Archives of Dermatological Research

, Volume 296, Issue 5, pp 203–211 | Cite as

A simple reconstructed human epidermis: preparation of the culture model and utilization in in vitro studies

  • Y. Poumay
  • F. Dupont
  • S. Marcoux
  • M. Leclercq-Smekens
  • M. Hérin
  • A. Coquette
Original Paper

Abstract

The preparation of a reconstructed human epidermis is described with examples of its utilization in in vitro studies. The model was obtained by culturing normal human keratinocytes at high cell density for 14 days in serum-free and high calcium (1.5 mM) medium on an inert polycarbonate filter at the air-liquid interface. These stratified cultures showed histological features similar to those observed in vivo in the epidermis: a proliferating basal layer and differentiating spinous, granular, and cornified layers. Electron microscopy illustrated lamellar bodies, junctions and keratohyalin granules. Immunofluorescent localization of epidermal markers (keratins 14 and 10, involucrin and filaggrin) revealed typical differentiation. This in vitro reconstructed tissue was used in studies of toxic effects of chemicals. The modelled tissue showed progressive cytotoxicity of a skin irritant (benzalkonium chloride) and a sensitizer (dinitrochlorobenzene) as assessed by MTT assay. Moreover, differential release of interleukin-1α and interleukin-8 were measured after 20 h of incubation allowing the irritant to be distinguished from the sensitizer. Permeation studies indicated efficient barrier function of the reconstructed epidermis, as well as metabolizing properties towards hormones. This model can be custom-made and is potentially useful for studies involving keratinocytes in the epidermis, in basic science, dermatology or toxicology.

Keywords

Reconstructed human epidermis Keratinocyte Barrier function Irritant Sensitizer 

Notes

Acknowledgements

The authors wish to thank Dr. B. Bienfait (Clinique Saint-Luc, Bouge) for providing samples of normal skin. The occasional technical assistance of R. Déom, D. Van Vlaender, F. Herphelin and C. Devignon is gratefully acknowledged. S. Marcoux was in receipt of a fellowship from the Fonds pour la Formation à la Recherche dans l’Industrie et l’Agriculture (FRIA). This work was partly supported by grant 2.4.506.01F from F.R.F.C. to Y. Poumay.

