminating cells, colony isolation, and/or passaging cells until all contaminating cells have been removed (Figure 1).
      • Homma S.
      • Nagamori S.
      • Fujise K.
      • Yamazaki K.
      • Hasumura S.
      • Sujino H.
      • Matsuura T.
      • Shimizu K.
      • Kameda H.
      • Takaki K.
      Human bile duct carcinoma cell line producing abundant mucin in vitro.
      ,
      • Yamaguchi N.
      • Morioka H.
      • Ohkura H.
      • Hirohashi S.
      • Kawai K.
      Establishment and characterization of the human cholangiocarcinoma cell line HChol-Y1 in a serum-free, chemically defined medium.
      ,
      • Miyagiwa M.
      • Ichida T.
      • Tokiwa T.
      • Sato J.
      • Sasaki H.
      A new human cholangiocellular carcinoma cell line (HuCC-T1) producing carbohydrate antigen 19/9 in serum-free medium.

      Need for More CCA Cell Lines

      Cell lines provide indispensable in vitro model systems for the assessment of features of cancer cells, based on the properties that make the cells direct descendants of the primary tumors.
      • Mirabelli P.
      • Coppola L.
      • Salvatore M.
      Cancer cell lines are useful model systems for medical research.
      Under the right conditions, most of the phenotypic and genotypic properties of the primary tumor are preserved in cancer cell lines.
      • Masters J.R.
      Human cancer cell lines: fact and fantasy.
      These features have allowed the establishment of thousands of cell lines from various malignancies, of which only 70 to 80 belong to the CCA family after >50 years of research (Tables 1 and 2). While each new cell line has not necessarily been superior to previous lines, collectively, they have been important for the understanding of common and differing features of CCA, including: i) antigenic variations, ii) new genotypes, and iii) enzymes that sustain tumorigenic growth to drug sensitivities or resistances.
      One reason that CCA remains difficult to cure is the lack of a precise understanding of oncogenesis.
      • Zabron A.
      • Edwards R.J.
      • Khan S.A.
      The challenge of cholangiocarcinoma: dissecting the molecular mechanisms of an insidious cancer.
      The established CCA cell lines can be utilized to identify the properties of cells that can help in predicting positive or negative responses to antigen- or pathway-targeted therapies. CCA cell lines could be powerful in determining many aspects of CCA phenotypes, especially when used in a 3D microenvironment in the presence of other liver cells to study the impact of adjacent cells in the tumor milieu.
      • Sato K.
      • Zhang W.
      • Safarikia S.
      • Isidan A.
      • Chen A.M.
      • Li P.
      • Francis H.
      • Kennedy L.
      • Baiocchi L.
      • Alvaro D.
      • Glaser S.
      • Ekser B.
      • Alpini G.
      Organoids and spheroids as models for studying cholestatic liver injury and cholangiocarcinoma.
      The 3D tumor organoid system can be used for the identification of candidate novel therapies for future clinical trials.
      • Neve R.M.
      • Chin K.
      • Fridlyand J.
      • Yeh J.
      • Baehner F.L.
      • Fevr T.
      • Clark L.
      • Bayani N.
      • Coppe J.P.
      • Tong F.
      • Speed T.
      • Spellman P.T.
      • DeVries S.
      • Lapuk A.
      • Wang N.J.
      • Kuo W.L.
      • Stilwell J.L.
      • Pinkel D.
      • Albertson D.G.
      • Waldman F.M.
      • McCormick F.
      • Dickson R.B.
      • Johnson M.D.
      • Lippman M.
      • Ethier S.
      • Gazdar A.
      • Gray J.W.
      A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes.
      In a recent study, an established IHCCA cell line was used for understanding the impact of RA190, a proteasome subunit ADRM1 inhibitor that induces cell apoptosis, in a pathway-targeted therapy model.
      • Yu G.Y.
      • Wang X.
      • Zheng S.S.
      • Gao X.M.
      • Jia Q.A.
      • Zhu W.W.
      • Lu L.
      • Jia H.L.
      • Chen J.H.
      • Dong Q.Z.
      • Lu M.
      • Qin L.X.
      RA190, a proteasome subunit ADRM1 inhibitor, suppresses intrahepatic cholangiocarcinoma by inducing NF-KB-mediated cell apoptosis.
      Although the outcomes of that in vitro study were encouraging, the study did not evaluate the roles of the tumor microenvironment and other CCA cell lines to determine whether a genotypic difference within the CCA lines may have modulated (increased or decreased) the response and sensitivity to RA190.
      • Yu G.Y.
      • Wang X.
      • Zheng S.S.
      • Gao X.M.
      • Jia Q.A.
      • Zhu W.W.
      • Lu L.
      • Jia H.L.
      • Chen J.H.
      • Dong Q.Z.
      • Lu M.
      • Qin L.X.
      RA190, a proteasome subunit ADRM1 inhibitor, suppresses intrahepatic cholangiocarcinoma by inducing NF-KB-mediated cell apoptosis.
      A counterexample is evidenced by the recent discovery in Italy of a novel IHCCA cell line characterized by the presence of genes encoding resistance to fluorouracil, carboplatin, and oxaliplatin—drugs actively and frequently used in CCA treatment.
      • Peraldo-Neia C.
      • Massa A.
      • Vita F.
      • Basiricò M.
      • Raggi C.
      • Bernabei P.
      • Ostano P.
      • Casorzo L.
      • Panero M.
      • Leone F.
      • Cavalloni G.
      • Aglietta M.
      A novel multidrug-resistant cell line from an Italian intrahepatic cholangiocarcinoma patient.

