Bioengineered organoids-on-a-chip to study enteric disease

用于研究肠道疾病的生物工程类器官芯片

• 批准号: 8855063
• 负责人: SHUICHI TAKAYAMA
• 金额: $ 22.57万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-01 至 2020-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8855063
• 关键词: 3-Dimensional; Antibiotic Therapy; Antibiotics; Bacteria; Biochemical; Biological Markers; Biomedical Engineering; Cells; Chemical Stimulation; Coculture Techniques; Colony-forming units; Complex; Computers; Cues; Data; Development; Devices; Disease; Drug Compounding; Drug Delivery Systems; Drug Transport; Electrical Resistance; Electrodes; Engineering; Enteral; Epithelial; Epithelium; Geometry; Goals; Growth; Harvest; Homeostasis; Human; Human Engineering; Immune; Immunofluorescence Immunologic; In Vitro; Inflammatory Bowel Diseases; Intestines; Lactobacillus; Life; Liquid substance; Maintenance; Measurement; Measures; Mechanical Stimulation; Mechanics; Metabolic; Methods; Microbe; Microfabrication; Microfluidics; Modeling; Molecular; Nutrient; Optics; Organoids; Oxygen; Peptides; Perfusion; Pharmaceutical Preparations; Physiological; Probiotics; Procedures; Production; Property; Proteins; Publishing; Pump; Regulation; Salmonella enterica; Salmonella typhimurium; Sampling; Shapes; Stem cells; Structure; System; Testing; Therapeutic; Time; Tissues; Villus; Virus; absorption; base; cytokine; design; experience; feeding; fluorescein isothiocyanate dextran; in vivo; innovation; insight; instrument; killings; lithography; man; microbial; novel; pathogen; pathogenic bacteria; self organization; sensor

PROJECT SUMMARY/ABSTRACT This project is a combined design-driven and hypothesis-driven project to bioengineer microscale models of enteric disease. Starting with the Spence lab's in vitro intestine system that accurately reflects both the complex cellular makeup and the appropriate layered organization of the human intestine, this project will provide these 3-Dimensional (3D) Human Intestinal Organoids (HIOs) with physiologicaly soft but confining mechanical cues as well as microscale fluid perfusion capabilities that will mimic luminal flow, to further induce physiological structures such as crypts and villi. Both of these properties (constraint, flow) have a significant impact on intestine development, differentiation and function. Our hypothesis is that by providing a mechanically confined culture condition and fluid perfusion, as opposed to the free expanding culture with a static, enclosed lumen as is currently used for HIO formation, that the epithelial layer will self-organize additional levels of physiological complexity, including as crypts and villi, along with associated spatial organization of intestinal stem cells (ISCs) in crypts and differentiated cells on the villi. Incorporation of microscale fluid perfusion capabilities in HIO culture devices will also allow precise regulation of intraluminal flow of nutrients, and long-term colonization with bacteria, and pathogens. Technologically, this project will be innovative in developing a method (“supersoft lithography”) for reproducibly creating supersoft PDMS structures with physiological moduli of 1-100 kPa. To enable closed-loop control for maintenance of tissue homeostasis as well as to provide readouts of tissue function, this project will also integrate miniature oxygen sensors and electrodes for trans-epithelial electrical resistance (TEER) measurements. Additionally, sampling capabilities from the interior and exterior of the HIO will be incorporated to enable off-line measures of fluid and drug absorption/secretion. HIO microscale culture devices will also facilitate measurement of cytokine production in integrated HIO-immune co-cultures. Finally, we will demonstrate modularity and utility of the bioengineered and instrumented HIO system by integrating NAMSED Projects 1, 2 and 3. Specifically, instrumented-HIOs with luminal flow will be generated, co-cultured with immune cells and colonized by probiotic microbes (Lactobacillus GG, LGG) and/or pathogens (S.typhimurium). In each co-culture, (probiotic/HIO/immune vs. probiotic/pathogen/HIO/immune), we will test the ability of the system to generate real-time physiological data by measuring epithelial barrier function (TEER, FITC-Dextran), oxygen concentration, cytokine production, and finally by examining epithelial invasion by S.typhimurium. We will also test the utility of this system to screen drugs/compounds by generating instrumented LGG/S.typhimurium/HIO/immune co-cultures and adding Cefoperazone, an antibiotic that will selectively target the pathogen S.typhimurium, but not the probiotic LGG. The ability of Cefoperazone to kill S.typhimurium will be examined by culturing the luminal effluent to determine S.typhimurium colony forming units before, during and after antibiotic treatment. Finally, when live cultures are terminated, we will harvest the system and examine cellular and molecular difference between the different groups using immunofluorescence or qRT-PCR on purified immune cells and epithelium.

项目摘要/摘要 该项目是一个组合设计驱动的和假设驱动的项目，用于生物工程师微观模型 肠道疾病。从Spence Lab的体外肠系统开始，该系统准确地反映了 复杂的蜂窝化妆和人类肠道的适当分层组织，该项目将 提供这些3维（3D）人肠癌（HIO），具有身体柔软但限制 机械提示以及微观流体灌注能力，这些功能将模仿腔流量，以进一步诱导 生理结构，例如地下室和绒毛。这两种属性（约束，流）都有很大的 对肠发展，分化和功能的影响。我们的假设是通过提供 机械限制的培养条件和流体灌注，而不是用 当前用于HIO形成的静态，封闭的管腔，上皮层会自组织 其他水平的物理复杂性，包括地crypts和Villi，以及相关的空间 在绒毛上的隐窝和分化细胞中的肠道干细胞（ISC）的组织。合并 HIO培养设备中的显微镜流体灌注能力还将允许精确调节腔内 养分流以及细菌和病原体的长期定植。从技术上讲，这个项目将是 开发一种可重复创建SuperSoft PDM的方法（“ SuperSoft Supersoft光刻”）的创新 物理模量为1-100 kPa的结构。 为了启用闭环控制以维持组织稳态以及提供组织的读数 功能，该项目还将整合跨上皮电气的微型氧气传感器和电极 电阻（TEER）测量。另外，从HIO的内部和外部进行采样功能 将合并以实现流体和药物滥用/分泌的离线测量。 HIO微观培养 设备还将促进综合hio-rmmune共培养中细胞因子产生的测量。 最后，我们将通过 集成名称的项目1、2和3。 与免疫细胞共培养，并通过益生菌微生物（Lactobacillus GG，LGG）和/或病原体培养 （S. typhimurium）。在每个共培养中（益生菌/HIO/免疫与益生菌/病原体/Hio/Immune），我们将测试 系统通过测量上皮屏障功能生成实时物理数据的能力 （TEER，FITC-DEXTRAN），氧气浓度，细胞因子产生，最后检查上皮浸润 由S. typhimurium。我们还将通过产生该系统的实用程序来筛选药物/化合物 仪器的LGG/s. typhimurium/hio/hio/免疫共培养和添加头孢心赛，这是一种抗生素 有选择地靶向病原体链球菌，但不是益生菌LGG。头孢唑酮杀死的能力 胰杆菌将通过培养腔效应来确定型胰菌群形成。 抗生素治疗前，期间和之后的单位。最后，当终止现场文化时，我们将恢复 系统并使用免疫荧光检查不同组之间的细胞和分子差异 或QRT-PCR纯化的免疫细胞和上皮。