GuMI: New In Vitro Platforms to Parse the Human Gut Epithelial-Microbiome-Immune Axis

 描述(申请人提供)：该项目致力于开发一种研究肠道微生物群对人类健康影响的新工具：肠道上皮-微生物群-免疫动态平衡的体外人体模型。虽然肠道微生物组已知对人类健康有巨大影响，但这些影响是复杂的，通常还不清楚，限制了对观察到的微生物组影响的有效治疗。目前还没有人类肠道上皮-微生物-免疫动态平衡的体外模型，因此，目前大多数关于这些影响的研究都是在灵生啮齿动物模型中进行的。一种受控的，在体外定义的 因此，人体系统捕捉关键的肠道微生物-上皮-免疫相互作用对于学术和工业、科学和医学研究界来说将是一个非常有价值的工具。该方法包括开发肠道免疫哺乳动物原代培养所需的必要硬件以及培养方案和生物材料，以及建立与肠道哺乳动物细胞和肠道共生微生物群体代表的微生物种群。重要的是，项目组已经共同努力，与共培养的简单微生物群落建立了基础流体培养平台和免疫能力生物肠道模型，从而展示了利用他们互补专业知识的独特组合并将项目推向商业化的能力。待开发的特定硬件(目标1)包括模拟循环系统和肠腔内容物流动的流体平台，这对于控制氧气浓度和维持肠道内微生物种群的动态平衡非常重要，以及氧气和肠道屏障功能(跨上皮电阻，TEER)的传感器。初级肠道培养系统(AIM 2)将以单分子层的形式方便地进入顶端粘膜表面，并将包括用于免疫功能的树突状细胞。具有可调化学功能和机械性能的聚乙二醇基生物材料将用于支持原代肠道培养，包括结合细胞 维持肠道内环境稳定的关键：肌成纤维细胞和肠神经胶质细胞。微生物联盟(目标3)将从人类样本中分离的菌株中培养出来，并在肠道培养中为维持体内平衡而进行筛选。整合的肠道上皮-微生物-免疫生物系统将维持在开发的流体平台上进行延长(2周)的培养，并将分析共生种群对肠道活性和功能指标的影响，以及提供细胞信号事件洞察的细胞因子释放谱。将使用TW案例研究来评估由此产生的硬件-生物复合系统捕获人类微生物组特征良好的反应的能力(目标4)。硬件培养系统将被刺激以引起“肠漏”和炎症，然后将被证明在这些条件下提供有益效果的特定细菌种类来改善。

 DESCRIPTION (provided by applicant): This project is focused on developing a novel tool for studying gut microbiome impact on human health: an in vitro human model of gut epithelium-microbiome-immune homeostasis. While the gut microbiome is known to have tremendous impact on human health, these effects are complex and generally not well understood, limiting translation of observed microbiome impact to effective therapies. There is currently no in vitro model of human gut epithelium-microbiome-immune homeostasis, and most studies of these effects are thus currently carried out in gnotobiotic rodent models. A controlled, defined in vitro human system capturing key gut microbiome-epithelium-immune interactions would thus be a tremendously valuable tool to academic and industrial scientific and medical research communities. The approach combines development of the necessary hardware as well as culture protocols and biomaterials required for primary intestinal-immune mammalian culture and establishment of microbial populations to be cultured with gut mammalian cells and representative of gut commensal microbe populations. Importantly, the project team has already worked together to establish base fluidic culture platforms and immune-competent biological gut models with co-cultured simple microbial communities, and thus has demonstrated the ability to utilize their unique combination of complementary expertise and move projects toward commercialization. Specific hardware to be developed (Aim 1) includes a fluidic platform for flow mimicking both the circulatory system and flow of gut luminal contents, highly important for controlling oxygen concentration and maintenance of gut homeostasis with a resident microbial population, as well as sensors for oxygen and intestinal barrier function (trans-epithelial electrical resistance, TEER). The primary intestinal culture system (Aim 2) will be in the form of a monolayer for facile access to the apical mucosal surface and will include dendritic cells for immune function. PEG-based biomaterials with tunable chemical functionality and mechanical properties will be used to support the primary intestinal culture, including incorporation of cells key to maintenance of intestinal homeostasis: myofibroblasts and enteric glia. Microbial consortia (Aim 3) will be developed from isolated strains from human samples, and screened for maintenance of homeostasis in gut culture. The integrated gut epithelium-microbiome-immune biological system will be maintained on the developed fluidic platform for extended (2 week) culture, and the impact of the commensal populations on metrics of gut viability and function, as well as cytokine release profiles providing insight into cell signaling events will be analyzed. Tw case studies will be used to assess the ability of the resulting hardware-biology composite system to capture well-characterized responses of the human microbiome (Aim 4). The hardware-culture system will be stimulated to induce "leaky gut" and inflammation, which will then be ameliorated with specific bacterial species demonstrated to provide beneficial effects in these conditions.