Co-Culture of THP-1 Cells and Normal Human Epidermal Keratinocytes (NHEK) for Modified Human Cell Line Activation Test (h-CLAT)

Abstract

To improve the accuracy of skin sensitization prediction of chemicals by conventional alternative methods using cells, it is important to reproduce the environment of skin in vitro, such as the crosstalk between keratinocytes and dendritic cells (DCs). We developed a skin sensitization test system based on the markers and criteria of the human cell line activation test (h-CLAT), which combines THP-1 cells as a surrogate for DCs and keratinized normal human epidermal keratinocytes (NHEK). After exposure to chemicals via keratinized NHEK, the cell surface expression of CD54 and CD86 on THP-1 was measured by flow cytometry. This co-culture system evaluated 2,4-dinitrochlorobenzene (DNCB), a typical sensitizer, as positive, lactic acid (LA), a non-sensitizer, as negative, and isoeugenol (IE), a prohapten that requires biological activation to acquire skin sensitization, as positive. However, the expression levels of CD54 and CD86 in DNCB-treated THP-1 were lower than those in normal h-CLAT. Therefore, we investigated the effects of the medium and secretion by NHEK cells on THP-1 cells. CD54 and CD86 expression was enhanced in monocultured THP-1 in the medium for keratinized NHEK and in the conditioned medium of keratinized NHEK. The increase in CD54 and CD86 by changes in the medium type was higher than that by the NHEK secretion; therefore, it was found that the medium composition has a large effect on the evaluation index among the experimental parameters in the co-culture system. It is necessary to find the optimal medium for immunotoxicity assessment in the co-culture system.