In the skin sensitization assessment, the in vivo method, specifically the Local Lymph Node Assay (LLNA), is regarded as the gold standard. However, to adhere to the 3R principle (Reduction, Refinement, Replacement), which advocates for the Reduction, Refinement, and Replacement of animal testing, and to enhance the ethical standards of risk assessment, a greater number of in vitro and in silico testing methods have been developed. While these in vitro and in silico methods offer a higher throughput and a more ethical approach to risk assessment, they each come with their own set of limitations. For instance, most in vitro tests can only be used for a single readout. On the other hand, in silico methods lack physiological and biological feedback. The adverse outcome pathway (AOP) for skin sensitization, as defined by the Organization for Economic Cooperation and Development (OECD) guidelines, involves four key events, which are protein binding (Key Event 1 – KE1), keratinocyte activation (Key Event 2 – KE 2), dendritic cell activation (Key Event 3 – KE 3), and T-lymphocyte activation (Key Event 4 – KE 4) (OECD, 2014). While animal-based assays are reasonably accurate, their skin is physiologically different than human skin. Therefore, this thesis aimed to develop the ‘ImmuSkin-MT’, a 3D-human skin model with immune cells, designed to address the limitations inherent in in vitro skin sensitization assays. The thesis began with 2D co-culture of keratinocytes and immune cells, which have shown to fall short in detecting a skin sensitizer. In parallel to the development of the 2D co-culture assay, the advantages of the autologous in comparison to the allogenic 2D co-culture were investigated. The results indicated that within the donor pool used, allogenic 2D co-culture did not lead to an alloreactive response in the cells. Therefore, whether autologous or allogenic cell pairings were used in the co-culture, they had no impact on the development of the assay. Later, the project progressed and the 'ImmuSkin-MT', created from human hair follicle-derived keratinocytes (HFDK), fibroblasts (HFDF), and immune cells were developed. monocyte-derived Langerhans cells (MoLC) and CD4+ naïve T-lymphocytes were integrated into the reconstructed human skin models (RHS). ImmuSkin-MT were topically exposed to various substances, including non-sensitizers and contact sensitizers. Using the MoLC activation and the stimulation index (SI) of T-lymphocytes, results demonstrate the effectiveness of ImmuSkin-MT in replicating KE 3 and 4 of skin sensitization in response to the skin sensitizers. This model presents a promising alternative to animal testing for contact sensitizers, contributing to more ethical and precise skin sensitization assessment techniques. In conclusion, ImmuSkin-MT represents a significant advancement in skin sensitization assessment by capturing multiple key events simultaneously. The project's findings have the potential to optimize skin sensitization testing methods, providing more accurate, reliable, and ethical alternatives to traditional animal-based assays.
