3D reconstruction of skin and spatial mapping of immune cell density, vascular distance and effects of sun exposure and aging

Mapping the human body at single cell resolution in three dimensions (3D) is an important step toward a “digital twin” model that digitizes organ structure and the dynamics of cell-cell interactions. Current approaches for 3D reconstruction of multiplexed serial sections are relatively cumbersome and not automated. We present a novel 3D reconstruction workflow for multiplexed sequential tissue sections: MATRICS-A (Multiplexed Image Three-D Reconstruction and Integrated Cell Spatial - Analysis). This combines a reproducible and streamlined method for two-dimensional (2D) cell segmentation and cell type classification, followed by 3D volume reconstruction. We also provide novel tools for 3D visualization of immune cell cluster density, cell-to-vasculature distance, and distance maps for cellular markers of UV damage/repair and proliferation to the skin surface. We used MATRICS-A to reconstruct 26 serial sections of fixed skin from 12 donors aged between 32-72 years. Samples were collected from six distinct anatomical regions with mild to marked sun exposure effects. Each tissue section was multiplexed with 18 fluorescently labeled antibodies covering 14 cell types in the epidermis and dermis. We demonstrate several new findings that are only possible in 3D, We present novel visualization of immune cell clusters and show that there are 10-70% more T cells (total) within 30 µm of a T helper cell in 3D vs 2D. Distances of cell markers of DNA damage (p53), DNA repair (DDB2) and proliferation (Ki67) to the skin surface were consistent across all ages/sun exposure. MATRICS-A provides a new powerful integrated open access approach to quantify 3D spatial cell relationships in healthy and aging organs and could be further extended to diseased organs.