Vitamin C enhances differentiation of a continuous keratinocyte cell line (REK) into epidermis with normal stratum corneum ultrastructure and functional permeability barrier

• Published in: Histochemistry and cell biology 2001-10, Vol.116 (4), p.287-297
• Main Authors: Pasonen-Seppänen, S, Suhonen, T M, Kirjavainen, M, Suihko, E, Urtti, A, Miettinen, M, Hyttinen, M, Tammi, M, Tammi, R
• Format: Article
• Language: English
• Subjects: Animals; Ascorbic acid; Ascorbic Acid - pharmacology; Cell culture; Cell differentiation; Cell Differentiation - drug effects; Cell Line; Collagen; Corticosterone; Corticosterone - pharmacokinetics; Epidermis; Epidermis - cytology; Epidermis - drug effects; Epidermis - metabolism; Filaggrin; Filaments; Gene Expression Regulation - drug effects; Granule cells; Humans; Intermediate Filament Proteins - drug effects; Intermediate Filament Proteins - genetics; Intermediate Filament Proteins - metabolism; Keratin; Keratin-10; Keratinocytes - cytology; Keratinocytes - drug effects; Keratinocytes - metabolism; Keratins - drug effects; Keratins - metabolism; Lamellae; Lipids; Mannitol; Membrane Lipids - metabolism; Microscopy, Electron; mRNA; Permeability; Protein Precursors - drug effects; Protein Precursors - genetics; Protein Precursors - metabolism; Rats; RNA, Messenger - drug effects; RNA, Messenger - genetics; RNA, Messenger - metabolism; Skin - cytology; Skin - drug effects; Skin - ultrastructure; Skin Physiological Phenomena - drug effects; Stratum corneum; Supplements; Time Factors; Ultrastructure; Vitamin C; Water Loss, Insensible - drug effects; Water Loss, Insensible - physiology; Western blotting
• ISSN: 0948-6143; 1432-119X
• DOI: 10.1007/s004180100312

A continuous rat epidermal cell line (rat epidermal keratinocyte; REK) formed a morphologically well-organized epidermis in the absence of feeder cells when grown for 3 weeks on a collagen gel in culture inserts at an air-liquid interface, and developed a permeability barrier resembling that of human skin. By 2 weeks, an orthokeratinized epidermis evolved with the suprabasal layers exhibiting the differentiation markers keratin 10, involucrin, and filaggrin. Granular cells with keratohyalin granules and lamellar bodies, and corneocytes with cornified envelopes and tightly packed keratin filaments were present. Morphologically, vitamin C supplementation of the culture further enhanced the normal wavy pattern of the stratum corneum, the number of keratohyalin granules present, and the quantity and organization of intercellular lipid lamellae in the interstices of the stratum corneum. The morphological enhancements observed with vitamin C correlated with improved epidermal barrier function, as indicated by reduction of the permeation rates of tritiated corticosterone and mannitol, and transepidermal water loss, with values close to those of human skin. Moreover, filaggrin mRNA was increased by vitamin C, and western blots confirmed higher levels of profilaggrin and filaggrin, suggesting that vitamin C also influences keratinocyte differentiation in aspects other than the synthesis and organization of barrier lipids. The unique REK cell line in organotypic culture thus provides an easily maintained and reproducible model for studies on epidermal differentiation and transepidermal permeation.