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

A continuous rat epidermal cell line (rat epidermal keratinocyte; REK) form ed a morphologically well-organized epidermis in the absence of feeder cell s when grown for 3 weeks on a collagen gel in culture inserts at an air-liq uid interface, and developed a permeability barrier resembling that of huma n skin. By 2 weeks, an orthokeratinized epidermis evolved with the suprabas al layers exhibiting the differentiation markers keratin 10, involucrin, an d filaggrin. Granular cells with keratohvalin granules and lamellar bodies, and corneocytes with cornified envelopes and tightly packed keratin filame nts were present. Morphologically, vitamin C supplementation of the culture further enhanced the normal wavy pattern of the stratum corneum, the numbe r of keratohyalin granules present, and the quantity and organization of in tercellular lipid lamellae in the interstices of the stratum corneum. The m orphological enhancements observed with vitamin C correlated with improved epidermal barrier function, as indicated by reduction of the permeation rat es of tritiated corticosterone and mannitol, and transepidermal water loss, with values close to those of human skin. Moreover, filaggrin mRNA was inc reased by vitamin C, and western blots confirmed higher levels of profilagg rin and filaggrin, suggesting that vitamin C also influences keratinocyte d ifferentiation in aspects other than the synthesis and organization of barr ier lipids. The unique REK cell line in organotypic culture thus provides a n easily maintained and reproducible model for studies on epidermal differe ntiation and transepidermal permeation.