## 4 Discussion
According to the comparison of the results, the co-cultured NHEK cells showed that the upregulation of CD54 and CD86 was more suppressed in the keratinized state than in the undifferentiated state. Keratinization was considered to reduce the amount of secretions that affects THP-1 cells. It was speculated that this was because it became stable as it \"matured\" [43]. As a further hypothesis, one of the differences between the undifferentiated state and the keratinized state of NHEK cells is the number of epidermal basal cells. Epidermal basal cells proliferate in KG2 medium, whereas proliferating cells and keratinized cells coexist in KI medium and the number of epidermal basal cells is expected to be lower after two weeks of air-liquid interface culture. Therefore, it was considered that the epidermal basal cells produce secretions that enhance the expression of CD54 and CD86. In addition, when co-cultured with undifferentiated NHEK cells, the expression levels of CD54 and CD86 in THP-1 cells varied widely, and there seemed to be considerable uncertainty about predicting sensitization. It was considered that reducing the stimulation of the keratinized NHEK cells with RPMI medium suppressed the production of factors that affect the expression of CD54 and CD86 in THP-1 cells. Therefore, we investigated the acclimatization of NHEK cells to RPMI medium. For the collected RPMI medium, in which only NHEK cells were cultured for 6, 12, 24, and 36 h, the longer the culture time of NHEK cells, the lower the expression levels of CD54 and CD86 in THP-1 cells. These results suggest that the amounts of factors produced by NHEK cells decrease over time. Keratinocyte growth factor (KGF) produced by fibroblasts is involved in keratinocyte proliferation; therefore, the primary keratinocytes can be maintained by culturing with feeder layer or a conditioned medium with fibroblasts [44]. Medium changes can be stressful for keratinocyte cultures, given that an environment containing the necessary nutrients or factors is required. Undernourishment stress, such as deficiency of pantothenic acid, a water-soluble vitamin, arrests cell proliferation and promotes differentiation [45], and keratinocytes, either constitutively or upon stimulation, express IL-1, -6, -7, -8, -10, -12, -15, -18, and -20 [46] and TGF-81 [47]. As an interpretation of our results, it was considered that the stress on NHEK cells caused by the change to RPMI medium was alleviated during 36 h of culture, and the secretion of factors that enhance the expression of CD54 and CD86 in THP-1 cells was decreased. It is generally known that signal proteins generated by cells are decomposed over time to maintain a dynamic equilibrium state [48]. We have revealed the effect of the medium. However, we have not yet optimized it to reproduce the in vivo sensitization response. It is necessary to verify whether it is optimal to increase or decrease the expression levels of CD54 and CD86 by changing the medium. We have shown that the base (CD54 and CD86 expression levels under control conditions) increases with changes in the medium, which leads to a decrease in evaluation sensitivity. First, we are thinking that it is necessary to develop the co-culture evaluation system to suppress the rise of the base by mixing the medium. In conclusion, this study showed that when producing a co-culture system for skin sensitization evaluation, it is necessary to select a medium that is optimal for culturing both cells and does not affect the functionality of the cells used as an evaluation index. In addition, it was reported that the medium stimulates THP-1 and NHEK cells, and through cell-cell interaction, the evaluation index of skin sensitization fluctuated in THP-1 cells. It is important to select a medium for THP-1 cells as well as NHEK cells that assist THP-1 cells in the co-culture system. The knowledge gained from this study is used to support the development and evaluation of novel alternative approaches for the evaluation of skin-sensitizing chemicals.