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使人衰弱并危及生命。最 用于研究这些病原体的常见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，这些模型通常不能显示发生肠道感染的真实结果， 在人类的肠道中。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。本项目旨在剖析复杂的宿主-病原体相互作用， 肠肠道病原体，霍乱弧菌和致病性假结核耶尔森氏菌，使用多细胞 具有绒毛和流动的3D体外人体组织模型。我们将开发和采用一种生物工程模型， 人肠图尼卡粘膜，其模拟肠的生理结构和功能， 将原代肠细胞、流动动力学和绒毛拓扑结构引入3D支架。具体来说，我们将使用 丝蛋白作为支架开发支持人肠上皮的3D多细胞基质系统 形成用于持续培养和用于肠道病原体感染。这个支架设计是基于我们的 先前开发的3D丝支架系统接种有培养的细胞系Caco-2和HT-29细胞， 原代人肌成纤维细胞。一旦我们将原代上皮细胞，绒毛拓扑结构和流动 动力学，并建立了三维人体肠道模型，我们将研究霍乱弧菌和Y。假结核 在人体肠道内定植并造成损害。因为这些病原体有着非常不同的 它们是用于探索人类肠道病理生理学结果的极好模型， 这些新型肠道系统3D生物模拟物的多功能性。我们的目标是（1）建立和表征一个 具有原代细胞、流动和绒毛的3D模型人小肠组织，以及（2）研究小肠组织的空间和结构。 病原体定殖和损害的暂时动态以及 宿主细胞对霍乱弧菌或Y.在这些三维组织中的假结核。最终的结果将是一个强大的， 易于处理且特征良好的3D小肠组织模型系统，其可由该领域用于 研究肠道病原体成功定植的具体机制步骤， 宿主肠道重要的是，这些研究将提供一个平台，在此基础上建立一个更大的计划， 多个研究者可以使用这些3D系统来探测与各种肠道病原体的相互作用， 微生物群和抗感染剂，以及进一步修饰这些系统以掺入其他细胞类型， 宿主因素

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.