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 该项目是一个设计驱动和假设驱动相结合的项目，旨在对微尺度模型进行生物工程 肠道疾病。从斯宾塞实验室的体外肠道系统开始，它准确地反映了 复杂的细胞构成和适当的人体肠道分层组织，这个项目将 为这些三维(3D)人体肠道器官(HIO)提供生理上柔软但受限的 机械信号以及微尺度流体灌流能力，将模拟管腔流动，以进一步诱导 生理结构，如隐窝和绒毛。这两个属性(约束、流)都具有重要的 对肠道发育、分化和功能的影响。我们的假设是，通过提供一个 机械受限的培养条件和液体灌流，而不是自由膨胀的培养 静态的、封闭的管腔，就像目前用于HIO形成的那样，上皮层将自组织 生理复杂性的额外水平，包括隐窝和绒毛，以及相关的空间 隐窝中的肠干细胞(ISCs)和绒毛上的分化细胞的组织。成立为法团 HIO培养设备中的微尺度液体灌流能力也将允许精确调节管腔内 营养物质的流动，以及细菌和病原体的长期定居。从技术上讲，这个项目将是 在开发可重复创建超软PDMS的方法(超软光刻)方面的创新 生理模数为1-100千帕的结构。 以实现用于维持组织内稳态以及提供组织读数的闭环控制 功能，该项目还将集成微型氧气传感器和电极，用于跨上皮电 电阻(TEER)测量。此外，来自HIO内部和外部的采样能力 将被纳入，以实现对液体和药物吸收/分泌的离线测量。HIO微型培养 设备还将有助于在整合的HIO-免疫共培养中测量细胞因子的产生。 最后，我们将通过以下方式演示生物工程和仪表化HIO系统的模块化和实用性 集成NAMSED项目1、2和3。具体地说，将生成具有管腔流动的仪表式HIO， 与免疫细胞共培养，并被益生菌(乳杆菌GG、LGG)和/或病原体定植 (小鼠沙门氏菌)。在每种共培养中(益生菌/HIO/免疫vs.益生菌/病原体/HIO/免疫)，我们将测试 该系统通过测量上皮屏障功能来生成实时生理数据的能力 (Teer，FITC-葡聚糖)，氧浓度，细胞因子产生，最后通过检测上皮侵袭 鼠伤寒沙门氏菌。我们还将测试该系统的效用，通过生成 仪器化的LGG/鼠伤寒沙门氏菌/HIO/免疫共培养，并添加头孢哌酮，一种将 选择性地以鼠伤寒沙门氏菌为靶标，而不是益生菌LGG。头孢哌酮的杀伤力 将通过培养腔内流出物来检测鼠伤寒沙门氏菌，以确定鼠伤寒沙门氏菌的集落形成。 抗生素治疗前、治疗中和治疗后。最后，当活的文化被终止时，我们将收获 系统并利用免疫荧光检测不同组之间的细胞和分子差异 或对纯化的免疫细胞和上皮细胞进行qRT-PCR。