Establishment of Biliary Epithelial Cell Lines from the Hamster

  • Takayuki Asakawa
  • Amane Kitasato
  • Tsutomu Tomioka
  • Tamotsu Kuroki
  • Ryuji Tsutsumi
  • Yoshitsugu Tajima
  • Takashi Kanematsu

We describe a method for the simultaneous culturing of biliary epithelial cells (BECs) from the gallbladder (GB), extrahepatic bile duct (EBD), and intrahepatic bile duct (IBD) of hamsters. The GB, EBD and IBD were excised from the biliary tree after collagenase perfusion of the liver. These biliary segments were minced into fragments, which were embedded in collagen gel and cultured in Dulbeccos Modified Eagle Medium/HamF12 Medium containing 10% fetal bovine serum. The various cells subsequently spread from the fragments and formed cellular sheets. After the fragments and flattened cells were removed under phase-contrast microscopy, the sheets remaining were found to be composed of cuboidal cells. These cuboidal cells expressed gamma glutamyl transpeptidase and cytokeratin 7, which are known to be specific markers of BECs. Ultrastructurally, there were many microvilli on the luminal surface and junctional complex and interdigitation was identifiable on the lateral surfaces. BEC cultures were subcultured by digestion with collagenase and dispase and then dissociated by subsequent digestion in trypsin and ethylenediaminetetraacetic acid. They were maintained in collagen gel for up to 8 weeks. After several passages, the BECs in the culture eventually grew and showed vacuoles in the cytoplasm. They demonstrated irreversible growth arrest at 9 weeks. The BECs tended to form cystic structures when they were transplanted with collagen gel into the interscapular fat pads of the syngeneic hamsters.


Nitric Oxide Comet Assay Biliary Tree Extrahepatic Bile Duct Intrahepatic Bile Duct 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Elsing C., Kassner A., Hubner C., Buhl H., Stremmel W. Absorptive and secretory mechanisms in biliary epithelial cells. J Hepatol 1996 24:121–127.PubMedGoogle Scholar
  2. 2.
    Nakanuma Y., Sasaki M. Expression of blood group-related antigens in the intrahepatic biliary tree and hepatocytes in normal livers and various hepatobiliary diseases. Hepatology 1989 10:174–178.PubMedCrossRefGoogle Scholar
  3. 3.
    Nakanuma Y., Ohta G. Histometric and serial section observations of the intrahepatic bile ducts in primary biliary cirrhosis. Gastroenterology 1979 76:1326–1332.PubMedGoogle Scholar
  4. 4.
    Auth M.K., Keitzer R.A., Scholz M., Blaheta R.A., Hottenrott E.C., Herrmann G., Encke A., Markus B. Establishment and immunological characterization of the cultured human gallbladder epithelial cells. Hepatology 1993 18:546–555.PubMedCrossRefGoogle Scholar
  5. 5.
    Strain A.J., Wallace L., Joplin R., Daikuhara Y., Ishii T., Kelly D.A., Neuberger J.M. Characterization of biliary epithelial cells isolated from needle biopsies of human liver in the presence of hepatocyte growth factor. Am J Pathol 1995 146:537–545.PubMedGoogle Scholar
  6. 6.
    Katayanagi K., Kono N., Nakanuma Y. Isolation, culture and characterization of biliary epithelial cells from different anatomical levels of the intrahepatic and extrahepatic biliary tree from a mouse. Liver 1998 18:90–98.PubMedGoogle Scholar
  7. 7.
    Mathis G.A., Walla S.A., Sirica A.E. Biochemical characteristics of hyperplastic rats bile ductular epithelial cells cultured “on top” and “inside” different extracellular matrix substitutes. Cancer Res 1988 48:6145–6153.PubMedGoogle Scholar
  8. 8.
    Paradis K., Sharp H.L. In vitro duct-like structure formation after isolation of the bile ductular cells from murine model. J Lab Clin Med 1989 113:689–694.PubMedGoogle Scholar
  9. 9.
    Yang L., Faris R.A., Hixson D.C. Long-term culture and characteristics of normal rat liver bile duct epithelial cells. Gastroenterology 1993 104:840–852.PubMedGoogle Scholar
  10. 10.
