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。比如说， 促炎、抗炎和促消退氧脂素对环境反应的合成 暴露或受伤是由免疫细胞严格控制的，免疫细胞可以随时改变氧脂素的产生。 免疫反应在真实的时间内进行。此外，相同的信号分子可以 一个完全不同的下游生物反应取决于微环境背景。 剖析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：每个信号分子的下游生物学功能是什么？怎么样 当分子存在于混合物中时，生物功能不同？微生物-比如细菌和 真菌存在于我们的身体-影响分子景观？我们的实验室正在开发新的工具来探测这些 问题包括（1）能够精确定位细胞类型的微尺度共培养和多培养方法， 研究信号传导，（2）整合微培养与小分子提取方法，用于下游 使用质谱法的代谢组学分析，（3）专门的培养平台和提取方法， 从复杂人-细菌-真菌多王国培养物中分离信号，和（4）新的基于细胞的行为 用于探测化学信号对生物功能（包括血管生成，粘液产生， 免疫细胞功能）。本提案扩展了我们实验室在领域（1）和（4）的能力。这项建议 将创建创新的功能测定来研究血管舒张（血管扩张， 炎症）和成纤维细胞肌分化（导致慢性炎症中有害的纤维化和重塑）。 炎症）。该提案的核心是使用“开放”微流体和自发毛细流动来雕刻 以亚毫米的精度制作三维凝胶结构。我们的实验室在以下方面取得了重大进展 使用开放式微流体引导凝胶流动，从而产生用户友好的、具有成本效益的方法来执行 在标准孔板内进行微规模多培养实验。拟议的工作建立在我们的能力之上 并接受了一个重大的工程挑战：生产可以动态扩张的血管模拟物 并且收缩，同时易于多路复用，以便研究大量的信号分子。拟议 这些方法将增强对有害的长期炎症信号的理解， 为许多炎症性疾病开发更好的治疗方法。此外，生物工程和 微流控方法的发展将转化为其他三维仿生培养平台。

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. For example, synthesis of pro-inflammatory, anti-inflammatory, and pro-resolution oxylipins in response to environmental exposures or wounding is tightly controlled by immune cells that can shift oxylipin production on the minute timescale as the immune response progresses in real time. Furthermore, the same signaling molecule can bring about an entirely different downstream biological response depending on microenvironmental context. 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? 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 signaling, (2) integration of microculture with small molecule extraction methods for downstream metabolomics analysis using mass spectrometry, (3) specialized culture platforms and extraction methods to isolate signals from complex human-bacteria-fungal multikingdom culture, and (4) novel cell-based behavioral assays to probe the effects of chemical signals on biological function (including angiogenesis, mucus production, and immune cell function). The present proposal expands our lab’s capabilities in areas (1) and (4). This proposal will create innovative functional assays to study vasodilation (blood vessel expansion, a hallmark of inflammation) and fibroblast myodifferentiation (which leads to harmful fibrosis and remodeling in chronic inflammation). Central to this proposal is the use of ‘open’ microfluidics and spontaneous capillary flow to sculpt gel structures in three dimensions with sub-millimeter precision. Our lab has made significant advances in directing gel flows using open microfluidics, resulting in user-friendly, cost-effective methods to perform microscale multiculture experiments within standard well plates. The proposed work builds on our capabilities and embraces a significant engineering challenge: producing blood vessel mimics that can dynamically dilate and contract while being easy to multiplex in order to study large sets of signaling molecules. 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. Further, the bioengineering and microfluidic approaches developed will translate to other three dimensional biomimetic culture platforms.