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Cell Biology and Toxicology

, Volume 10, Issue 5–6, pp 353–359 | Cite as

A new three-dimensional culture of human keratinocytes: optimization of differentiation

  • J. Font
  • F. Braut-Boucher
  • J. Pichon
  • M. S. Noel-Hudson
  • M. P. Muriel
  • M. Bonnet
  • J. Wepierre
  • M. Aubery
Article

Abstract

Many attempts have been made to obtain reconstructed human epidermis comprised of keratinocytes and extracellular-matrix constituents (essentially collagen) in the presence or absence of fibroblasts. A simple model of cultured human keratinocytes grown at the air-liquid interface of a noncoated artificial membrane, has been developed. This culture system offers many advantages: easy control of environmental factors and routine examination using optical or electronic microscopy, immunohistochemistry and indirect immunofluorescence techniques. This model enables the analysis of well-known differentiation markers and also intregrins, a family of cell-surface molecules involved in cell-cell and cell-extracellular matrix interactions, whose receptors are expressed on all basal keratinocytes.

In our culture system, the expression of the different integrin subunits (α2, α3, α5, α6, β1) was studied as a function of the differentiation state in two different media (K-SFM or DMEM/Ham's F12) supplemented with 5% fetal calf serum and adjusted to 1.5 mmol/L calcium. The most significant data are the preponderant expression of the α2 and α3 subunits in the basal and suprabasal layers, with membrane expression differing according to the culture medium; terminal differentiation was obtained in DMEM/Ham's F12. The use of membrane inserts represents a significant technological advance in culturing keratinocytes and is an easy-to-handle and valid model for determining the influence of physiological or pharmacological factors on cell proliferation or differentiation.

Keywords

differentiation human keratinocytes integrins in vitro three-dimensional model 

Abbreviations

DMEM

Dulbecco's Modified Eagle's Medium

EGF

epidermal growth factor

FCS

fetal calf serum

K-SFM

keratinocyte serum free medium

MAb

monoclonal antibody

NHEK

normal human epidermal keratinocytes

PBS

Dulbecco's phosphate-buffered saline

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Copyright information

© Kluwer Academic Publishers 1994

Authors and Affiliations

  • J. Font
    • 1
  • F. Braut-Boucher
    • 1
  • J. Pichon
    • 1
  • M. S. Noel-Hudson
    • 2
  • M. P. Muriel
    • 1
  • M. Bonnet
    • 2
  • J. Wepierre
    • 2
  • M. Aubery
    • 1
  1. 1.Laboratoire de Glycobiologie et Reconnaissance Cellulaire, INSERM U 180UFR Biomédicale des Saints-PèresParisFrance
  2. 2.Laboratoire de Pharmacologie, Unité de DermopharmacologieFaculté de PharmacieChâtenay-Malabry CédexFrance

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