P07-13 3D imaging of multiple markers of human reconstructed skin model based on tissue clearing method skin-DISCO

Immunofluorescence staining of 2D slices has been widely used in the biological effects evaluation of cosmetic ingredients, especially in efficacy and toxicology assessments. Biological markers such as reactive oxygen species (ROS), advanced glycation end-products (CML), and skin barrier-associated proteins (e.g., ZO-1, Filaggrin) are commonly used to assess oxidative stress, protein glycation, proliferation, and barrier integrity, which are key endpoints in dermal toxicity evaluation. However, 2D slice staining may have limitations such as unclear spatial relationships and local slice not reflecting the whole tissue. In this study, we developed a 3D tissue clearing approach, skin-DISCO, optimized from the neurological research method iDISCO, to visualize and quantify multiple biomarkers in full-thickness reconstructed human skin model (T-skin ™) exposed to irritants or active compounds. Immunofluorescence analysis was performed both on 2D sections and on skin-DISCO to compare sensitivity and spatial accuracy. The following key-markers were included: Oxidative stress & damage: ROS, CML; Proliferation: Ki67; Extracellular matrix integrity: Fibrillin-1, Collagen IV, Collagen XVII; Barrier function: TGM1, ZO-1, Filaggrin. For slices immunostaining, each group harvested 3 (samples) x 1 (slices) x 5 images, the mean fluorescence intensity or positive cell numbers was used for statistical analysis. The expression level of Ki67, Collagen IV, Collagen XVII, ZO-1 and Filaggrin were significantly increased in Vitamin C and retinol group. The expression level of TGM1 was significantly reduced in Vitamin C and retinol group. For 3D tissue clearing method, each group harvested 3 whole-tissue images. The whole tissue expression pattern of the markers mentioned above was harvested, the changes were in line with the 2D sections. 3D images provided some spatial information like some of the tissue was not homogeneity, the sections may not show this. The videos of 3D images also provided a more attractive approach for data presenting. In summary, the 3D visualization provided by skin-DISCO offers enhanced insight into toxicological marker distribution, tissue-level variability, and data visualization quality, supporting its application in advanced safety and risk assessment for dermal exposure studies.