Modeling immunity with a hybrid lymph node tissue-chip

项目总结/摘要 免疫系统中的局部相互作用决定了免疫应答是保护性的还是 破坏性的，对抗感染或启动自身免疫。适应性免疫始于淋巴结（LN）， 高度组织化和动态的组织目前，很难解析炎症介质或 合理设计治疗慢性炎症性疾病，如关节炎，类风湿性关节炎， 多发性硬化症，共影响5 - 7%的西方人口。我们假设分析当地的 完整组织中的离体响应将提供从当前方法（体外/体内）不容易获得的信息 体内）。这样的实验需要新的工具来分析免疫系统的动态，我们开发了这种工具， 结合生物分析化学、微流体学和免疫学的专业知识。 在这个项目中，我们将开发一种新的离体免疫模型，使用微流体的混合物， 培养物和LN切片。在目标1中，我们将建立长期培养，并结合分析方法， 鼠和人LN切片。切片培养提供了保存细胞外基质的优点， 微环境和任何基质结合信号。我们将优化小鼠LN的长期培养（7-21天） 切片和人扁桃体切片，以维持高活力、低静止细胞活化标记物，以及 对炎症和抗原特异性刺激有反应。在目标2中，我们将开发一种新的微流体系统， LN切片的按需局部刺激。我们将提高我们以前开发的空间分辨率 设备，使用短期和长期靶向直径为2 - 10个细胞的簇（20 - 100 μm横向分辨率） 刺激.我们还将通过使用移动的端口，使 整个组织可通过最少的操作进入。在目标3中，我们将验证混合微流体-组织切片 用于分析炎症反应和抗炎治疗的系统。我们将比较 切片与细胞培养物和体内对促炎细胞因子TNF-α的炎症反应 系统.最后，我们将测试该模型在多大程度上提供了指导免疫治疗的新信息， 通过使用小鼠和人组织的混合组织芯片系统来比较竞争性 TNF-α抑制剂（抗TNF-α单克隆抗体或可溶性TNF-α受体）。 将局部微流体刺激与组织切片技术相结合， 用于分析活LN组织中的空间组织化信号传导和细胞-细胞相互作用的平台。这一创新 该平台将推进基础和转化生物医学研究：局部递送的细胞因子将作为 急需的急性或慢性炎症模型，以及局部免疫治疗将指导 靶向载药纳米颗粒的设计。这项技术广泛适用于许多炎症性疾病， 这些疾病包括类风湿性关节炎、克罗恩病、多发性硬化症、阿尔茨海默病和癌症。

PROJECT SUMMARY/ABSTRACT Local interactions in the immune system determine whether an immune response is protective or destructive, fighting infection or initiating autoimmunity. Adaptive immunity begins in the lymph node (LN), a highly organized and dynamic tissue. Currently, it is difficult to parse the role of inflammatory mediators or rationally design therapies for chronic inflammatory disease such as artherosclerosis, rheumatoid arthritis and multiple sclerosis, which together affect 5 – 7% of the Western population. We hypothesize that analyzing local responses ex vivo in intact tissue will provide information not easily obtained from current methods (in vitro/in vivo). Such experiments require new tools to analyze dynamics in the immune system, which we develop by combining expertise in bioanalytical chemistry, microfluidics, and immunology. In this project, we will develop a novel ex vivo model of immunity, using a hybrid of microfluidic culture and LN slices. In Aim 1, we will establish long-term culture coupled with analysis methods for live murine and human LN slices. Slice culture offers the advantage of preservation of the extracellular microenvironment and any matrix-bound signals. We will optimize long-term culture (7-21 days) for murine LN slices and human tonsil slices to maintain high viability, low cellular activation markers at rest, and ability to respond to inflammatory and antigen-specific stimuli. In Aim 2, we will develop a novel microfluidic system for on-demand local stimulation of LN slices. We will improve the spatial resolution of our previously developed device, to target clusters 2 – 10 cells in diameter (20 – 100 μm lateral resolution) using short- and long-term stimulation. We will also enable on-demand selection of delivery zone by using a mobile port, making the whole tissue accessible with minimal handling. In Aim 3, we will validate the hybrid microfluidic-tissue slice system for analysis of inflammatory responses and anti-inflammatory therapies. We will compare the inflammatory response to a pro-inflammatory cytokine, TNF-α, in slices versus cell cultures and in vivo systems. Finally, we will test the extent to which the model provides new information to guide immunotherapy, by using the hybrid tissue-chip system with mouse and human tissue to compare the effects of competing TNF-α inhibitors (anti-TNF-α monoclonals or soluble TNF-α receptor). Combining local microfluidic stimulation with tissue slice technology produces the first experimental platform for analysis of spatially organized signaling and cell-cell interactions in live LN tissue. This innovative platform will advance both basic and translational biomedical research: locally delivered cytokines will serve as a much-needed model of acute or chronic inflammation, and locally delivered immunotherapies will guide the design of targeted drug-loaded nanoparticles. This technology is broadly applicable for a host of inflammatory diseases, including rheumatoid arthritis, Chron’s disease, multiple sclerosis, Alzheimer’s disease, and cancer.