    Takahashi M., Pour P., Althoff J., Donnelly T. The panceras of the Syrian hamster (Mesocricetus auratus). 1 Anatomical study. Lab Anim Sci 1977 27: 336–342.Google Scholar
  11. 11.
    Peralman B.J., Bonorris G.G., Philips M.J., Chung A., Vimadalal S., Marks J.W., Schoenfield L.J. Cholesterol gallstone formation and prevention by chenodeoxycholic and ursodeoxycholic acids. A new hamster model. Gastroenterology 1979 77:634–641.Google Scholar
  12. 12.
    Rinderknecht H., Maset R., Collias K., Carmack C. Panceratic secretory profiles of protein, digestive, and lysosomal enzymes in Syrian golden hamster. Effect of secretin and cholecystokinin. Dig Dis Sci 1983 28:518–525.PubMedCrossRefGoogle Scholar
  13. 13.
    Fukahori T., Tomioka T., Inoue K., Tajima Y., Tsunoda T., Kanematsu T. Establishment of a transplantable carcinoma arising from the intrahepatic bile duct in Syrian golden hamster. Virchows Arch A Pathol Anat 1993 422:233–238.CrossRefGoogle Scholar
  14. 14.
    Ikematsu Y., Tomioka T., Tajima Y., Tsunoda T., Kanematsu T. Enhancement of biliary carcinogenesis in hamsters by cholecystokinin. World J Surg 1995 19:847–851.PubMedCrossRefGoogle Scholar
  15. 15.
    Ikematsu Y., Tomioka T., Yamanaka S., Tajima Y., Tsunoda T., Kanematsu T. Bilioenterostomy enhances biliary carcinogenesis in hamsters. Carcinogenesis 1996 17:1505–1509.PubMedCrossRefGoogle Scholar
  16. 16.
    Inoue K., Tomioka T., Tajima Y., Fukahori T., Eto T., Tsunoda T., Kanematsu T. Characterization of an established transplantable adenocarcinoma of the gallbladder in Syrian golden hamster. J Surg Oncol 1994 56:269–276.PubMedCrossRefGoogle Scholar
  17. 17.
    Tajima Y., Eto T., Tsunoda T., Tomioka T., Inoue K., Fukahori T., Kanematsu T. Induction of extrahepatic biliary carcinoma by N-nitrosobis(2-oxopropyl)amine in hamsters given cholecystoduodenostomy with dissection of the common duct. Jpn J Cancer Res 1994 85:780–788.PubMedGoogle Scholar
  18. 18.
    Yamanaka S., Tomioka T., Tajima Y., Okada K., Shiku H., Kanematsu T. K-ras gene mutations in intrahepatic bile duct tumors of Syrian golden hamsters. J Surg Oncol 1997 66:97–103.PubMedCrossRefGoogle Scholar
  19. 19.
    Rutenburg A.M., Kim H., Fishbein J.W., Hander J.S., Wasserkrug H.L., Seligman A.M. Histochemical and ultrastructural demonstration of E-glutamyl transpeptidase activity. J Histochem Cytochem 1969 17:517–526PubMedGoogle Scholar
  20. 20.
    Hall P.A., Lemoine N.R. Models of pancreatic cancer. Cancer Surv 1993 16:135–155.PubMedGoogle Scholar
  21. 21.
    Mangold K.A., Hubchak S., Mangino M.M., Laconi S., Scarpelli D.G. In vitro carcinogenesis of hamster pancreatic duct cells: cellular and molecular alterations. Carcinogenesis 1994 15(9):1979–1984.PubMedCrossRefGoogle Scholar
  22. 22.
    Takeuchi Y., Takahashi M., Sakano K., Mutoh M., Niho N., Yamamoto M., Sato H., Sugimura T., Wakabayashi K. Suppression of N-nitrosobis(2-oxopropyl)amine-induced pancreatic carcinogenesis in hamsters by pioglitazone, a ligand of peroxisome proliferatior-activated receptor gamma. Carcinogenesis 2007 28(8):1692–1696.PubMedCrossRefGoogle Scholar
  23. 23.
