Optimization of Tissue Digestion Methods for Characterization of Photoaged Skin by Single Cell RNA Sequencing Reveals Preferential Enrichment of T Cell Subsets. Optimization of Tissue Digestion Methods for Characterization of Photoaged Skin by Single Cell RNA Sequencing Reveals Preferential Enrichment of T Cell Subsets

Healthy human skin tissue is often used as a control for comparison to diseased skin in patients with skin pathologies, including skin cancers or other inflammatory conditions such as atopic dermatitis or psoriasis. Although non-affected skin from these patients is a more appropriate choice for comparison, there is a paucity of studies examining such tissue. This lack is exacerbated by the difficulty of processing skin tissue for experimental analysis. In addition, choosing a processing protocol for skin tissue which preserves cell viability and identity while sufficiently dissociating cells for single-cell analysis is not a trivial task. Here, we compare three digestion methods for human skin tissue, evaluating the cell yield and viability for each protocol. We find that the use of a sequential dissociation method with multiple enzymatic digestion steps produces the highest cell viability. Using single-cell sequencing, we show this method results in a relative increase in the proportion of non-antigen-presenting mast cells and CD8 T cells as well as a relative decrease in the proportion of antigen-presenting mast cells and KYNU+ CD4 T cells. Overall, our findings support the use of this sequential digestion method on freshly processed human skin samples for optimal cell yield and viability. Overall design: Surgically resected human skin tissue for single-cell sequencing was digested using one of two protocols prior to bead column isolation of CD45+ cells, and CD45+ and CD45- fractions were analyzed by single-cell RNA sequencing and protein profiling.

健康人类皮肤组织常被用作对照样本，以对比皮肤疾病患者的病变皮肤，这类皮肤疾病包括皮肤癌，或特应性皮炎（atopic dermatitis）、银屑病（psoriasis）等炎症性病症。尽管取自这些患者的未受累皮肤是更为合适的对照选择，但针对此类组织的相关研究仍较为匮乏。而皮肤组织的实验分析处理难度进一步加剧了这一研究缺口：既要筛选出能够维持细胞活性与表型特征、同时又能充分解离细胞以支持单细胞分析的处理方案，这并非一项易事。
本研究针对人类皮肤组织的三种消化方法展开对比评估，分别测定各处理方案的细胞得率与细胞活性。研究发现，采用包含多步酶消化步骤的序贯解离法可获得最高的细胞活性。通过单细胞测序（single-cell sequencing）分析，我们证实该方法可使非抗原呈递肥大细胞与CD8阳性T细胞的相对占比升高，同时使抗原呈递肥大细胞以及KYNU+ CD4阳性T细胞的相对占比降低。综上，本研究结果支持在新鲜处理的人类皮肤样本中采用该序贯解离法，以获得最优的细胞得率与细胞活性。
实验整体设计：手术切除的人类皮肤组织用于单细胞测序实验，实验前采用两种处理方案之一进行消化，随后通过磁珠柱分选（bead column isolation）获取CD45阳性细胞组分，最终对CD45阳性与CD45阴性组分分别开展单细胞RNA测序（single-cell RNA sequencing）与蛋白质谱分析（protein profiling）。