Co-Culture of THP-1 Cells and Normal Human Epidermal Keratinocytes (NHEK) for Modified Human Cell Line Activation Test (h-CLAT)  
[PERSON]  
1 Department of Chemistry and Life Science, Graduate School of Engineering Science, Yokohama National University, 79-5 Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan; [EMAIL_ADDRESS]  
[PERSON]  
2 Department of Chemical and Energy Engineering, Graduate School of Engineering, Yokohama National University, 79-5 Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan; [EMAIL_ADDRESS]  
[PERSON]  
3 Division of Materials Science and Chemical Engineering, Faculty of Engineering, Yokohama National University, 79-5 Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan; [EMAIL_ADDRESS] (J.F. ); [EMAIL_ADDRESS] (H.I.) [PERSON]  
3 Division of Materials Science and Chemical Engineering, Faculty of Engineering, Yokohama National University, 79-5 Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan; [EMAIL_ADDRESS] (J.F. ); [EMAIL_ADDRESS] (H.I.) [PERSON]  
4 Itagaki Cosmetics Safety Consulting, 5-11-19-2504 Minamidai, Minami-ku, Sagamihara 252-0314, Japan  
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these KEs. The KE1 of AOP [2]: Direct Peptide Reactivity Assay (DPRA) and Amino Acid Derivative Reactivity Assay (ADRA), KE2 [3]: KeratinoSens(tm) and LuSens, KE3 [4]: the human cell line activation test (h-CLAT), U937 cell line activation test (U-SENSTM), and the interleukin-8 reporter gene assay (IL-8 Luc assay). However, it is not possible to completely replace conventional animal testing to predict the skin-sensitizing potential with the single test of these alternative methods because skin sensitization is a complex immune response [5,6]. Therefore, the Organization for Economic Co-operation and Development (OECD) reported the guidance on integrated approaches to testing and assessment (IATA) based on AOP [7]. Many testing strategies combining above alternative methods or other novel methods including in silico methods using the physicochemical index of the chemical substance have been proposed as \"defined approaches\" (DAs) that form part of IATA of skin sensitization. Some of the reported DAs show at least comparable performance in predicting the Local Lymph Node Assay (LLNA) hazard (sensitizer/non-sensitizer) [8]. Although several studies on the combination of approaches are underway, there are restrictions on the scope of application of chemical substances to alternatives to animal testing, for example, testing of pro-/pre-hapten, lipophilic materials, and those of low solubility are still challenging. Therefore, one of the studies for solving the above problem is an immune-competent in vitro co-culture system as an approach for skin sensitization assessment. It attempts to solve the above problem by observing a series of molecular or cell interactions between KEs within a single method. To date, co-culture systems combining keratinocytes and THP-1 cells used as a surrogate for DCs in h-CLAT have been reported to be useful as an alternative method for evaluating skin sensitization [9]. For example, [PERSON] et al. directly co-cultured the epithelial-like cell line NCTC2544 with THP-1 cells and showed that keratinocytes promote DC maturation [10]. In addition, it was reported that the co-culture system of human epidermal keratinocyte cell line HaCaT and THP-1 cells detected the dependence of the concentration of skin sensitizers and showed a possibility to predict the sensitization intensity [11,12]. In contrast to the above system of homogeneous co-culture of two types of cells, [PERSON] et al.