    Hubchak S., Mangino M.M., Reddy M.K., Scarpelli D.G. Characterization of differentiated Syrian golden hamster pancreatic duct cells maintained in extended monolayer culture. In Vitro Cell Dev Biol 1990 26(9):889–897.PubMedCrossRefGoogle Scholar
  24. 24.
    Sirica A.E., Mathis G.A., Sano N., Elmore L.W. Isolation, culture, and transplantation of intrahepatic biliary epithelial cells and oval cells. Pathobiology 1990 58(1):44–64.PubMedCrossRefGoogle Scholar
  25. 25.
    De Lü M., Miyazaki K., Yoshitomi S., Nakayama F. DNA repair synthesis in primary culture of bovine bile duct epithelial cells induced by chemical agents in relation to bile duct cancer. Mutat Res 1988 194(1):73–79.PubMedGoogle Scholar
  26. 26.
    Ohshima H., Bartsh H. Chronic infections and inflammatory process as cancer risk factors: possible role of nitric oxide in carcinogenesis. Mutat Res 1994 305:253–264.PubMedGoogle Scholar
  27. 27.
    Fitzpatrick F.A. Inflammation, carcinogenesis and cancer. Int Immunopharmacol 2001 1:1651–1667.PubMedCrossRefGoogle Scholar
  28. 28.
    Baumann R., Uettwiller H., Duclos B., Jouin H., Kerschen A., Adloff M., Weill J.P. Congenital cystic dilatation of the common bile duct, anomaly of the biliopancreatic junction and cancer of the bile ducts. Gastroenterol Clin Biol 1987 11:849–855.PubMedGoogle Scholar
  29. 29.
    Fujii H., Yang Y., Tang R., Kunitomo K., Itakura J., Mogaki M., Matsuda M., Suda K., Nobukawa B., Matsumoto Y. Epithelial cell proliferation activity of the biliary ductal system with congenital biliary malformations. J Hepatobiliary Pancr Surg 1999 6:294–302.CrossRefGoogle Scholar
  30. 30.
    Tocchi A., Mazzoni G., Liotta G., Lepre L., Cassini D., Miccini M. Late development of bile duct cancer in patients who had biliary-enteric drainage for benign disease: a follow up study of more than 1,000 patients. Ann Surg. 2001 234:210–214.PubMedCrossRefGoogle Scholar
  31. 31.
    Hakamada K., Sasaki M., Endoh M., Itoh T., Morita T., Konn M. Late development of bile duct cancer after sphincteroplasty: a ten- to twenty-two-year follow up study. Surgery 1997 121:488–492.PubMedCrossRefGoogle Scholar
  32. 32.
    Tanaka M., Takahata S., Konomi H., Matsunaga H., Yokohata K., Takeda T., Utsunomia N., Ikeda S. Long-term consequence of endoscopic sphincterotomy for bile duct stones. Gastrointest Endosc 1998 48:465–469.PubMedCrossRefGoogle Scholar
  33. 33.
    Kitajima T., Tajima Y., Onizuka S., Matsuzaki S., Matsuo K., Kanematsu T. Linkage of persistent cholangitis after bilioenterostomy with biliary carcinogenesis in hamsters. J Exp Clin Cancer Res 2000 19:453–458.PubMedGoogle Scholar
  34. 34.
    Kitajima T., Tajima Y., Matsuzaki S., Kuroki T., Fukuda K., Kanematsu T. Acceleration of spontaneous biliary carcinogenesis in hamsters by bilioenterostomy. Carcinogenesis 2003 24:133–137.PubMedCrossRefGoogle Scholar
  35. 35.
    Asakawa T., Tomioka T., Kanematu T. A method for culturing and transplanting biliary epithelial cell from Syrian golden hamster. Virchows Arch 2000 436:140–146.PubMedCrossRefGoogle Scholar
  36. 36.
    Facchetti F., Vermi W., Fiorentini S., Chilosi M., Caruso A., Duse M., Notarangelo L.D., Badolato R. Expression of inducible nitric oxide synthase in human granulomas and histiocytic reactions. Am J Pathol 1999 154:145–152.PubMedGoogle Scholar
  37. 37.
