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

项目总结/摘要 该项目是一个结合设计驱动和假设驱动的项目，以生物工程的微尺度模型， 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 复杂的细胞组成和人类肠道的适当分层组织，该项目将 为这些三维（3D）人类肠道类器官（HIO）提供生理上柔软但限制性的 机械提示以及将模拟管腔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。这两个属性（约束、流动）都有显著的 影响肠道发育、分化和功能。我们的假设是，通过提供 机械限制的培养条件和流体灌注，而不是自由扩增培养， 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，包括作为隐窝和绒毛，沿着与相关的空间结构， 肠干细胞（ISCs）在隐窝中的组织和绒毛上的分化细胞。掺入 HIO培养装置中的微尺度流体灌注能力也将允许精确调节管腔内的微循环。 营养物质的流动，以及细菌和病原体的长期定植。从技术上讲，这个项目将是 创新地开发了一种用于可再现地产生超软PDMS的方法（“超软光刻”）， 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电极，用于跨上皮电 电阻（TEER）测量。此外，HIO内部和外部的采样能力 将被合并以实现流体和药物吸收/分泌的离线测量。HIO微量培养 这些装置还将有助于在整合的HIO-免疫共培养物中测量细胞因子的产生。 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。具体而言，将生成具有管腔流的仪器化HIO， 与免疫细胞共培养并被益生菌微生物（乳杆菌GG，LGG）和/或病原体定殖 （鼠伤寒沙门氏菌）。在每种共培养物中（益生菌/HIO/免疫vs.益生菌/病原体/HIO/免疫），我们将测试 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 鼠伤寒沙门氏菌我们还将测试该系统的实用性，以筛选药物/化合物，通过生成 仪器LGG/鼠伤寒沙门氏菌/HIO/免疫共培养物，并加入头孢哌酮，一种抗生素， 选择性地靶向病原体鼠伤寒沙门氏菌，而不是益生菌LGG。头孢哌酮的致死能力 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 在抗生素治疗之前、期间和之后。最后，当活培养物被终止时，我们将收获 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。

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.