Skip to main content
Log in

Long-term culture of normal and cystic fibrosis epithelial cells grown under serum-free conditions

  • Regular Papers
  • Published:
In Vitro Cellular & Developmental Biology Aims and scope Submit manuscript

Summary

The understanding of pathways associated with differentiated function in human epithelial cells has been enhanced by the development of methods for the short-term culture of human epithelial cells. In general these methods involve the use of serum. The subculture and maintenance of epithelial cells in long-term culture has been more problematic. A serum-free medium developed for human bronchial epithelial cells was slightly modified and found to be useful for the subculture and long-term maintenance of not only bronchial epithelial cells, but also tracheal, nasal polyp, and sweat gland epithelial cells from either normal or cystic fibrosis individuals. The cells maintained epithelial-specific characteristics after multiple subcultures. Monolayers of epithelial cells showed junctional complex formation, the presence of keratin, and micro villi. Functional studies with Ussing chambers showed short circuit current (Isc) responses to isoproterenol, bradykinin, or calcium ionophore (A23187) in subcultured tracheal and bronchial cells.

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

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Banks-Schlegel, S. P. Isolation, cultivation, and characterization of normal human esophageal epithelial cells. J. Tissue Cult. Methods 9:95–105; 1985.

    Article  CAS  Google Scholar 

  2. Boucher, R. C.; Larsen, E. H. Comparison of ion transport by cultured secretory and absorptive canine airway epithelia. Am. J. Physiol. 254:C535-C547; 1988.

    PubMed  CAS  Google Scholar 

  3. Boyce, S. T.; Ham, R. G. Cultivation, frozen storage, and clonal growth on normal human epidermal keratinocytes in serum-free media. J. Tissue Cult. Methods 9:83–93; 1985.

    Article  Google Scholar 

  4. Coleman, D. L.; Tuet, I. K.; Widdicombe, J. H. Electrical properties of dog tracheal epithelial cells grown in monolayer culture. Am. J. Physiol. 246:C355-C359; 1984.

    PubMed  CAS  Google Scholar 

  5. Cristofalo, V. J.; Sharf, B. B. Cellular senescence and DNA synthesis. Exp. Cell Res. 76:419–427; 1973.

    Article  PubMed  CAS  Google Scholar 

  6. Gibson-D'Ambrosio, R. E.; Samuel, M.; Chang, C. C., et al. Characteristics of long-term human epithelial cell cultures derived from normal human fetal kidney. In Vitro Cell. Dev. Biol. 23:279–287; 1987.

    PubMed  Google Scholar 

  7. Gruenert, D. C. Differentiated properties of human epithelial cells transformed in vitro. Bio Techniques 5:740–749; 1987.

    CAS  Google Scholar 

  8. Gruenert, D. C.; Basbaum, C. B.; Welsh, M. J., et al. Characterization of human tracheal epithelial cells transformed by an origin-defective simian virus 40. Proc. Natl. Acad. Sci. USA 85:5951–5955; 1988.

    Article  PubMed  CAS  Google Scholar 

  9. Knowles, M.; Murray, G.; Shallal, J., et al. Bioelectric properties and ion flow across excised human bronchi. J. Applied Physiol. 56:868–877; 1984.

    CAS  Google Scholar 

  10. Kraus, M. E.; Hagiwara, G.; Chen, J., et al. Ion channels in normal human and cystic fibrosis cultured sweet gland cells. Am. J. Physiol. 257:C129-C140; 1989.

    Google Scholar 

  11. Lechner, J. F.; Babcock, M. S.; Marnell, M., et al. Normal human prostatic epithelial cell cultures. Methods Cell. Biol. 21B:195–225; 1980.

    Article  PubMed  CAS  Google Scholar 

  12. Lechner, J. F.; LaVeck, M. A. A serum-free method for culturing normal human bronchial epithelial cells at clonal density. J. Tissue Cult. Methods 9:43–48; 1985.

    Article  Google Scholar 

  13. Li, M.; McCann, J. D.; Liedtke, C., et al. Cyclic AMP-dependent protein kinase opens chloride channels in normal but not cystic fibrosis airway epithelium. Nature 331:358–360; 1988.

    Article  PubMed  CAS  Google Scholar 

  14. Masui, T.; Wakefield, L. M.; Lechner, J. F., et al. Type B transforming growth factor is the primary differentiation-inducing serum factor for normal human bronchial epithelial cells. Proc. Natl. Acad. Sci. USA 83:2438–2442; 1986.

