Skip to main content

Culture and Immortalization of Pancreatic Ductal Epithelial Cells

  • Protocol
Pancreatic Cancer

Part of the book series: Methods in Molecular Medicineā„¢ ((MIMM,volume 103))

Abstract

Some populations of the epithelial cells from the duct and ductular network of the mammalian pancreas have been isolated and maintained in vitro for up to 3 mo. These cells express many of the surface factors that are unique to them in vivo. They also retain significant drug-and carcinogen-metabolizing capacity in vitro. In this chapter we review the progression of the methods for the isolation, culture and maintenance in vitro for these cells from the earliest when only duct/ductular fragments were obtainable to the current ones which provide cells. The critical steps in the isolation process are identified and strategies are provided to facilitate these steps. These include the selection of tissue digestive enzymes, the importance of extensive mincing before culture and the importance of roles of some co-factors used in the culture medium.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Jones, R. T., Hudson, E. A., and Resau, J. H. (1981) A review of in vitro and in vivo culture techniques for the study of pancreatic carcinogenesis. Cancer 47, 1490ā€“1496.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  2. Githens, S., Holmquist, D. R., Whelan, J. F., and Ruby, J. R. (1981) Morphologic and biochemical characteristics of isolated and cultured pancreatic ducts. Cancer 47, 1505ā€“1512.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  3. Githens, S. and Whelan, J. F. (1983) Isolation and culture of hamster pancreatic ducts. J. Tissue Cult. Methods 8, 97ā€“103.

    ArticleĀ  Google ScholarĀ 

  4. Richards, J., Pasco, D., Yang, J., Guzman, R., and Nandi, S. (1983) Comparison of the growth of normal and neoplastic mouse mammary cells on plastic, on collagen gels and in collagen gels. Exp. Cell Res. 146, 1ā€“14.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  5. Hootman, S. R. and Logsdon, C. D. (1998) Isolation and monolayer culture of guinea pig pancreatic duct epithelial cells. In Vitro 24, 566ā€“574.

    Google ScholarĀ 

  6. Lefebvre, V. H., Otonkoski, T., Ustinov, J., Huotari, M. A., Pipeleers, D. G, and Bouwens, L. (1998) Culture of adult human islet preparations with hepatocyte growth factor and 804G matrix is mitogenic for duct cells but not for beta cells. Diabetes 47, 134ā€“137.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  7. Langhofer, M., Hopkinson, S. B., and Jones, J. C. R. (1993) The matrix secreted by 804G cells contains laminin-related components that participate in hemidesmosome assembly in vitro. J. Cell Sci. 105, 753ā€“764.

    PubMedĀ  CASĀ  Google ScholarĀ 

  8. Parsa, I., Marsh, W. H., and Sutton, A. L. (1980) An in vitro model of pancreas carcinogenesis. Am. J. Pathol. 98, 649ā€“662.

    PubMedĀ  CASĀ  Google ScholarĀ 

  9. Parsa, I., Marsh, W. H., Sutton, A. L., and Butt, K. M. H. (1981) Effects of dimethylnitrosamine on organ-cultured adult human pancreas. Am. J. Pathol. 102, 403ā€“411.

    PubMedĀ  CASĀ  Google ScholarĀ 

  10. Resau, J. H., Cottrell, J. R., Elligett, K. A., and Hudson, E. A. (1987) Cell injury and regeneration of human epithelium in organ culture. Cell Biol. Toxicol. 3, 441ā€“458.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  11. Harris, A. and Coleman, L. (1988) Cultured epithelial cells derived from foetal pancreas as a model for the study of cystic fibrosis: Further analysis on the origins and nature of the cell types. J. Cell Sci. 90, 73ā€“77.

    PubMedĀ  Google ScholarĀ 

  12. Harris, A., Chalkley, G., Goodman, S., and Coleman, L. (1991) Expression of the cystic fibrosis gene in human development. Development 113, 305ā€“310.

    PubMedĀ  CASĀ  Google ScholarĀ 

  13. Githens, S., Patke, C. L., and Schexnayder, J. A. (1994) Isolation and culture of rhesus monkey pancreatic ductules ductule-like epithelium. Pancreas 9, 20ā€“31.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  14. Verme, T. B. and Hootman, S. R. (1990) Regulation of pancreatic duct epithelial growth in vitro. Am. J. Physiol. 258, G833ā€“G840.

