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

 描述（由申请人提供）：该项目的重点是开发一种用于研究肠道微生物组对人类健康影响的新工具：肠道上皮-微生物组-免疫稳态的体外人体模型。虽然已知肠道微生物组对人类健康有巨大影响，但这些影响是复杂的，通常还没有得到很好的理解，这限制了将观察到的微生物组影响转化为有效的治疗方法。目前还没有人肠道上皮-微生物组-免疫稳态的体外模型，因此目前对这些作用的大多数研究都是在无菌啮齿动物模型中进行的。一种受控的、定义的体外 因此，捕获关键的肠道微生物组-上皮-免疫相互作用的人类系统对于学术和工业科学以及医学研究团体来说将是非常有价值的工具。该方法结合了必要硬件的开发以及培养方案和原代肠道免疫哺乳动物培养所需的生物材料，以及待与肠道哺乳动物细胞一起培养的微生物种群的建立和肠道微生物种群的代表。重要的是，该项目团队已经共同努力建立了基础流体培养平台和具有免疫能力的生物肠道模型，共培养了简单的微生物群落，从而证明了利用其互补专业知识的独特组合并将项目推向商业化的能力。待开发的具体硬件（目标1）包括用于模拟循环系统和肠腔内容物流动的流体平台，这对于控制氧浓度和维持肠道稳态与常驻微生物种群以及用于氧和肠屏障功能（跨上皮电阻，TEER）的传感器非常重要。原代肠道培养系统（目标2）将采用单层形式，便于接近顶端粘膜表面，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PEG基生物材料将用于支持原代肠道培养，包括细胞掺入 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。微生物聚生体（目标3）将从人样本中分离的菌株中开发，并筛选维持肠道培养物中的稳态。整合的肠道上皮-微生物组-免疫生物系统将在开发的流体平台上维持延长（2周）培养，并将分析肠道菌群对肠道活力和功能指标的影响，以及提供对细胞信号传导事件的洞察的细胞因子释放谱。两个案例研究将用于评估所得硬件-生物复合系统捕获人类微生物组的充分表征的响应的能力（目标4）。硬件培养系统将被刺激以诱导“肠漏”和炎症，然后将用被证明在这些条件下提供有益效果的特定细菌物种来改善。

 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.