References

  1. Altenburger R, Kissel T (1998) Biotransformation of estradiol in the human keratinocyte cell line HaCaT: metabolism kinetics and the inhibitory effect of ethanol. Pharm Res 15:1684–1689CrossRefPubMedGoogle Scholar
  2. Bernard FX, Barrault C, Deguercy A, De Wever B, Rosdy M (2000a) Expression of type-1 5α-reductase and metabolism of testosterone in reconstructed human epidermis (SkinEthic): a new model for screening skin-targeted androgen modulators. Int J Cosmetic Sci 22:397–407CrossRefGoogle Scholar
  3. Bernard FX, Barrault C, Deguercy A, De Wever B, Rosdy M (2000b) Development of a highly sensitive in vitro phototoxicity assay using the SkinEthic reconstructed human epidermis. Cell Biol Toxicol 16:391–400CrossRefPubMedGoogle Scholar
  4. Boyce ST, Ham RG (1983) Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free serial culture. J Invest Dermatol 81:33S–40SCrossRefPubMedGoogle Scholar
  5. Coquette A, Berna N, Vandenbosch A, Rosdy M, Poumay Y (1999) Differential expression and release of cytokines by an in vitro reconstructed human epidermis following exposure to skin irritant and sensitizing chemicals. Toxicol In Vitro 13:867–877CrossRefGoogle Scholar
  6. Coquette A, Berna N, Poumay Y, Pittelkow MR (2000) The keratinocyte in cutaneous irritation and sensitization. In: Kydonieus AF, Wille JJ (eds) Biochemical modulation of skin reactions. Transdermals, topicals, cosmetics. CRC Press, Boca Raton, pp 125–143Google Scholar
  7. Coquette A, Berna N, Vandenbosch A, Rosdy M, De Wever B, Poumay Y (2003) Analysis of interleukin-1α (IL-1α) and interleukin-8 (IL-8) expression and release in in vitro reconstructed human epidermis for the prediction of in vivo skin irritation and/or sensitisation. Toxicol In Vitro 17:311–321PubMedGoogle Scholar
  8. De Brugerolle de Fraisinette A, Picarles V, Chibout S, Kolopp M, Medina J, Burtin P, Ebelin ME, Osborne S, Mayer FK, Spake A, Rosdy M, De Wever B, Ettlin RA, Cordier A (1999) Predictivity of an in vitro model for acute and chronic skin irritation (SkinEthic) applied to the testing of topical vehicles. Cell Biol Toxicol 15:121–135CrossRefPubMedGoogle Scholar
  9. Doucet O, Garcia N, Zastrow L (1998) Skin culture model: a possible alternative to the use of excised human skin for assessing in vitro percutaneous absorption. Toxicol In Vitro 12:423–430CrossRefGoogle Scholar
  10. Franz TJ, Lehman PA (2000) The skin as a barrier: structure and function. In: Kydonieus AF, Wille JJ (eds) Biochemical modulation of skin reactions. Transdermals, topicals, cosmetics. CRC Press, Boca Raton, pp 15–33Google Scholar
  11. Gysler A, Kleuser B, Sippl W, Lange K, Korting HC, Höltje HD, Schäfer-Korting M (1999) Skin penetration and metabolism of topical glucocorticoids in reconstructed epidermis and excised human skin. Pharm Res 16:1386–1391CrossRefPubMedGoogle Scholar
  12. Hennings H, Michael D, Cheng C, Steinert PM, Holbrook K, Yuspa SH (1980) Calcium regulation of growth and differentiation of mouse epidermal cells in culture. Cell 19:245–254CrossRefPubMedGoogle Scholar
  13. Hobbs RM, Watt FM (2003) Regulation of interleukin-1α expression by integrins and epidermal growth factor receptor in keratinocytes from a mouse model of inflammatory skin disease. J Biol Chem 278:19798–19807CrossRefPubMedGoogle Scholar
  14. Kobayashi H, Aiba S, Yoshino Y, Tagami H (2003) Acute cutaneous barrier disruption activates epidermal p44/42 and p38 mitogen-activated protein kinases in human and hairless guinea pig skin. Exp Dermatol 12:734–746PubMedGoogle Scholar
  15. Pasonen-Seppänen S, Suhonen TM, Kirjavainen M, Suihko E, Urtti A, Miettinen M, Hyttinen M, Tammi M, Tammi R (2001) Vitamin C enhances differentiation of a continuous keratinocyte cell line (REK) into epidermis with normal stratum corneum ultrastructure and functional permeability barrier. Histochem Cell Biol 116:287–297CrossRefPubMedGoogle Scholar
  16. Pittelkow MR, Scott RE (1986) New techniques for the in vitro culture of human skin keratinocytes and perspectives on their use for grafting of patients with extensive burns. Mayo Clin Proc 61:771–777PubMedGoogle Scholar
  17. Ponec M, Weerheim A, Kempenaar J, Mulder A, Gooris GS, Bouwstra J, Mommaas AM (1997) The formation of competent barrier lipids in reconstructed human epidermis requires the presence of vitamin C. J Invest Dermatol 109:348–355CrossRefPubMedGoogle Scholar
  18. Potts RO, Bommannan DB, Guy RH (1992) Percutaneous absorption. In: Muhktar H (ed) Pharmacology of the skin. CRC Press, Boca Raton, pp 13–28Google Scholar
  19. Poumay Y, Leclercq-Smekens M (1998) In vitro models of epidermal differentiation. Folia Medica (Plovdiv) 40:5–12Google Scholar
  20. Poumay Y, Pittelkow MR (1995) Cell density and culture factors regulate keratinocyte commitment to differentiation and expression of suprabasal K1/K10 keratins. J Invest Dermatol 104:271–276CrossRefPubMedGoogle Scholar
  21. Poumay Y, Herphelin F, Smits P, De Potter IY, Pittelkow MR (1999) High-cell-density phorbol ester and retinoic acid upregulate involucrin and downregulate suprabasal keratin 10 in autocrine cultures of human epidermal keratinocytes. Mol Cell Biol Res Commun 2:138–144CrossRefPubMedGoogle Scholar
  22. Pruniéras M, Regnier M, Woodley D (1983) Methods for cultivation of keratinocytes with an air-liquid interface. J Invest Dermatol 81:28S–33SCrossRefPubMedGoogle Scholar
  23. Regnier M, Caron D, Reichert U, Schaefer H (1992) Reconstructed human epidermis: a model to study in vitro the barrier function of the skin. Skin Pharmacol 5:49–56PubMedGoogle Scholar
  24. Rosdy M, Claus LC (1990) Terminal epidermal differentiation of human keratinocytes grown in chemically defined medium on inert filter substrates at the air-liquid interface. J Invest Dermatol 95:409–414CrossRefPubMedGoogle Scholar
  25. Southee JA, McPherson JP, Osborne R, Carr GJ, Rasmussen E (1999) The performance of the tissue equivalent assay using the skin2TM ZK1200 model in the COLIPA international validation study on alternatives to the Draize eye irritation test. Toxicol In Vitro 13:355–373CrossRefGoogle Scholar
  26. Stinchcomb AL (2003) Xenobiotic bioconversion in human epidermis models. Pharm Res 20:1113–1118CrossRefPubMedGoogle Scholar
  27. Wille JJ, Pittelkow MR, Shipley GD, Scott RE (1984) Integrated control of growth and differentiation of normal human prokeratinocytes cultured in serum-free medium: clonal analyses, growth kinetics, and cell cycle studies. J Cell Physiol 121:31–44PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Y. Poumay
    • 1
  • F. Dupont
    • 1
  • S. Marcoux
    • 1
  • M. Leclercq-Smekens
    • 1
  • M. Hérin
    • 1
  • A. Coquette
    • 2
  1. 1.Département Histologie-EmbryologieFacultés Universitaires Notre-Dame de la PaixNamurBelgium
  2. 2.SGS Biopharma S.A.WavreBelgium

Personalised recommendations