A tunable 3D human small intestinal tissue model for study of enteric pathogens

摘要 由细菌病原体引起的肠道感染通常会使人虚弱并危及生命。最多的 研究这些病原体的常用模型是体内啮齿动物模型和体外肠上皮细胞模型 单层。然而，这些模型往往不能显示发生的肠道感染的真实结果。 在人的肠道里。因此，这些病原体与人类之间相互作用的许多方面 主机仍然未知。该项目旨在剖析复杂的宿主-病原体相互作用 肠道病原体，霍乱弧菌和致病性假结核耶尔森氏菌，使用多细胞 具有绒毛和血流的三维体外人体组织模型。我们将开发和应用一种生物工程模型 人的肠黏膜，通过以下方式模拟肠道的生理结构和功能 将原代肠道细胞、血流动力学和绒毛拓扑引入3D支架。具体地说，我们将使用 以丝蛋白为支架构建支持人体肠道上皮的三维多细胞基质系统 用于持续培养和肠道病原体感染的形成。这个脚手架设计是基于我们的 先前开发的3D丝素支架系统种植于培养的细胞系Caco-2和HT-29细胞和 原代人肌成纤维细胞。一旦我们合并了初级上皮细胞，绒毛-拓扑和血流 动力学，并建立这个3D人体肠道模型，我们将研究霍乱弧菌和假结核杆菌如何 定植并对人体肠道造成损害。因为这些病原体有非常不同的 对人体肠道的病理生理结果它们是探索 这些新的肠道系统的3D生物模拟的多功能性。我们的目标是(1)建立和描述一个 用原代细胞、血流和绒毛建立人体小肠组织的三维模型；(2)研究小肠组织的空间和 病原菌定植和损害的暂时性动态以及病原菌的病理生理反应 在这些3D组织中，宿主细胞可感染霍乱弧菌或假结核杆菌。最终的结果将是一个强大的， 易于处理、特点良好的3D小肠组织模型系统，可供现场使用 研究对肠道病原体成功定植至关重要的具体机制步骤 宿主肠道。重要的是，这些研究将提供一个平台，在此平台上建立一个更大的计划，其中 多名研究人员可以使用这些3D系统来探测与各种肠道病原体的相互作用， 微生物区系和抗感染药物，以及进一步修改这些系统，以纳入其他类型的细胞和 寄主因素。

ABSTRACT Enteric infections caused by bacterial pathogens are often debilitating and life-threatening. The most common models for studying these pathogens are in vivo rodent models and in vitro intestinal epithelial cell monolayers. However, these models often do not manifest the true outcomes of enteric infections that occurred in the human intestine. Therefore, many aspects of the interactions between these pathogens and the human host remain unknown. This project aims to dissect the intricate host-pathogen interactions for two important intestinal enteric pathogens, Vibrio cholerae and pathogenic Yersinia pseudotuberculosis, using a multicellular 3D in vitro human tissue model that has villi and flow. We will develop and employ a bioengineered model of the human intestine tunica mucosa that mimics the physiological structures and functions of the intestine by introducing primary intestinal cells, flow dynamics, and villus topology to a 3D scaffold. Specifically, we will use silk proteins as scaffolds to develop a 3D multicellular matrix system to support human intestinal epithelium formation for sustained cultivation and for infection by enteric pathogens. This scaffold design is based on our previously developed 3D silk scaffold system seeded with the cultured cell lines Caco-2 and HT-29 cells and primary human myofibroblast cells. Once we incorporate primary epithelial cells, villus-topology and flow dynamics and build this 3D human intestinal model, we will study how V. cholerae and Y. pseudotuberculosis colonize and cause damage to the human intestine. Because these pathogens have very different pathophysiological outcomes on the human intestine they are excellent models to use in exploring the versatility of these novel 3D bio-mimetics of the intestinal system. Our aims are (1) to build and characterize a 3D model human small intestinal tissue with primary cells, flow and villi and (2) investigate the spatial and temporary dynamics of pathogen colonization and damage as well as the pathophysiological responses of the host cells to V. cholerae or Y. pseudotuberculosis in these 3D tissues. The end result will be a robust, tractable, and well-characterized 3D small intestinal tissue model system that can be used by the field for studying the specific mechanistic steps that are important for enteric pathogens to successfully colonize the host intestine. Importantly, these studies will provide a platform upon which to build a larger program in which multiple investigators can use these 3D systems to probe interactions with various enteric pathogens, microbiota, and anti-infectives as well as to further modify these systems to incorporate other cell types and host factors.