Serial culturing of human bronchial epithelial cells derived from biopsies

  • Petra M. de Jong
  • Marianne A. J. A. van Sterkenburg
  • Johanna A. Kempenaar
  • Joop H. Dijkman
  • Maria Ponec
Cellular Models

Summary

In the present study we describe the establishment of serial cultures of human bronchial epithelial cells derived from biopsies obtained by fiberoptic bronchoscopy. The cell cultures were initiated from small amounts of material (2 mm forceps biopsies) using either explants or epithelial cell suspensions in combination with a feeder-layer technique. The rate of cell proliferation and the number of passages (up to 8 passages) achieved were similar, irrespective of whether the explants or dissociated cells were used. To modulate the extent of differentiation, the bronchial epithelial cells were cultured either under submerged, low calcium (0.06 mM) (proliferating), normal calcium (1.6 mM) (differentiation enhancing) conditions, or at the air-liquid interface. Characterization of the bronchial epithelial cell cultures was assessed on the basis of cell morphology, cytokeratin expression, and ciliary activity. The cells cultured under submerged conditions formed a multilayer consisting of maximally three layers of polygonal-shaped, small cuboidal cells, an appearance resembling the basal cells in vivo. In the air-exposed cultures, the formed multilayer consisted of three to six layers exhibiting squamous metaplasia. The cytokeratin profile in cultured bronchial epithelial cells was similar in submerged and air-exposed cultures and comparable with the profile found in vivo. In addition to cytokeratins, vimentin was co-expressed in a fraction of the subcultured cells. The ciliary activity was observed in primary culture, irrespective of whether the culture had been established from explants or from dissociated cells. This activity was lost upon subculturing and it was not regained by prolongation of the culture period. In contrast to submerged cultures and despite the squamous metaplasia appearance, the cells showed a reappearance of cilia when cultured at the air-liquid interface. Human bronchial epithelial cell cultures can be a representative model for controlling the mechanisms of regulation of bronchial epithelial cell function.

