Deciphering the role of chemical signals in inflammation with open microfluidic functional assays

项目摘要 小分子和蛋白质信号为细胞通讯提供了丰富的词汇。制作和制作 这些信号的后果对细胞环境和微环境非常敏感。解剖学研究 细胞类型之间的分子对话是具有挑战性的，需要新的方法来解决基本问题 问：每个信号分子的下游生物学功能是什么？生物功能如何？ 当分子存在于混合物中或当不同类型的细胞存在于 微环境？微生物--就像我们体内的细菌和真菌--是如何影响分子的 风景？我们的实验室正在开发新的工具来探索这些问题，包括(1)微尺度联合和 能够精确定位细胞类型以研究细胞信号的多种培养方法，(2)专门培养 复杂的人-细菌-真菌多王国培养平台，(3)微生物和真菌的整合 采用挥发性有机化合物(VOC)采样的多培养系统研究挥发物如何中介微生物 对话，以及(4)家庭生物流体采样和稳定平台，以探测人类免疫反应 随着时间的推移。目前的建议扩大了我们实验室在(1)和(3)方面的能力，有可能扩大 (2)和(4)在今后的工作中。这项提案将探索人类细胞信号传递的两个领域的悬而未决的问题： (I)嗜酸性粒细胞和成纤维细胞之间的旁分泌信号和(Ii)旁分泌/物理接触介导的信号 中性粒细胞、单核细胞和B细胞之间。此外，我们将发展新颖的文化平台，使 微生物和多领域(如细菌、真菌)的VOC通信和气体的综合采样 色质联用(GC-MS)分析。这一文化体系将首次使 多个微生物培养物的受控空间定位，一个用户友好的设置，可以通过简单的 移液器、提供介质和化学刺激的流体通道以及固相微萃取集成 (SPME)用于VOC样本采集的纤维。这项提议的核心是使用“开放式”微流控技术和 自发性毛细管流。我们在开放式微流控领域处于领先地位，并在开发方面有着良好的记录 使用方便、经济高效的方法在标准井板内进行微型多品种培养实验 以及生物学家所熟悉的文化。拟议的工作建立在我们的能力基础上，并包含了重要的 利用敏感原代细胞进行三种培养和创新一种全新方法的工程挑战 用于研究微生物培养物中的挥发性有机化合物。建议的方法将加强对信号的理解 牵涉到有害的长期炎症，对开发更好的治疗方法至关重要 炎症条件；它们还将使研究微生物群落对维持 人类健康(共生微生物)和导致疾病的微生物(病原体)。此外，生物工程 开发的微流控技术将转化为其他仿生培养平台和基础 信号调查。

PROJECT ABSTRACT Small molecule and protein signals provide a rich vocabulary for cellular communication. The production and consequences of these signals are exquisitely sensitive to cellular context and microenvironment. Dissecting the molecular dialogue between cell types is challenging, and new methods are required to address fundamental questions: What is the downstream biological function of each signaling molecule? How is the biological function different when molecules are present in mixtures or when different cell types are present in the microenvironment? How do microbes—like the bacteria and fungi present in our bodies—affect the molecular landscape? Our lab is developing new tools to probe these questions including (1) microscale co- and multiculture methods that enable precise positioning of cell types to study cell signaling, (2) specialized culture platforms for complex human-bacteria-fungal multikingdom culture, (3) integration of microbial co- and multiculture systems with volatile organic compound (VOC) sampling to study how volatiles mediate microbial dialogue, and (4) at-home biofluid sampling and stabilization platforms to probe the human immune response over time. The present proposal expands our lab’s capabilities in areas (1) and (3), with the possibility to extend to (2) and (4) in future work. This proposal will probe unanswered questions in two areas of human cell signaling: (i) paracrine signaling between eosinophils and fibroblasts and (ii) paracrine/physical contact-mediated signaling between neutrophils, monocytes, and B cells. Further, we will develop novel culture platforms that enable microbial and multikingdom (e.g., bacteria, fungi) VOC communication and integrated sampling for gas chromatography-mass spectrometry (GC-MS) analysis. This culture system will, for the first time, enable controlled spatial positioning of multiple microbial cultures, a user-friendly setup that can be operated with simple pipettes, fluidic channels to deliver media and chemical stimulation, and integration of solid phase micoextraction (SPME) fibers for VOC sample collection. Central to this proposal is the use of ‘open’ microfluidics and spontaneous capillary flow. We are leaders in open microfluidics and have a strong track record of developing user-friendly, cost-effective methods to perform microscale multiculture experiments within standard well plates and cultureware familiar to biologists. The proposed work builds on our capabilities and embraces significant engineering challenges in doing triculture with sensitive primary cells and innovating an entirely new approach for study VOCs in microbial cultures. The proposed methods will enhance the understanding of the signals involved in detrimental prolonged inflammation, critical to the development of better therapies for numerous inflammatory conditions; they will also enable study of microbial communities that are essential to maintaining human health (commensal microbes) and those that lead to disease (pathogens). Further, the bioengineering and microfluidic approaches developed will translate to other biomimetic culture platforms and fundamental signaling investigations.