## 4 Discussion  
Since the epidermis model used in this study uses an in-house model instead of the existing commercial model, this quality evaluation was performed prior to the sensitization test. It was confirmed that this model meets the criteria for barrier function described in the standardized test method (TG439) for skin irritation tests using the three-dimensional cultured epidermis model shown by the OECD [25]. In addition, the quality of the epidermis model used was ensured by measuring the transdermal electrical resistance (TEER), which is an index of barrier function, and observing the cell layer by microscopic observation after HE staining of the immobilized section. The materials and methods of the experiment and the details of the results are described in Supplemental Materials. Observing crosstalk between skin cells and immune cells is necessary for predicting skin sensitization of chemical compounds. However, it was considered that cell functionality such as the immune response used for an evaluation index is very sensitive to culture conditions. The main purpose of this study was to obtain expertise about the effects of the co-culture system on the evaluation index of h-CLAT. First, we confirmed that DNCB was evaluated as positive (Figure 2B) and LA as negative (Figure 3B) in co-culture h-CLAT. This study demonstrated that IE, which was evaluated as negative in normal h-CLAT (Figure 4A), was evaluated as positive in the co-culture system (Figure 4B). The difference between monoculture and co-culture in IE results can be explained by the metabolization of IE by NHEK cells. For a pre- or pro-hapten, the formation of reactive hapten by spontaneous air-oxidation (prehapten) or enzyme-mediated activation (prohapten) is needed to obtain the skin sensitization potential [26,27]. It has been reported that keratinocytes express metabolic enzymes such as the cytochrome P450 (CYP) superfamily [28]. Similar to the report by [PERSON] et al. that the HaCaT/THP-1 cell co-culture system improved the evaluation of pro-/pre-hapten [12], it was considered that the effect of metabolism by NHEK cells was observed in this study as well. It is considered that co-culture system allows to observe the effects of the parent compound and the metabolite simultaneously. In contrast, it is difficult to examine these separately in a co-culture system; therefore, it is important to break down the pathways individually and examine them as in  
Figure 6: The effect of conditioned medium on cell viability and basal CD54 and CD86 expression on monocultured THP-1 cells. THP-1 cells were cultured in the conditioned medium in which keratinized NHEK cells were cultured for 6, 12, 24, and 36 h, and cell viability and their expression of CD54 and CD86 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. * \(p\) < 0.05, significantly different from the control (“normal”) (by ANOVA followed by Dunnett’s test). the study in which THP-1 cells were treated with prohapten bioactivated by S9 fraction [29]. Concluding from the results of Figure 1 to Figure 4, we showed the possibility of evaluating the effect of prohapten, which acquires sensitization for the first time by being metabolized by the epidermis model. Therefore, we also emphasize the importance of developing a co-culture system. While demonstrating its usefulness for predicting skin sensitization, co-cultured THP-1 cells showed a lower rate of increase in CD54 and CD86 expression levels upon DNCB exposure (Figure 2B) than monocultured THP-1 cells (Figure 2A). In the control cells in the co-culture system (THP-1 cells co-cultured with NHEK cells in 20:7 mixed medium), the MFI values of CD54 and CD86 were higher than those in the control cells in normal h-CLAT (THP-1 cells monocultured in RPMI medium). Therefore, it was speculated that the difference between the control cells and the DNCB-treated cells was difficult to observe in the co-culture system.