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信提供了丰富的词汇。生产和 这些信号的结果对细胞环境和微环境非常敏感。解剖 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的 问题：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时或当不同细胞类型存在于混合物中时是不同的。 微环境？微生物--比如我们体内的细菌和真菌--是如何影响 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）微尺度的合作， 能够精确定位细胞类型以研究细胞信号传导的多培养方法，（2）专门培养 用于复杂的人-细菌-真菌多王国培养的平台，（3）微生物协同和 采用挥发性有机化合物（VOC）采样的多元培养系统，以研究挥发物如何介导微生物 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 随着时间本提案扩展了我们实验室在领域（1）和（3）的能力，并有可能扩展 （2）和（4）在今后的工作中。该提案将探索人类细胞信号传导的两个领域中未回答的问题： (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，我们将开发新的文化平台， 微生物和多界（例如，细菌、真菌）VOC通信和气体集成采样 色谱-质谱（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，用户友好的设置， 移液器、输送介质和化学刺激的流体通道，以及固相微萃取的集成 （SPME）纤维用于VOC样品收集。这项提议的核心是使用“开放”微流体技术， 自发毛细流动我们是开放式微流体领域的领导者，在开发 在标准孔板中进行微规模多培养实验的用户友好、成本效益高的方法 和生物学家熟悉的培养皿。拟议的工作建立在我们的能力之上， 在用敏感的原代细胞进行三代培养和创新一种全新的方法方面， 用于研究微生物培养物中的VOC。所提出的方法将增强对信号的理解 参与有害的长期炎症，这对开发更好的治疗方法至关重要。 炎症条件;他们也将使研究微生物群落，是必不可少的维持 人类健康（肠道微生物）和那些导致疾病（病原体）。此外，生物工程 微流控方法的发展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.