## 3 Results  
### Comparison of Normal h-CLAT and Co-Culture System h-CLAT  
The expression of CD54 and CD86 in THP-1 cells monocultured and co-cultured with keratinized NHEK cells as a skin model was evaluated. First, according to the h-CLAT protocol of the concentration setting test, the cell viability 75% (CV75) doses for each chemical were determined. CV75 doses for DNCB, LA, and IE of monocultured THP-1 cells were 5.1 mg/mL, 3.1 \(\times\) 10\({}^{3}\) mg/mL, and 1.1 \(\times\) 10\({}^{2}\) mg/mL, respectively. In contrast, CV75 doses for DNCB and LA under co-culture conditions were 6.1 mg/mL and 3.1 \(\times\) 10\({}^{3}\) mg/mL, respectively. The CV75 dose for IE in the co-culture condition could not be determined because THP-1 cell viability was maintained over 80% under the examined conditions (data not shown). Based on CV75 doses, the expression of CD54 and CD86 in THP-1 cells after exposure to each chemical at eight concentrations (common ratio of 1.2) was measured. In the case of IE in the co-culture condition, THP-1 cells were exposed to IE solution of CV75 dose for IE under monoculture conditions (111 \(\upmu\)g/mL) and seven higher concentrations (commonratio of 1.2). DNCB treatment upregulated CD54 and CD86 beyond the h-CLAT criteria (CD54 \(\geq\) 200% and/or CD86 \(\geq\) 150%) (Figure 2A). THP-1 cells co-cultured with keratinized NHEK cells also showed a positive response using h-CLAT criteria (Figure 2B). However, the RFIs of CD54 and CD86 in co-cultured THP-1 cells were lower than that of the normal h-CLAT (Figure 2A). For LA, the RFIs of CD54 and CD86 were under 200 and under 150, respectively, on both mono-cultured (Figure 3A) and co-cultured (Figure 3B) THP-1 cells. IE showed a negative response in the normal h-CLAT (monocultured THP-1 cells, Figure 4A), whereas IE upregulated CD54 and CD86 beyond the h-CLAT criteria in the THP-1 cells co-cultured with keratinized NHEK cells (Figure 4B). It was shown that co-culture could evaluate IE, which is a prohapten. Effects of Medium and Co-Culturing with NHEK Cells on CD54 and CD86 Expression Levels of THP-1 Cells  
The effect of medium and co-culture with keratinized NHEK cells on the expression of CD54 and CD86 in THP-1 cells was analyzed. First, the expression of CD54 and CD86 in THP-1 cells monocultured in RPMI medium, KI medium, and 1:1 and 20:7 mixed (vol.) medium of RPMI medium and KI medium was examined. Figure 5 shows the effects of each medium on the expression of CD54 and CD86 in monocultured THP-1 cells. In KI  
Figure 2: CD54 and CD86 expression induced by DNCB on THP-1 cells in monocultured (**A**) and co-cultured with keratinized NHEK cells (**B**). DNCB solution at eight concentrations prepared based on the CV75 determined for each exposure route was directly exposed to THP-1 cells (**A**), on the other hand, was applied on the surface of keratinized NHEK cells in cell culture insert (**B**). After 24 h of incubation, the expression of CD54 and CD86 and cell viability were analyzed using flow cytometry. Data are shown as relative fluorescence intensity (RFI, % of control). Mean values of RFI \(\pm\) SD for three individual experiments are shown. The black and gray dashed lines visually indicate the positive criteria for skin sensitization in h-CLAT for CD54 and CD86, respectively (CD54 \(\geq\) 200%, CD86 \(\geq\) 150%). medium, the expression levels of CD54 and CD86 were increased two-fold and three-fold, respectively, compared to the RPMI medium, which was a normal condition of h-CLAT (Figure 5A,B, monoculture). The reduction of the KI medium ratio to 50% (1:1 mixed medium) and 35% (20:7 mixed medium) did not suppress the expression of CD54 and CD86. In the case of the co-culture system with keratinized NHEK cells, the expression levels of CD54 and CD86 in THP-1 cells in RPMI medium were increased by 2-fold and 2.5-fold, respectively, compared to the normal h-CLAT conditions (monocultured THP-1 cells in RPMI). In the case of KI medium and 1:1 and 20:7 mixed (vol.)