    Geller D.A., Billiar T.R. Molecular biology of nitric oxide synthases. Cancer Metastasis Rev 1998 17:7–23.PubMedCrossRefGoogle Scholar
  38. 38.
    Marletta M. Nitric oxide synthase: aspects concerning structure and catalysis. Cell 1994 78:927–930.PubMedCrossRefGoogle Scholar
  39. 39.
    Michel T., Feron O. Perspective series: Nitric oxide and nitric oxide synthases. J Clin Invest 1997 100:2146–2152.PubMedCrossRefGoogle Scholar
  40. 40.
    Mannick J.B., Asano K., Izumi K., Kieff E., Stamler J.S. Nitric oxide produced by human B lymphocytes inhibits apoptosis and Epstein-Barr virus reactivation. Cell 1994 79:1137–1146.PubMedCrossRefGoogle Scholar
  41. 41.
    Hoffman R.A., Zhang G.S., Nussler N.C., Gleixner S.L., Ford H.R., Simmons R.L., Watkins S.C. Constitutive expression of inducible nitric oxide synthase in the mouse ileal mucosa. Am J Phys 1997 35:G383–G392.Google Scholar
  42. 42.
    Geller D.A., Nussler A.K., Di Silvio M.A., Lowenstein C.J., Shapiro R., Wang S.C., Simmons R.L., Billiar T.R. Cytokines, endotoxin and glicocorticoids regulate the expression of inducible nitric oxide synthase in hepatocytes. Proc Natl Acad Sci U S A 1992 90:522–526.CrossRefGoogle Scholar
  43. 43.
    Geller D.A., Di Silvio M., Nussler A.K., Wang S.C., Shapiro R.A., Simmons M.D., Billiar T.R. Nitric oxide synthase expression is induced in hepatocytes in vivo during hepatic inflammation. J Surg Res 1993 55:427–432.PubMedCrossRefGoogle Scholar
  44. 44.
    Tamir S., Burney S., Tannenbaum S.R. DNA damage by nitric oxide. Chem Res Toxicol 1996 9(5):821–827.PubMedCrossRefGoogle Scholar
  45. 45.
    Lachances S., Chan J.S. Nitric oxide co-operates with hydrogen peroxide in inducing DNA fragmentation and cell lysis in murine lymphoma cells. Biochem J 1997 321:897–901.Google Scholar
  46. 46.
    Nussler A.K., Geller D.A., Aweetland M.A., Di Silvio M., Billiar T.R., Madariaga J.B., Simmons R.L., Lancaster J.R. Induction of nitric oxide synthesis and its reactions in cultured human and rat hepatocytes stimulated with cytokines plus lipopolysaccharides. Biochem Biophys Res Commun 1993 194:820–835.CrossRefGoogle Scholar
  47. 47.
    Archer S. Measurement of nitric oxide in biological models. FASEB J 1993 7:349–360.PubMedGoogle Scholar
  48. 48.
    Ramirez-Emiliano J., Gonzalez-Hernandes A., Arias-Negrete S. Expression of inducible nitric oxide synthase mRNA and nitric oxide production during the development of liver abscess in hamster inoculated with Entamoeba histolytica. Curr Microbiol 2005 50:299–308.PubMedCrossRefGoogle Scholar
  49. 49.
    Cox R., Mariano T., Heck D., Laskin J., Stegeman J. Nitric oxide synthase sequences in the marine fish Stenotomus chrysops and the sea urchin Arbacia punctulata, and phylogenetic analysis of nitric oxide synthase calmodulin-binding domains. Comp Biochem Physiol B Biochem Mol Biol 2001 130:479–49.PubMedCrossRefGoogle Scholar
  50. 50.
    Liu S., Adcock I., Old R., Barnes P., Evans T. Lipopolysaccharide treatment in vivo induces widespread tissue expression of inducible nitric oxide synthase mRNA. Biochem Biophys Res Commun 1993 196:1208–1213.PubMedCrossRefGoogle Scholar
  51. 51.
