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

• Published in: Cell biology and toxicology 1994-12, Vol.10 (5-6), p.353-359
• Main Authors: FONT, J, BRAUT-BOUCHER, F, PICHON, J, NOEL-HUDSON, M. S, MURIEL, M. P, BONNET, M, WEPIERRE, J, AUBERY, M
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Cell Adhesion - physiology; Cell Differentiation - genetics; Cell Differentiation - physiology; Cell Division - genetics; Cell Division - physiology; Cells, Cultured; Culture Media - chemistry; Extracellular Matrix Proteins - metabolism; Extracellular Matrix Proteins - physiology; Fluorescent Antibody Technique; Fundamental and applied biological sciences. Psychology; Humans; Integrins - biosynthesis; Integrins - metabolism; Keratinocytes - cytology; Keratinocytes - metabolism; Keratinocytes - secretion; Keratinocytes - ultrastructure; Membranes, Artificial; Microscopy, Electron; Skin - growth & development; Vertebrates: skin, associated glands, phaneres, light organs, various exocrine glands (salt gland, uropygial gland...), adipose tissue, connective tissue
• ISSN: 0742-2091; 1573-6822
• DOI: 10.1007/BF00755782

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 integrins, 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 (alpha 2, alpha 3, alpha 5, alpha 6, beta 1) 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 alpha 2 and alpha 3 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.