    Article  PubMed  CAS  Google Scholar 

  15. Noyes, I.; Milo, G.; Cunningham, C. Establishment of proliferating human epithelial cells in vitro from cell suspensions of neonatal foreskin. Tissue Cult. Manual 5:1173–1175; 1980.

    Article  Google Scholar 

  16. Peehl, D. M. Serial culture of adult human prostatic epithelial cells. J. Tissue Cult. Methods 9:53–60; 1985.

    Article  Google Scholar 

  17. Price, F. M.; Taylor, W. G.; Camalier, R. F. et al. Approaches to enhanced proliferation of human epidermal keratinocytes in mass culture. JNCI 70:853–861; 1983.

    PubMed  CAS  Google Scholar 

  18. Quinton, P. M. Chloride impermeanbility in cystic fibrosis. Nature 301:421–422; 1983.

    Article  PubMed  CAS  Google Scholar 

  19. Rheinwald, J. G.; Green, H. Serial cultivation of strains of human epidermal keratinocytes. The formation of keratinizing colonies from single cells. Cell 6:331–343; 1975.

    Article  PubMed  CAS  Google Scholar 

  20. Schoumacher, R. A.; Shoemaker, R. L.; Holm, D. R., et al. Phosphorylation fails to activate chloride channels from cystic fibrosis airway cells. Nature 330:752–754; 1987.

    Article  PubMed  CAS  Google Scholar 

  21. Stampfer, M.; Hallowes, R. C.; Hackett, A. J. Growth of normal human mammary cells in culture. In Vitro 16:415–425; 1980.

    Article  PubMed  CAS  Google Scholar 

  22. Stampfer, M. R. Isolation and growth of human mammary epithelial cells. J. Tissue Cult. Methods 9:107–115; 1985.

    Article  Google Scholar 

  23. Talamo, R. C.; Rosenstein, B. T.; Berninger, R. W. Cystic fibrosis. In: Stanbury, J. B.; Wyngaarden, J. B., eds. Metabolic basis of inherited disease. New York: McGraw-Hill; 1983:1889–1917.

    Google Scholar 

  24. Van Scott, M. R.; Yankaskas J. R.; Boucher, R. C. Culture of airway epithelial cells: research techniques. Exp. Lung Res. 11:75–94; 1986.

    PubMed  Google Scholar 

  25. Welsh, M. J. Electrolyte transport by airway epithelia. Physiol. Rev. 67:1143–1184; 1985.

    Google Scholar 

  26. Wheelock, M. J.; Buck, C. A.; Bechtol, K. B. et al. Soluble 80-kd fragment of cell-CAM 120/80 disrupts cell-cell adhesion. J. Cell. Biochem. 34:187–202; 1987.

    Article  PubMed  CAS  Google Scholar 

  27. Widdicombe, J. H.; Coleman, D. L.; Finkbeiner, W. E., et al. Electrical properties of monolayers cultured from cells of human mucosa. J. Appl. Physiol. 58:1729–1735; 1985.

    PubMed  CAS  Google Scholar 

  28. Widdicombe, J. H.; Welsh, M. J.; Finkbeiner, W. E. Cystic fibrosis decreases the apical membrane chloride permeability of monolayers cultured from cells of tracheal epithelium. Proc. Natl. Acad. Sci. USA 82:6167–6171; 1985.

    Article  PubMed  CAS  Google Scholar 

  29. Wu, R.; Yankaskas, J.; Cheng, E., et al. Growth and differentiation of human nasal epithelial cells in culture. Serum-free, hormone-supplemented medium and proteoglycan synthesis. Am. Rev. Respir. Dis. 132:311–320; 1985.

    PubMed  CAS  Google Scholar 

  30. Yankaskas, J. R.; Cotton, C. U.; Knowles, M. R. et al. Culture of human nasal epithelial cells on collagen matrix supports. Am. Rev. Respir Dis. 132:1281–1287; 1985.

    PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Additional information

This work is supported by grants HL41928 and DK39619 (DCG), HL24136 (CBB), and HL42368 (JHW and DCG) from the National Institutes of Health, Bethesda, MD.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gruenert, D.C., Basbaum, C.B. & Widdicombe, J.H. Long-term culture of normal and cystic fibrosis epithelial cells grown under serum-free conditions. In Vitro Cell Dev Biol 26, 411–418 (1990). https://doi.org/10.1007/BF02623833

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02623833

Key words

Navigation