Pediatric Surgery International

, Volume 29, Issue 3, pp 249–256 | Cite as

Optimizing in vitro culture conditions leads to a significantly shorter production time of human dermo-epidermal skin substitutes

  • Luca Pontiggia
  • Agnieszka Klar
  • Sophie Böttcher-Haberzeth
  • Thomas Biedermann
  • Martin Meuli
  • Ernst Reichmann
Original Article



Autologous dermo-epidermal skin substitutes (DESS) generated in vitro represent a promising therapeutic means to treat full-thickness skin defects in clinical practice. A serious drawback with regard to acute patients is the relatively long production time of 3–4 weeks. With this experimental study we aimed to decrease the production time of DESS without compromising their quality.


Two in vitro steps of DESS construction were varied: the pre-cultivation time of fibroblasts in hydrogels (1, 3, and 6 days), and the culture time of keratinocytes (3, 6, and 12 days) before transplantation of DESS on nude rats. Additionally, the impact of the air–liquid interface culture during 3 days before transplantation was investigated. 3 weeks after transplantation, the macroscopic appearance was evaluated and histological sections were produced to analyze structure and thickness of epidermis and dermis, the stratification of the epidermis, and the presence of a basal lamina.


Optimal DESS formation was obtained with a fibroblast pre-cultivation time of 6 days. The minimal culture time of keratinocytes on hydrogels was also 6 days. The air–liquid interface culture did not improve graft quality.


By optimizing our in vitro culture conditions, it was possible to very substantially reduce the production time for DESS from 21 to 12 days. However, pre-cultivation of fibroblasts in the dermal equivalent and proliferation of keratinocytes before transplantation remain crucial for an equilibrated maturation of the epidermis and cannot be completely skipped.


Tissue engineering Dermo-epidermal skin substitutes Skin reconstruction Air-liquid interface Collagen hydrogels 


