Protocol

Stem Cells in Regenerative Medicine

Volume 482 of the series Methods in Molecular Biology pp 233-256

Regeneration of Skin and Cornea by Tissue Engineering

  • Danielle LaroucheAffiliated withExperimental Organogenesis Laboratory/LOEX and Department of Surgery and Ophthalmology, Laval University
  • , Claudie PaquetAffiliated withExperimental Organogenesis Laboratory/LOEX and Department of Surgery and Ophthalmology, Laval University
  • , Julie FradetteAffiliated withExperimental Organogenesis Laboratory/LOEX and Department of Surgery and Ophthalmology, Laval University
  • , Patrick CarrierAffiliated withExperimental Organogenesis Laboratory/LOEX and Department of Surgery and Ophthalmology, Laval University
  • , François A. AugerAffiliated withExperimental Organogenesis Laboratory/LOEX and Department of Surgery and Ophthalmology, Laval University
  • , Lucie GermainAffiliated withExperimental Organogenesis Laboratory/LOEX and Department of Surgery and Ophthalmology, Laval University

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Abstract

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