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.

项目概要/摘要 该项目是一个设计驱动和假设驱动相结合的项目，旨在生物工程微尺度模型 从 Spence 实验室的体外肠道系统开始，它准确地反映了肠道疾病的情况。 复杂的细胞组成和人体肠道的适当分层组织，该项目将 为这些 3 维 (3D) 人类肠道类器官 (HIO) 提供生理柔软但受限的 机械线索以及模拟腔内流动的微尺度流体灌注能力，以进一步诱导 生理结构，如隐窝和绒毛，这两种特性（约束、流动）都具有重要意义。 我们的假设是通过提供一种对肠道发育、分化和功能的影响。 机械限制的培养条件和液体灌注，而不是自由扩张培养 目前用于 HIO 形成的静态、封闭腔，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 隐窝中肠干细胞（ISC）的组织和绒毛上分化细胞的结合。 HIO 培养装置中的微尺度流体灌注能力也将允许精确调节腔内 从技术上讲，该项目将导致营养物质的流动以及细菌和病原体的长期定殖。 创新开发方法（“超软光刻”）可重复地创建超软 PDMS 生理模量为1-100 kPa的结构。 实现闭环控制以维持组织稳态并提供组织读数 功能，该项目还将集成微型氧传感器和跨上皮电电极 此外，还具有 HIO 内部和外部的采样功能。 将纳入液体和药物吸收/分泌的离线测量。 设备还将有助于测量集成 HIO 免疫共培养物中细胞因子的产生。 最后，我们将通过以下方式展示生物工程和仪器化 HIO 系统的模块化和实用性 整合 NAMSED 项目 1、2 和 3。具体来说，将生成具有管腔流量的仪表化 HIO， 与免疫细胞共培养并被益生菌微生物（乳杆菌 GG、LGG）和/或病原体定植 （鼠伤寒沙门氏菌），（益生菌/HIO/免疫与益生菌/病原体/HIO/免疫），我们将进行测试。 系统通过测量上皮屏障功能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，最后通过检查上皮侵袭 我们还将测试该系统通过生成来筛选药物/化合物的效用。 仪器化 LGG/鼠伤寒沙门氏菌/HIO/免疫共培养物并添加头孢哌酮（一种抗生素）， 选择性针对病原体鼠伤寒沙门氏菌，但不针对益生菌 LGG 头孢哌酮的杀灭能力。 将通过培养管腔流出物来检查鼠伤寒沙门氏菌以确定鼠伤寒沙门氏菌菌落形成 最后，当活培养终止时，我们将收获抗生素治疗之前、期间和之后的单位。 系统并使用免疫荧光检查不同组之间的细胞和分子差异 或对纯化的免疫细胞和上皮进行 qRT-PCR。