Key words

human bronchial epithelial cell culture cytokeratins ciliary activity 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Barnes, P. J.; Cuss, F. M.; Palmer, J. B. The effect of airway epithelium on smooth muscle contractility in bovine trachea. Br. J. Pharmacol. 86:685–691; 1985.PubMedGoogle Scholar
  2. 2.
    Broers, J. L. V.; Ramaekers, F. C. S.; Klein Rot, M., et al. Cytokeratins in different types of human lung cancer as monitored by chain-specific monoclonal antibodies. Cancer Res. 48:3221–3229; 1988.PubMedGoogle Scholar
  3. 3.
    Chopra, D. P.; Sullivan, J.; Wille, J. J., et al. Propagation of differentiating normal human tracheobronchial epithelial cells in serum-free medium. J. Cell. Physiol. 130:173–181; 1987.PubMedCrossRefGoogle Scholar
  4. 4.
    Church, M. K.; Lai, C.; Beasley, R., et al. The mediator and cellular basis of the allergic response. Allergy 43 (suppl 8):26–29; 1988.PubMedCrossRefGoogle Scholar
  5. 5.
    Crandall, E. D.; Kim, K. J. Protein traffic across lung epithelia. Am. J. Respir. Cell. Mol. Biol. 1:255; 1989.PubMedGoogle Scholar
  6. 6.
    Dairkee, S. H.; Blayney, C. M.; Asarnow, D. M., et al. Early expression of vimentin in human mammary cultures. In Vitro Cell. Dev. Biol. 21:321–327; 1985.PubMedGoogle Scholar
  7. 7.
    Flavahan, N. A.; Aarhus, L. L.; Rimele, T. J., et al. Respiratory epithelium inhibits bronchial smooth muscle tone. J. Appl. Physiol. 58:834–838; 1985.PubMedGoogle Scholar
  8. 8.
    Gray, T. E.; Thomassen, D. G.; Mass, M. J., et al. Quantitation of cell proliferation, colony formation, and carcinogen induced cytotoxicity of rat tracheal epithelial cells grown in culture on 3T3 feeder layers. In Vitro 19:559–570; 1983.PubMedCrossRefGoogle Scholar
  9. 9.
    Green, H.; Kehinde, O.; Thomas, J. Growth of cultured human epidermal cells into multiple epithelia suitable for grafting. Proc. Natl. Acad. Sci. USA 76:5665–5668; 1979.PubMedCrossRefGoogle Scholar
  10. 10.
    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.PubMedCrossRefGoogle Scholar
  11. 11.
    Hennings, H.; Michael, D.; Cheng, C., et al. Calcium regulation of growth and differentiation of epidermal cells in culture. Cell 19:245–254; 1980.PubMedCrossRefGoogle Scholar
  12. 12.
    Hogg, J. C.; Eggleston, P. A. Is asthma an epithelial disease? Am. Rev. Respir. Dis. 129:207–208; 1984.PubMedGoogle Scholar
  13. 13.
    Holroyde, M. C. The influence of epithelium on the responsiveness of guinea-pig isolated trachea. Br. J. Pharmacol. 87:501–507; 1986.PubMedGoogle Scholar
  14. 14.
    Ishida, K.; Kelly, L. J.; Thomson, R. J., et al. Repeated antigen challenge induces airway hyperresponsiveness with tissue eosinophilia in guinea pigs. J. Appl. Physiol. 67:1133–1139; 1989.PubMedGoogle Scholar
  15. 15.
    Jacoby, D. B.; Nadel, J. A. Airway epithelial metabolism and airway smooth muscle hyperresponsiveness. In: Coburn, R. F., ed. Airway smooth muscle in health and disease. New York: Plenum Publishing Corporation; 1989.Google Scholar
  16. 16.
    Jorissen, M.; Van der Schueren, B.; Van den Berghe, H., et al. Contribution of in vitro culture methods for respiratory epithelial cells to the study of the physiology of the respiratory tract. Eur. Respir. J. 4:210–217; 1991.PubMedGoogle Scholar
  17. 17.
    Kelsen, S. G.; Mardini, I. A.; Zhou, S., et al. A technique to harvest viable tracheobronchial epithelial cells from living human donors. Am. J. Respir. Cell Mol. Biol. 7:66–72; 1992.PubMedGoogle Scholar
  18. 18.
    Lane, E. B.; Bártek, J.; Purkis, P. E., et al. Keratin antigens in differentiating skin. Ann. NY Acad. Sci. 455:241–258; 1985.PubMedCrossRefGoogle Scholar
  19. 19.
    Lechner, J. F.; Haugen, A.; Autrup, H., et al. Clonal growth of epithelial cells from normal adult human bronchus. Cancer Res. 41:2294–2304; 1981.PubMedGoogle Scholar
  20. 20.
    Lechner, J. F.; Haugen, A.; McClendon, I. A., et al. Clonal growth of normal adult human bronchial epithelial cells in a serum-free medium. In Vitro 18:633–642; 1982.PubMedGoogle Scholar
  21. 21.
    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.CrossRefGoogle Scholar
  22. 22.
    Liu, S. C.; Karasek, M. Isolation and growth of adult human epidermal keratinocytes in cell culture. J. Invest. Dermatol. 71:157–162; 1978.PubMedCrossRefGoogle Scholar
  23. 23.