    PubMedĀ  CASĀ  Google ScholarĀ 

  15. Vila, M. R., Lloreta, J., Schussler, M. H., Berrozpe, G, Welt, S., and Real, F. X. (1995) New pancreas cancers cell lines that represent distinct stages of ductal differentiation. Lab. Invest. 72, 395ā€“404.

    PubMedĀ  CASĀ  Google ScholarĀ 

  16. Chambers, J. A. and Harris, A. (1993) Expression of the cystic fibrosis gene and the major pancreatic mucin gene, MUC1, in human ductal epithelial cells. J. Cell Sci. 105, 417ā€“422.

    PubMedĀ  CASĀ  Google ScholarĀ 

  17. Kolar, C, Caffrey, T., Hollingsworth, M., et al. (1997) Duct epithelial cells cultured from human pancreas processed for transplantation retain differentiated ductal characteristics. Pancreas 15, 265ā€“271.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  18. Kerr-Conte, J., Pattou, F., Lecomte-Houcke, M., et al. (1996) Ductal cyst formation in collagen-embedded adult human islet preparations. A means to the reproduction of nesidioblastosis in vitro. Diabetes 45, 1108ā€“1114.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  19. Trautmann, B., Schlitt, H.-J., Hahn, E. G, and Lohr, M. (1993) Isolation, culture, and characterization of human pancreatic duct cells. Pancreas 8, 248ā€“254.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  20. Ryu, B., Jones, J., Hollingsworth, M. A., Hruban, R. H., and Kern, S. E. (2001) Invasion-specific genes in malignancy: Serial analysis of gene expression comparisons of primary and passaged cancers. Cancer Res. 61, 1833ā€“1838.

    PubMedĀ  CASĀ  Google ScholarĀ 

  21. Harris, A. and Coleman, L. (1987) Establishment of a tissue culture system for epithelial cells derived from human pancreas: A model for the study of cystic fibrosis. J. Cell Sci. 87, 695ā€“703.

    PubMedĀ  Google ScholarĀ 

  22. Singer, K. H., Scearce, R. M., Tuck, D. T., Whichard, L. P., Denning, S. M., and Haynes, B. F. (1989) Removal of fibroblasts form human epithelial cell cultures with use of a complement fixing monoclonal antibody reactive with human fibroblasts and monocytes/macrophages. J. Invest. Dermatol. 92, 166ā€“170.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  23. Harris, C. C, Autrup, H., Stoner, G, et al. (1977) Metabolism of benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene in cultured human bronchus and pancreatic duct. Cancer Res. 37, 3349ā€“3355.

    PubMedĀ  CASĀ  Google ScholarĀ 

  24. Jones, R. T., Barrett, L. A., van Haaften, C, Harris, C. C, and Trump, B. F. (1977) Carcinogenesis in the pancreas. I. Long-term explant culture of human and bovine pancreatic ducts. J. Natl. Cancer Inst. 58, 557ā€“565.

    PubMedĀ  CASĀ  Google ScholarĀ 

  25. Lawson, T. and Kolar, C. (1994) Mutagenicity of heterocyclic amines when activated by pancreas tissue. Mutation Res. 325, 125ā€“128.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  26. Hyde, K, Harrison, D., Hollingsworth, M. A., and Harris, A. (1999) Chloride-bicarbonate exchangers in human fetal pancreas. Biochem. Biophys. Res. Commun. 263, 315ā€“321.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  27. de Lisle, R. C. and Logsdon, C. D. (1990) Pancreatic acinar cells in culture: Expression of acinar and ductal antigens in a growth-related manner. Eur. J. Cell Biol. 51, 64ā€“75.

    PubMedĀ  Google ScholarĀ 

  28. Hall, P. A. and Lemoine, N. R. (1993) Rapid acinar to ductal transdifferentiation in cultured human exocrine pancreas. J. Pathol. 166, 97ā€“103.

    ArticleĀ  Google ScholarĀ 

  29. VilĆ”, M. R., Lloreta, J., and Real, F. X. (1994) Normal human pancreas cultures display functional ductal characteristics. Lab. Invest. 71, 423ā€“431.

    PubMedĀ  Google ScholarĀ 

  30. Yuan, S., Rosenberg, L., Paraskevas, S., Agapitos, D., and Duguid, W. P. (1996) Transdifferentiation of human islets to pancreatic ductal cells in collagen matrix culture. Differentiation 61, 67ā€“75.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  31. Paddenberg, R., Flocke, K., Elsasser, H. P., Lesch, G., Heidtmann, H. H., and Mannherz, H. G. (1998) Phenotypical changes of a human pancreatic adenocarcinoma cell line after selection on laminin-1/nidogen (LM/Ng) substratum. Eur. J. Cell Biol. 76, 51ā€“64.