      Future Perspectives

      The establishment of new, well-defined and well-characterized CCA cell lines is crucial in enhancing the understanding of this aggressive disease. The establishment of oncogenesis through the cancer cells forms their existence, the drug-sensitivity and -resistance mechanism, and helps to identify possible novel drug targets. The inclusion of tumor stromal cells, such as hepatic stellate cells, liver sinusoidal endothelial cells, and tumor-associated macrophages, in the CCA cell lines makes these culture models more representative of the CCA tumor microenvironment (ie, 3D tumor organoids)
      • Sato K.
      • Zhang W.
      • Safarikia S.
      • Isidan A.
      • Chen A.M.
      • Li P.
      • Francis H.
      • Kennedy L.
      • Baiocchi L.
      • Alvaro D.
      • Glaser S.
      • Ekser B.
      • Alpini G.
      Organoids and spheroids as models for studying cholestatic liver injury and cholangiocarcinoma.
      (Figure 2). Therefore, the inclusion of multiple other cell types will help to determine the ways in which CCA cells interact with other neighboring cells and alter their genotypes, phenotypes, and transcriptomic profiles.
      Figure thumbnail gr2
      Figure 2CCA organoids with multiple cell lines. Three-dimensional (3D) CCA organoids include multiple cell lines, such as CCA cell lines (cholangiocytes; CHOL), liver sinusoidal endothelial cells (LSECs), and hepatic stellate cells (HSCs) (representative). Other liver cells, such as hepatocytes and Kupffer cells, can also be added, if needed. These organoids can be made scaffold-free without any biomaterial (eg, Matrigel) in a low-binding plate using culture mediums and can be kept for 14 to 28 days. There is good evidence that CCA cells express cytokeratin (CK)-7, a cholangiocyte marker, and organoids can be stained with other cellular markers (eg, vascular endothelial cadherin; VE-Cad) that are present in organoids. CCA organoids can be exposed to immune cells. An example is seen at the bottom right. Bright-field microscopy shows a 3D CCA organoid exposed to mast cells. Scale bar = 500 μm. Original magnification, ×10.
      The establishment of well-characterized CCA cell lines with a close resemblance to primary tumors, especially in a 3D microenvironment, will provide an infinite capacity for replicability, limiting the use of small animal in vivo studies and making them prime materials for CCA research.

      Author Contributions

      A.I. drafted the manuscript. A.Y., D.S., and E.A. helped in the writing and reviewing the manuscript. B.E. developed the concept and helped in the writing and critically reviewing the manuscript. All of the authors critically reviewed and participated in the writing of the manuscript, and approved the final version.

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