Inflammatory Bowel Disease Center, Division of Gastroenterology, University of California San Diego, La Jolla, California.
VA San Diego Healthcare System, San Diego, California.
Inflamm Bowel Dis. 2018 Oct 12;24(11):2366-2376. doi: 10.1093/ibd/izy214.
Novel therapeutics for inflammatory bowel disease (IBD) are under development, yet mechanistic readouts at the tissue level are lacking. Techniques to assess intestinal immune composition could represent a valuable tool for mechanism of action (MOA) studies of novel drugs. Mass cytometry enables analysis of intestinal inflammatory cell infiltrate and corresponding molecular fingerprints with unprecedented resolution. Here, we aimed to optimize the methodology for isolation and cryopreservation of cells from intestinal tissue to allow for the potential implementation of mass cytometry in MOA studies.
We investigated key technical issues, including minimal tissue requirements, cell isolation protocols, and cell storage, using intestinal biopsies and peripheral blood from healthy individuals. High-dimensional mass cytometry was employed for the analyses of biopsy-derived intestinal cellular subsets.
Dithiothreitol and mechanical dissociation decreased epithelial cell contamination and allowed for isolation of adequate cell numbers from 2 to 4 colonic or ileal biopsies (6 × 104±2 × 104) after a 20-minute collagenase digestion, allowing for reliable detection of most major immune cell subsets. Biopsies and antibody-labeled mononuclear cells could be cryopreserved for later processing and acquisition (viability > 70%; P < 0.05).
Mass cytometry represents a unique tool for deep immunophenotyping intestinal cell composition. This technique has the potential to facilitate analysis of drug actions at the target tissue by identifying specific cellular subsets and their molecular signatures. Its widespread implementation may impact not only IBD research but also other gastrointestinal conditions where inflammatory cells play a role in pathogenesis.
新型治疗炎症性肠病（IBD）的药物正在研发中，但在组织水平上缺乏机制研究。评估肠道免疫组成的技术可能是新型药物作用机制（MOA）研究的有价值工具。液质流式细胞术可以前所未有的分辨率分析肠道炎症细胞浸润和相应的分子特征。在此，我们旨在优化从肠道组织中分离和冷冻保存细胞的方法，以便有可能将液质流式细胞术应用于 MOA 研究。
我们使用健康个体的肠道活检和外周血，研究了包括最小组织需求、细胞分离方案和细胞储存等关键技术问题。使用高维液质流式细胞术分析活检衍生的肠道细胞亚群。
二硫苏糖醇和机械解离可减少上皮细胞污染，并允许从 2 至 4 个结肠或回肠活检中分离出足够数量的细胞（6×104±2×104），经过 20 分钟的胶原酶消化后，可可靠地检测到大多数主要免疫细胞亚群。活检和标记有抗体的单核细胞可冷冻保存以备后续处理和采集（存活率>70%；P<0.05）。
液质流式细胞术代表了一种用于深入免疫表型分析肠道细胞组成的独特工具。该技术有可能通过鉴定特定的细胞亚群及其分子特征，促进对靶向组织中药物作用的分析。它的广泛应用可能不仅会影响 IBD 研究，还会影响其他胃肠道疾病，因为炎症细胞在发病机制中起作用。