  1. 1.
    Rheinwald JG, Green H (1975) Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6:331–343PubMedCrossRefGoogle Scholar
  2. 2.
    O’Connor NE, Mulliken JB, Banks-Schlegel S, Kehinde O, Green H (1981) Grafting of burns with cultured epithelium prepared from autologous epidermal cells. Lancet 1:75–78CrossRefGoogle Scholar
  3. 3.
    Gallico GG 3rd, O’Connor NE, Compton CC, Kehinde O, Green H (1984) Permanent coverage of large burn wounds with autologous cultured human epithelium. N Engl J Med 311:448–451PubMedCrossRefGoogle Scholar
  4. 4.
    Munster AM (1996) Cultured skin for massive burns. A prospective, controlled trial. Ann Surg 224:372–375 discussion 375–377PubMedCrossRefGoogle Scholar
  5. 5.
    Krupp S, Benathan M, Meuli M, Deglise B, Holzer E, Wiesner L, Delacretaz F, Chiolero R (1992) Current concepts in pediatric burn care: management of burn wounds with cultured epidermal autografts. Eur J Pediatr Surg 2:210–215PubMedCrossRefGoogle Scholar
  6. 6.
    Cuono C, Langdon R, McGuire J (1986) Use of cultured epidermal autografts and dermal allografts as skin replacement after burn injury. Lancet 1:1123–1124PubMedCrossRefGoogle Scholar
  7. 7.
    Boyce ST, Goretsky MJ, Greenhalgh DG, Kagan RJ, Rieman MT, Warden GD (1995) Comparative assessment of cultured skin substitutes and native skin autograft for treatment of full-thickness burns. Ann Surg 222:743–752PubMedCrossRefGoogle Scholar
  8. 8.
    Gobet R, Raghunath M, Altermatt S, Meuli-Simmen C, Benathan M, Dietl A, Meuli M (1997) Efficacy of cultured epithelial autografts in pediatric burns and reconstructive surgery. Surgery 121:654–661PubMedCrossRefGoogle Scholar
  9. 9.
    Biedermann T, Pontiggia L, Bottcher-Haberzeth S, Tharakan S, Braziulis E, Schiestl C, Meuli M, Reichmann E (2010) Human eccrine sweat gland cells can reconstitute a stratified epidermis. J Invest Dermatol 130:1996–2009PubMedCrossRefGoogle Scholar
  10. 10.
    Costea DE, Loro LL, Dimba EA, Vintermyr OK, Johannessen AC (2003) Crucial effects of fibroblasts and keratinocyte growth factor on morphogenesis of reconstituted human oral epithelium. J Invest Dermatol 121:1479–1486PubMedCrossRefGoogle Scholar
  11. 11.
    Pontiggia L, Biedermann T, Meuli M, Widmer D, Bottcher-Haberzeth S, Schiestl C, Schneider J, Braziulis E, Montano I, Meuli-Simmen C, Reichmann E (2009) Markers to evaluate the quality and self-renewing potential of engineered human skin substitutes in vitro and after transplantation. J Invest Dermatol 129:480–490PubMedCrossRefGoogle Scholar
  12. 12.
    Braziulis E, Diezi M, Biedermann T, Pontiggia L, Schmucki M, Hartmann-Fritsch F, Luginbuhl J, Schiestl C, Meuli M, Reichmann E (2012) Modified plastic compression of collagen hydrogels provides an ideal matrix for clinically applicable skin substitutes. Tissue Eng Part C Methods 18(6):464–475PubMedCrossRefGoogle Scholar
  13. 13.
    Armour AD, Powell HM, Boyce ST (2008) Fluorescein diacetate for determination of cell viability in tissue-engineered skin. Tissue Eng Part C Methods 14:89–96PubMedCrossRefGoogle Scholar
  14. 14.
    Candi E, Schmidt R, Melino G (2005) The cornified envelope: a model of cell death in the skin. Nat Rev Mol Cell Biol 6:328–340PubMedCrossRefGoogle Scholar
  15. 15.
    Stark HJ, Baur M, Breitkreutz D, Mirancea N, Fusenig NE (1999) Organotypic keratinocyte cocultures in defined medium with regular epidermal morphogenesis and differentiation. J Invest Dermatol 112:681–691PubMedCrossRefGoogle Scholar
  16. 16.
    Borradori L, Sonnenberg A (1999) Structure and function of hemidesmosomes: more than simple adhesion complexes. J Invest Dermatol 112:411–418PubMedCrossRefGoogle Scholar
  17. 17.
    Supp AP, Wickett RR, Swope VB, Harriger MD, Hoath SB, Boyce ST (1999) Incubation of cultured skin substitutes in reduced humidity promotes cornification in vitro and stable engraftment in athymic mice. Wound Repair Regen 7:226–237PubMedCrossRefGoogle Scholar
  18. 18.
    Wilkins LM, Watson SR, Prosky SJ, Meunier SF, Parenteau NL (1994) Development of a bilayered living skin construct for clinical applications. Biotechnol Bioeng 43:747–756PubMedCrossRefGoogle Scholar
  19. 19.
    Parenteau NL, Nolte CM, Bilbo P, Rosenberg M, Wilkins LM, Johnson EW, Watson S, Mason VS, Bell E (1991) Epidermis generated in vitro: practical considerations and applications. J Cell Biochem 45:245–251PubMedCrossRefGoogle Scholar
  20. 20.
    Wood FM (2003) Clinical potential of autologous epithelial suspension. Wounds 15:16–22Google Scholar
  21. 21.
    Marionnet C, Pierrard C, Vioux-Chagnoleau C, Sok J, Asselineau D, Bernerd F (2006) Interactions between fibroblasts and keratinocytes in morphogenesis of dermal epidermal junction in a model of reconstructed skin. J Invest Dermatol 126:971–979PubMedCrossRefGoogle Scholar
  22. 22.
    Nolte CJ, Oleson MA, Hansbrough JF, Morgan J, Greenleaf G, Wilkins L (1994) Ultrastructural features of composite skin cultures grafted onto athymic mice. J Anat 185(Pt 2):325–333PubMedGoogle Scholar
  23. 23.
    Okamoto E, Kitano Y (1993) Expression of basement membrane components in skin equivalents–influence of dermal fibroblasts. J Dermatol Sci 5:81–88PubMedCrossRefGoogle Scholar
  24. 24.
    Auger FA, Rouabhia M, Goulet F, Berthod F, Moulin V, Germain L (1998) Tissue-engineered human skin substitutes developed from collagen-populated hydrated gels: clinical and fundamental applications. Med Biol Eng Comput 36:801–812PubMedCrossRefGoogle Scholar
  25. 25.
    Stark HJ, Willhauck MJ, Mirancea N, Boehnke K, Nord I, Breitkreutz D, Pavesio A, Boukamp P, Fusenig NE (2004) Authentic fibroblast matrix in dermal equivalents normalises epidermal histogenesis and dermoepidermal junction in organotypic co-culture. Eur J Cell Biol 83:631–645PubMedCrossRefGoogle Scholar
  26. 26.
    Lamme EN, Van Leeuwen RT, Brandsma K, Van Marle J, Middelkoop E (2000) Higher numbers of autologous fibroblasts in an artificial dermal substitute improve tissue regeneration and modulate scar tissue formation. J Pathol 190:595–603PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Luca Pontiggia
    • 1
  • Agnieszka Klar
    • 1
  • Sophie Böttcher-Haberzeth
    • 1
    • 2
  • Thomas Biedermann
    • 1
  • Martin Meuli
    • 2
  • Ernst Reichmann
    • 1
  1. 1.Tissue Biology Research UnitUniversity Children’s Hospital ZurichZurichSwitzerland
  2. 2.Department of Pediatric SurgeryUniversity Children’s Hospital ZurichZurichSwitzerland

Personalised recommendations