    Jaiswal M., LaRusso N.F., Burgart L.J., Gores G.J. Inflammatory cytokines induce DNA damage and inhibit DNA repair in cholangiocarcinoma cells by nitric oxide-dependent mechanism. Cancer Res 2000 60:184–190.PubMedGoogle Scholar
  52. 52.
    Duthie S.J., McMillian P. Uracil misincorporation in human DNA detected using single cell gel electrophoresis. Carcinogenesis 1997 18:1709–1714.PubMedCrossRefGoogle Scholar
  53. 53.
    Helma C., Uhl M. A public domain image-analysis program for the single-cell gel-electrophoresis (comet) assay. Mutat Res 2000 466:9–15.PubMedGoogle Scholar
  54. 54.
    Szabo C., Oshima H. DNA damage induced by peroxynitrite: subsequent biological effects. Nitric Oxide Biol Chem 1997 1:373–385.CrossRefGoogle Scholar
  55. 55.
    Ambs S., Bennett W.P., Merriam W.G., Ogunfusika M.O., Oser S.M., Harrington A.M., Shields P.G., Felly-Bosco E., Hussain S.P., Harris C.C. Relationship between p53 mutations and inducible nitric oxide synthase expression in human colorectal cancer. J Natl Cancer Inst 1999 91:86–88.PubMedCrossRefGoogle Scholar
  56. 56.
    Murakami A., Ohigashi H. Targeting NOX, INOS and COX-2 in inflammatory cells: chemoprevention using food phytochemicals. Int J Cancer 2007 121(11):2357–2363.PubMedCrossRefGoogle Scholar
  57. 57.
    Jaiswal M., LaRusso N.F., Shapiro R.A., Billiar T.R., Gores G.J. Nitric oxide-mediated inhibition of DNA repair potentiates oxidative DNA damage in cholangiocytes. Gastroenterology 2001 120:190–199.PubMedCrossRefGoogle Scholar
  58. 58.
    Perez-Sala D., Rebollo A. Novel aspects of Ras proteins biology: regulation and implications. Cell Death Differ 1999 6:722–728.PubMedCrossRefGoogle Scholar
  59. 59.
    Garcia-Cardena G., Folkman J. Is there a role for nitric oxide in tumor angiogenesis? J Natl Cancer Inst 1998 90:560–561.PubMedCrossRefGoogle Scholar
  60. 60.
    Wood R.D., Mitchell M., Sgouros J., Lindahl T. Human DNA repair genes. Science 2001 291(5507):1284–1289.PubMedCrossRefGoogle Scholar
  61. 61.
    Ambs S., Hussain P., Harris C.C. Interactive effects of nitric oxide and the p53 tumor suppressor gene in carcinogenesis and tumor progression. FASEB J 1997 11:443–448.PubMedGoogle Scholar
  62. 62.
    Calmels S., Hainaut P., Oshima H. Nitric oxide induces conformational and functional modifications of wild-type p53 tumor suppressor protein. Cancer Res 1997 57:3365–3369.PubMedGoogle Scholar
  63. 63.
    Kitasato A, Tajima Y, Kuroki T, Tsutsumi R, Adachi T, Mishima T, Kanematsu T. Inflammatory cytokines promote inducible nitric oxide synthase-mediated DNA damage in hamster gallbladder epithelial cells. World J Gastroenterol 2007 13(47):6379–84.PubMedCrossRefGoogle Scholar

Copyright information

© Springer 2009

Authors and Affiliations

  • Takayuki Asakawa
    • 1
  • Amane Kitasato
    • 2
  • Tsutomu Tomioka
    • 3
  • Tamotsu Kuroki
    • Ryuji Tsutsumi
      • 4
    • Yoshitsugu Tajima
      • Takashi Kanematsu
        1. 1.Department of SurgeryNational Hospital Organization Saga National HospitlSagaJapan
        2. 2.Department of SurgeryNational Hospital Organization Nagasaki Medical CenterNagasakiJapan
        3. 3.Department of SurgeryNagasaki Yurino HospitalNishisonogiJapan
        4. 4.Department of SurgeryNagasaki Prefectural Shimabara HospitalShimabaraJapan

        Personalised recommendations