Regeneration of Skin and Cornea by Tissue Engineering

  • Danielle Larouche
  • Claudie Paquet
  • Julie Fradette
  • Patrick Carrier
  • François A. Auger
  • Lucie Germain
Part of the Methods in Molecular Biology book series (MIMB, volume 482)


Progress in tissue engineering has led to the development of technologies allowing the reconstruction of autologous tissues from the patient’s own cells. Thus, tissue-engineered epithelial substitutes produced from cultured skin epithelial cells undergo long-term regeneration after grafting, indicating that functional stem cells were preserved during culture and following grafting. However, these cultured epithelial sheets reconstruct only the upper layer of the skin and lack the mechanical properties associated to the connective tissue of the dermis. We have designed a reconstructed skin entirely made from human cutaneous cells comprising both the dermis and the epidermis, as well as a well-organized basement membrane by a method named the self-assembly approach. In this chapter, protocols to generate reconstructed skin and corneal epithelium suitable for grafting are described in details. The methods include extraction and culture of human skin keratinocytes, human skin fibroblasts as well as rabbit and human corneal epithelial cells, and a complete description of the skin reconstructed by the self-assembly approach and of corneal epithelium reconstructed over a fibrin gel.

Key words

Stem cells keratin 19 epidermis human cornea skin substitute reconstructed skin corneal epithelial cell tissue engineering fibrin gel 


  1. 1.
    Benitah, S. A., Frye, M., Glogauer, M., and Watt, F. M. (2005) Stem cell depletion through epidermal deletion of Rac1 Science 309, 933–935.CrossRefPubMedGoogle Scholar
  2. 2.
    Bickenbach, J. R., McCutecheon, J., and Mackenzie, I. C. (1986) Rate of loss of tritiated thymidine label in basal cells in mouse epithelial tissues Cell Tissue Kinet 19, 325–333.PubMedGoogle Scholar
  3. 3.
    Cotsarelis, G., Sun, T. T., and Lavker, R. M. (1990) Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis Cell 61, 1329–1337.CrossRefPubMedGoogle Scholar
  4. 4.
    Kruse, F. E. (1994) Stem cells and corneal epithelial regeneration Eye 8 (Pt 2), 170–183.PubMedGoogle Scholar
  5. 5.
    Blanpain, C., Lowry, W. E., Geoghegan, A., Polak, L., and Fuchs, E. (2004) Self-renewal, multipotency, and the existence of two cell populations within an epithelial stem cell niche Cell 118, 635–648.CrossRefPubMedGoogle Scholar
  6. 6.
    Morris, R. J., Liu, Y., Marles, L., Yang, Z., Trempus, C., Li, S., Lin, J. S., Sawicki, J. A., and Cotsarelis, G. (2004) Capturing and profiling adult hair follicle stem cells Nat Biotechnol 22, 411–417.CrossRefPubMedGoogle Scholar
  7. 7.
    Tumbar, T., Guasch, G., Greco, V., Blanpain, C., Lowry, W. E., Rendl, M., and Fuchs, E. (2004) Defining the epithelial stem cell niche in skin Science 303, 359–363.CrossRefPubMedGoogle Scholar
  8. 8.
    Michel, M., Torok, N., Godbout, M. J., Lussier, M., Gaudreau, P., Royal, A., and Germain, L. (1996) Keratin 19 as a biochemical marker of skin stem cells in vivo and in vitro: keratin 19 expressing cells are differentially localized in function of anatomic sites, and their number varies with donor age and culture stage J Cell Sci 109 (Pt 5), 1017–1028.PubMedGoogle Scholar
  9. 9.
    Auger, F. A., Rémy-Zolghadri, M., Grenier, G., and Germain, L. (2000) The self-assembly approach for organ reconstruction by tissue engineering e-biomed 1, 75–86.CrossRefGoogle Scholar
  10. 10.
    Germain, L., Berthod, F., Moulin, V., Goulet, F., and Auger, F. A. (2004) Principles of living organ reconstruction by tissue engineering in "Tissue Engineering and Novel Delivery Systems" (Yaszemski, M. J., Trantolo, D. J., Lewandrowski, K.-W., Hasirci, V., Altobelli, D. E., and Wise, D. L., Eds.), pp. 197–228, Marcel Dekker, New-York.Google Scholar
  11. 11.
    Michel, M., L'Heureux, N., Pouliot, R., Xu, W., Auger, F. A., and Germain, L. (1999) Characterization of a new tissue-engineered human skin equivalent with hair In Vitro Cell Dev Biol Anim 35, 318–326.CrossRefPubMedGoogle Scholar
  12. 12.
    Pouliot, R., Larouche, D., Auger, F. A., Juhasz, J., Xu, W., Li, H., and Germain, L. (2002) Reconstructed human skin produced in vitro and grafted on athymic mice Transplantation 73, 1751–1757.CrossRefPubMedGoogle Scholar
  13. 13.
    Gaudreault, M., Carrier, P., Larouche, K., Leclerc, S., Giasson, M., Germain, L., and Guerin, S. L. (2003) Influence of sp1/sp3 expression on corneal epithelial cells proliferation and differentiation properties in reconstructed tissues Invest Ophthalmol Vis Sci 44, 1447–1457.CrossRefPubMedGoogle Scholar
  14. 14.
    Masson-Gadais, B., Fugere, C., Paquet, C., Leclerc, S., Lefort, N. R., Germain, L., and Guerin, S. L. (2006) The feeder layer-mediated extended lifetime of cultured human skin keratinocytes is associated with altered levels of the transcription factors Sp1 and Sp3 J Cell Physiol 206, 831–842.CrossRefPubMedGoogle Scholar
  15. 15.
    Robitaille, H., Proulx, R., Robitaille, K., Blouin, R., and Germain, L. (2005) The mitogen-activated protein kinase kinase kinase dual leucine zipper-bearing kinase (DLK) acts as a key regulator of keratinocyte terminal differentiation J Biol Chem 280, 12732–12741.CrossRefPubMedGoogle Scholar
  16. 16.
    Rama, P., Bonini, S., Lambiase, A., Golisano, O., Paterna, P., De Luca, M., and Pellegrini, G. (2001) Autologous fibrin- cultured limbal stem cells permanently restore the corneal surface of patients with total limbal stem cell deficiency Transplantation 72, 1478–1485.CrossRefPubMedGoogle Scholar
  17. 17.
    Talbot, M., Carrier, P., Giasson, C. J., Deschambeault, A., Guerin, S. L., Auger, F. A., Bazin, R., and Germain, L. (2006) Autologous transplantation of rabbit limbal epithelia cultured on fibrin gels for ocular surface reconstruction Mol Vis 12, 65–75.PubMedGoogle Scholar
  18. 18.
    Germain, L., Rouabhia, M., Guignard, R., Carrier, L., Bouvard, V., and Auger, F. A. (1993) Improvement of human keratinocyte isolation and culture using thermolysin Burns 19, 99–104.CrossRefPubMedGoogle Scholar
  19. 19.
    Germain, L., Auger, F. A., Grandbois, E., Guignard, R., Giasson, M., Boisjoly, H., and Guerin, S. L. (1999) Reconstructed human cornea produced in vitro by tissue engineering Pathobiology 67, 140–147.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Danielle Larouche
    • 1
  • Claudie Paquet
    • 1
  • Julie Fradette
    • 1
  • Patrick Carrier
    • 1
  • François A. Auger
    • 1
  • Lucie Germain
    • 1
  1. 1.Experimental Organogenesis Laboratory/LOEX and Department of Surgery and OphthalmologyLaval UniversitySainte-FoyCanada

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