    Google ScholarĀ 

  32. Bonner-Weir, S., Taneja, M., Weir, G C, et al. (2000) In vitro cultivation of human islets from expanded ductal tissue. Proc. Natl. Acad. Sci. USA 97, 7999ā€“8004.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  33. Gmyr, V., Kerr-Conte, J., Vanderwalle, B., Proye, C, Lefebvre, J., and Pattou, F. (2001) Human pancreatic ductal cells: Large scale isolation and expansion. Cell Transplant. 10, 109ā€“121.

    PubMedĀ  CASĀ  Google ScholarĀ 

  34. Gmyr, V., Kerr-Conte, J., Belaich, S., et al. (2000) Adult human cytokeratin 19-positive cells reexpress insulin promoter factor 1 in vitro: Further evidence for pluripotent pancreatic stem cells in humans. Diabetes 49, 1671ā€“1680.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  35. Ramiya, V. K., Maraist, M., Arfors, K. E., Schatz, D. A., Peck, S. B., and Cornelius, J. G (2000) Reversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells. Nat. Med. 6, 278ā€“282.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  36. Ouellette, M. M. and Lee, K. (2001) Telomerase: Diagnostics, cancer therapeutics and tissue engineering. Drug Discov. Today 6, 1231ā€“1237.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  37. Bodnar, A. G., Ouellette, M., Frolkis, M., et al. (1998) Extension of life-span by introduction of telomerase into normal human cells. Science 279, 349ā€“352.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  38. Morales, C. P., Holt, S. E., Ouellette, M., et al. (1999) Absence of cancer-associated changes in human fibroblasts immortalized with telomerase. Nat. Genet. 21, 115ā€“118.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  39. Jiang, X. R., Jimenez, G, Chang, E., et al. (1999) Telomerase expression in human somatic cells does not induce changes associated with a transformed phenotype. Nat. Genet. 21, 111ā€“114.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  40. Shay, J. W. and Wright, W. E. (2001) Aging. When do telomeres matter? Science 291, 839ā€“840.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  41. Furukawa, T., Duguid, W. P., Rosenberg, L., Viallet, J., Galloway, D. A., and Tsao, M. S. (1996) Long-term culture and immortalization of epithelial cells from normal adult human pancreatic ducts transfected by the E6E7 gene of human papilloma virus 16. Am. J. Pathol. 148, 1763ā€“1770.

    PubMedĀ  CASĀ  Google ScholarĀ 

  42. Ouyang, H., Mou, L. J., Luk, C, et al. (2000) Immortal human pancreatic duct epithelial cell lines with near normal genotype and phenotype. Am. J. Pathol. 157, 1623ā€“1631.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  43. Jesnowski, R., Muller, P., Schareck, W., Liebe, S., and Lohr, M. (1999) Immortalized pancreatic duct cells in vitro and in vivo. Ann. NY Acad. Sci. 880, 50ā€“65.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  44. Halbert, C. L., Demers, G. W., and Galloway, D. A. (1992) The E6 and E7 genes of human papillomavirus type 6 have weak immortalizing activity in human epithelial cells. J. Virol. 66, 2125ā€“2134.

    PubMedĀ  CASĀ  Google ScholarĀ 

  45. Ouellette, M. M., Aisner, D. L., Savre-Train, I., Wright, W. E., and Shay, J. W. (1999) Telomerase activity does not always imply telomere maintenance. Biochem. Biophys. Res. Commun. 254, 795ā€“803.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  46. Counter, C. M., Hahn, W. C, Wei, W., et al. (1998) Dissociation among in vitro telomerase activity, telomere maintenance, and cellular immortalization. Proc. Natl. Acad. Sci. USA 95, 14723ā€“14728.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

Ā© 2005 Humana Press Inc.

About this protocol

Cite this protocol

Lawson, T., Ouellette, M., Kolar, C., Hollingsworth, M. (2005). Culture and Immortalization of Pancreatic Ductal Epithelial Cells. In: Su, G.H. (eds) Pancreatic Cancer. Methods in Molecular Medicineā„¢, vol 103. Humana Press. https://doi.org/10.1385/1-59259-780-7:113

Download citation

  • DOI: https://doi.org/10.1385/1-59259-780-7:113

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-107-3

  • Online ISBN: 978-1-59259-780-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics