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.

以单细胞分辨率开展三维（3D）人体图谱绘制，是构建数字孪生（digital twin）模型的关键一步——该模型可将器官结构与细胞间互作动态进行数字化复刻。当前针对多重标记连续切片的三维重建方法普遍较为繁琐，且无法实现自动化操作。本研究针对多重标记连续组织切片提出了一种全新的三维重建工作流：MATRICS-A（Multiplexed Image Three-D Reconstruction and Integrated Cell Spatial Analysis，即多重成像三维重建与细胞空间整合分析系统）。该工作流整合了可复现且流程简化的二维（2D）细胞分割与细胞类型分类方法，后续可直接用于三维体积重建。本研究同时开发了全新的三维可视化工具，可用于分析免疫细胞簇密度、细胞与脉管系统的距离，以及紫外线损伤/修复相关细胞标记物、增殖相关标记物距皮肤表面的距离图谱。本研究使用MATRICS-A对12名年龄介于32至72岁的捐赠者的26例固定皮肤连续切片进行了重建。样本采集自6个不同的解剖部位，其日光暴露程度从轻度至重度不等。每一张组织切片均使用18种荧光标记抗体进行多重标记，可覆盖表皮与真皮中的14种细胞类型。本研究揭示了多项仅能通过三维分析获取的全新发现：我们首次实现了免疫细胞簇的可视化，并证实相较于二维分析，在三维空间中，辅助性T细胞30微米范围内的总T细胞数量高出10%至70%。DNA损伤标记物（p53）、DNA修复标记物（DDB2）以及增殖标记物（Ki67）距皮肤表面的距离，在所有年龄组与日光暴露条件下均保持一致。MATRICS-A为量化健康与衰老器官的三维空间细胞互作关系提供了一种全新且功能强大的整合式开放获取工具，未来还可进一步拓展至疾病器官的相关研究。