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Primary Epithelial Cell Models for Cystic Fibrosis Research

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Cystic Fibrosis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 742))

Abstract

When primary human airway epithelial (hAE) cells are grown in vitro on porous supports at an air–liquid interface (ALI), they recapitulate in vivo morphology and key physiologic processes. These cultures are useful for studying respiratory tract biology and diseases and for testing new cystic fibrosis (CF) therapies. This chapter gives protocols enabling creation of well-differentiated primary CF and non-CF airway epithelial cell cultures with non-proprietary reagents. We also discuss the production of retroviral and lentiviral vectors, the derivation of hAE cell lines, reporter gene assays, and the evolving science of gene overexpression and knockdown in ALI hAE cultures.

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References

  1. Lechner, J. F., Haugen, A., McLendon, I. A., and Pettis, E. W. (1982) Clonal growth of normal adult human bronchial epithelial cells in a serum-free medium. In Vitro 18, 633–642.

    Article  PubMed  CAS  Google Scholar 

  2. Fulcher, M. L., Gabriel, S., Burns, K. A., Yankaskas, J. R., and Randell, S. H. (2005) Well-differentiated human airway epithelial cell cultures. Methods Mol Med 107, 183–206.

    PubMed  CAS  Google Scholar 

  3. Lechner, J. F., and LaVeck, M. A. (1985) A serum-free method for culturing normal human bronchial epithelial cells at clonal density. J Tiss Cult Methods 9, 43–48.

    Article  Google Scholar 

  4. Gray, T. E., Guzman, K., Davis, C. W., Abdullah, L. H., and Nettesheim, P. (1996) Mucociliary differentiation of serially passaged normal human tracheobronchial epithelial cells. Am J Respir Cell Mol Biol 14, 104–112.

    PubMed  CAS  Google Scholar 

  5. Gazdar, A. F., and Minna, J. D. (1996) NCI series of cell lines: an historical perspective. J Cell Biochem Suppl 24, 1–11.

    Article  PubMed  CAS  Google Scholar 

  6. Boers, J. E., Ambergen, A. W., and Thunnissen, F. B. (1998) Number and proliferation of basal and parabasal cells in normal human airway epithelium. Am J Respir Crit Care Med 157, 2000–2006.

    PubMed  CAS  Google Scholar 

  7. Stoner, G. D., Katoh, Y., Foidart, J. M., Myers, G. A., and Harris, C. C. (1980) Identification and culture of human bronchial epithelial cells. Methods Cell Biol 21A, 15–35.

    Article  PubMed  CAS  Google Scholar 

  8. Lundberg, A. S., Randell, S. H., Stewart, S. A., Elenbaas, B., Hartwell, K. A., Brooks, M. W., et al. (2002) Immortalization and transformation of primary human airway epithelial cells by gene transfer. Oncogene 21, 4577–4586.

    Article  PubMed  CAS  Google Scholar 

  9. Zabner, J., Karp, P., Seiler, M., Phillips, S. L., Mitchell, C. J., Saavedra, M., et al. (2003) Development of cystic fibrosis and noncystic fibrosis airway cell lines. Am J Physiol 284, L844–L854.

    CAS  Google Scholar 

  10. Gruenert, D. C., Basbaum, C. B., Welsh, M. J., Li, M., Finkbeiner, W. E., and Nadel, J. A. (1988) Characterization of human tracheal epithelial cells transformed by an origin-defective simian virus 40. Proc Natl Acad Sci USA 85, 5951–5955.

    Article  PubMed  CAS  Google Scholar 

  11. Masui, T., Lechner, J. F., Yoakum, G. H., Willey, J. C., and Harris, C. C. (1986) Growth and differentiation of normal and transformed human bronchial epithelial cells. J Cell Physiol Suppl 4, 73–81.

    Article  PubMed  CAS  Google Scholar 

  12. Ramirez, R. D., Sheridan, S., Girard, L., Sato, M., Kim, Y., Pollack, J., et al. (2004) Immortalization of human bronchial epithelial cells in the absence of viral oncoproteins. Cancer Res 64, 9027–9034.

    Article  PubMed  CAS  Google Scholar 

  13. Gruenert, D. C., Willems, M., Cassiman, J. J., and Frizzell, R. A. (2004) Established cell lines used in cystic fibrosis research. J Cyst Fibros 3 Suppl 2, 191–196.

    Article  PubMed  CAS  Google Scholar 

  14. Fulcher, M. L., Gabriel, S. E., Olsen, J. C., Tatreau, J. R., Gentzsch, M., Livanos, E., et al. (2009) Novel human bronchial epithelial cell lines for cystic fibrosis research. Am J Physiol Lung Cell Mol Physiol 296, L82–L91.

