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Development of skin inflammation test model by co-culture of reconstituted 3D skin and RAW264. 7 cells

Chung, E;Choi, H;Lim, JE;Son, Y;

We developed in vitro test model for assessing skin inflammation. This model consisted with co-culture of a stratified 3D skin and RAW264.7 cells in transwell. The defined expression of Keratin 5 and 10 was observed in the basal layer and suprabasal layer of the epidermis in the 3D skin, respectively. Skin barrier, determined by immunoreactivity of involucrin and filaggrin was well developed in the 3D skin. Importantly, elevated IL-1 which plays an essential role in the maintenance of skin barrier function and TNF- in the dermal layer was detected, suggesting immunologically active reconstitution of the skin. RAW264.7 cells were morphologically transformed in response to lipopolysaccharides (LPS) either vacuolated or spindle shaped, which was accompanied with the induced expression of inducible nitric oxide synthase (iNOS), cycloxygenase-2 (COX-2), and cytokines. However, topical administration of LPS to the co-culture system reduced LPS-responding cells, which accompanied with COX-2 reduction. In addition, impaired production of TNF- and reduction of IL-1 were observed in co-culture system. It represents stratified 3D skin actively played as a skin barrier and provided innate immunity in response to LPS. Eventually, the immune modulating activity of 3D skin alleviated LPS-induced immune responses of RAW264.7 cells alone. We propose that this co-culture system for assessing skin inflammation in vitro and this model could serve as an alternative to animal experiments for predicting skin sensitization potential. Key words co-cultureRAW264.7 cells3D skininnate immunityskin inflammation