    Mendelsohn, M. G.; Dilorenzo, T. P.; Abramson, A. L., et al. Retinoic acid regulates, in vitro, the two normal pathways of differentiation of human laryngeal keratinocytes. In Vitro Cell. Dev. Biol. 27A:137–141; 1991.PubMedGoogle Scholar
  24. 24.
    Moll, R.; Franke, W. W.; Schiller, D. L., et al. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell 31:11–24; 1982.PubMedCrossRefGoogle Scholar
  25. 25.
    Munakata, M.; Huang, I.; Mitzner, W., et al. Protective role of epithelium in the guinea pig airway. J. Appl. Physiol. 66:1547–1552; 1989.PubMedGoogle Scholar
  26. 26.
    Nakane, P. K.; Pierce, G. B. Enzyme-labeled antibodies: preparation and application for the localization of antigens. J. Histochem. Cytochem. 14:929–931; 1966.PubMedGoogle Scholar
  27. 27.
    Oomen, L. C.; Ten Have-Opbroek, A. A.; Hageman, P. C., et al. Fetal mouse alveolar type II cells in culture express several type II cell characteristics found in vivo, together with major histocompatibility antigens. Am. J. Respir. Cell. Mol. Biol. 3:325–339; 1990.PubMedGoogle Scholar
  28. 28.
    Ponec, M.; Weerheim, A.; Kempenaar, J., et al. Lipid composition of cultured human keratinocytes in relation to their differentiation. J. Lipid Res. 29:949–961; 1988.PubMedGoogle Scholar
  29. 29.
    Powell, D. W. Barrier function of epithelia. Am. J. Physiol. 241:G275-G288; 1981.PubMedGoogle Scholar
  30. 30.
    Régnier, M.; Pruniéras, M.; Woodley, D. Growth and differentiation of adult human epidermal cells on dermal substrates. Front. Matrix Biol. 9:4–35; 1981.Google Scholar
  31. 31.
    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.PubMedCrossRefGoogle Scholar
  32. 32.
    Richard, M. H.; Viac, J.; Reano, A., et al. Vimentin expression in normal human keratinocytes grown in serum-free defined MCDB 153 medium. Arch. Dermatol. Res. 282:512–515; 1990.PubMedCrossRefGoogle Scholar
  33. 33.
    Salari, H.; Schellenberg, R. R. Stimulation of human airway epithelial cells by platelet activating factor (PAF) and arachidonic acid produces 15-hydroxyeicosatetraenoic acid (15-HETE) capable of contracting bronchial smooth muscle. Pulmonary Pharmacol. 4:1–7; 1991.CrossRefGoogle Scholar
  34. 34.
    Schaafsma, H. E.; Ramaekers, F. C. S.; van Muijen, G. N. P., et al. Distribution of cytokeratin polypeptides in epithelia of the adult human urinary tract. Histochemistry 91:151–159; 1989.PubMedCrossRefGoogle Scholar
  35. 35.
    Sigal, E.; Nadel, J. A. The airway epithelium and arachidonic acid 15-lipoxygenase. Am. Rev. Respir. Dis. 143:S71-S74; 1991.PubMedGoogle Scholar
  36. 36.
    Stuart-Smith, K.; Vanhoutte, P. M. Heterogeneity in the effects of epithelium removal in the canine bronchial tree. J. Appl. Physiol. 63:2510–2515; 1987.PubMedGoogle Scholar
  37. 37.
    Van Muijen, G. N. P.; Warnaar, S. O.; Ponec, M. Differentiation-related changes of cytokeratin expression in cultured keratinocytes and in fetal, newborn, and adult epidermis. Exp. Cell Res. 171:331–345; 1987.PubMedCrossRefGoogle Scholar
  38. 38.
    Van Muijen, G. N. P.; Ruiter, D. J.; Franke, W. W., et al. Cell type heterogeneity of cytokeratin expression in complex epithelia and carcinomas as demonstrated by monoclonal antibodies specific for cytokeratins nos. 4 and 13. Exp. Cell. Res. 162:97–113; 1986.PubMedCrossRefGoogle Scholar
  39. 39.
    Watt, F. M. Terminal differentiation of epidermal keratinocytes. Curr. Opinion Cell Biol. 1:1107–1115; 1989.PubMedCrossRefGoogle Scholar
  40. 40.
    Wu, R.; Martin, W. R.; Robinson, C. B., et al. Expression of mucin synthesis and secretion in human tracheobronchial epithelial cells grown in culture. Am. J. Respir. Cell Mol. Biol. 3:467–478; 1990.PubMedGoogle Scholar
  41. 41.
    Wu, R.; Nolan, E.; Turner, C. Expression of tracheal differentiated functions in serum-free, hormone-supplemented medium. J. Cell Physiol. 127:167–181; 1985.CrossRefGoogle Scholar

Copyright information

© Tissue Culture Association 1993

Authors and Affiliations

  • Petra M. de Jong
    • 2
  • Marianne A. J. A. van Sterkenburg
    • 2
  • Johanna A. Kempenaar
    • 3
  • Joop H. Dijkman
    • 2
  • Maria Ponec
    • 3
  1. 1.Dept. of Pulmonology, C3PUniversity HospitalLeidenThe Netherlands
  2. 2.Department of Respiratory DiseasesUniversity HospitalLeidenThe Netherlands
  3. 3.the Department of DermatologyUniversity HospitalLeidenThe Netherlands

Personalised recommendations