    Article  PubMed  CAS  Google Scholar 

  15. Coyne, C. B., Kelly, M. M., Boucher, R. C., and Johnson, L. G. (2000) Enhanced epithelial gene transfer by modulation of tight junctions with sodium caprate. Am J Respir Cell Mol Biol 23, 602–609.

    PubMed  CAS  Google Scholar 

  16. Wu, Q., Lu, Z., Verghese, M. W., and Randell, S. H. (2005) Airway epithelial cell tolerance to Pseudomonas aeruginosa. Respir Res 6, 26.

    Article  PubMed  CAS  Google Scholar 

  17. Luo, J., Deng, Z. L., Luo, X., Tang, N., Song, W. X., Chen, J., et al. (2007) A protocol for rapid generation of recombinant adenoviruses using the AdEasy system. Nat Protoc 2, 1236–1247.

    Article  PubMed  CAS  Google Scholar 

  18. Cockrell, A. S., and Kafri, T. (2007) Gene delivery by lentivirus vectors. Mol Biotechnol 36, 184–204.

    Article  PubMed  CAS  Google Scholar 

  19. Shin, K. J., Wall, E. A., Zavzavadjian, J. R., Santat, L. A., Liu, J., Hwang, J. I., et al. (2006) A single lentiviral vector platform for microRNA-based conditional RNA interference and coordinated transgene expression. Proc Natl Acad Sci USA 103, 13759–13764.

    Article  PubMed  CAS  Google Scholar 

  20. Jones, L. C., Wonsetler, R. L., Olsen, J., Davis, W. C., Randell, S. H., Stutts, M., and O’Neal, W. K. (2008) Short hairpin RNA knockdown in well-differentiated human bronchial epithelial cells: A method for querying gene function relevant to cystic fibrosis. Pediatr Pulmonol Suppl 31, 284. [Abstract].

    Google Scholar 

  21. Randell, S. H., Walstad, L., Schwab, U. E., Grubb, B. R., and Yankaskas, J. R. (2001) Isolation and culture of airway epithelial cells from chronically infected human lungs. In Vitro Cell Dev Biol Anim 37, 480–489.

    Article  PubMed  CAS  Google Scholar 

  22. Dull, T., Zufferey, R., Kelly, M., Mandel, R.J., Nguyen, M., Trono, D., et al. (1998) A third-generation lentivirus vector with a conditional packaging system. J Virol 72, 8463–8471.

    PubMed  CAS  Google Scholar 

  23. Johnson, L. G., Mewshaw, J. P., Ni, H., Friedmann, T., Boucher, R. C., and Olsen, J. C. (1998) Effect of host modification and age on airway epithelial gene transfer mediated by a murine leukemia virus-derived vector. J Virol 72, 8861–8872.

    PubMed  CAS  Google Scholar 

  24. Salmon, P., Oberholzer, J., Occhiodoro, T., Morel, P., Lou, J., and Trono, D. (2000) Reversible immortalization of human primary cells by lentivector-mediated transfer of specific genes. Mol Ther 2, 404–414.

    Article  PubMed  CAS  Google Scholar 

  25. Sanlioglu, S., Williams, C. M., Samavati, L., Butler, N. S., Wang, G., and McCray, P. B., Jr. et al. (2001) Lipopolysaccharide induces Rac1-dependent reactive oxygen species formation and coordinates tumor necrosis factor-alpha secretion through IKK regulation of NF-kappa B. J Biol Chem 276, 30188–30198.

    Article  PubMed  CAS  Google Scholar 

  26. Cullen, B. R. (2006) Enhancing and confirming the specificity of RNAi experiments. Nat Methods 3, 677–681.

    Article  PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Cystic Fibrosis/Pulmonary Research and Treatment Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Scott H. Randell, M. Leslie Fulcher, Wanda O’Neal & John C. Olsen

Authors
  1. Scott H. Randell
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  2. M. Leslie Fulcher
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  3. Wanda O’Neal
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  4. John C. Olsen
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Corresponding author

Correspondence to Scott H. Randell .

Editor information

Editors and Affiliations

  1. Faculty of Sciences, Centre for Biodiversity & Functional, University of Lisboa, Campo Grande, Lisboa, 1749-016, Portugal

    Margarida D. Amaral

  2. , Department of Physiology, University of Regensburg, Universitätsstr. 31, Regensburg, 93053, Germany

    Karl Kunzelmann

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Randell, S.H., Fulcher, M.L., O’Neal, W., Olsen, J.C. (2011). Primary Epithelial Cell Models for Cystic Fibrosis Research. In: Amaral, M., Kunzelmann, K. (eds) Cystic Fibrosis. Methods in Molecular Biology, vol 742. Humana Press. https://doi.org/10.1007/978-1-61779-120-8_18

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  • DOI: https://doi.org/10.1007/978-1-61779-120-8_18

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-119-2

  • Online ISBN: 978-1-61779-120-8

  • eBook Packages: Springer Protocols

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