Development of functional 3D human intestinal mucosae to replace rodent models and improve the predictive accuracy of existing in vitro systems.

开发功能性 3D 人类肠粘膜以取代啮齿动物模型并提高现有体外系统的预测准确性。

• 批准号: 1804540
• 金额: --
• 依托单位: Durham University
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1804540
• 关键词: Development; functional; 3D; human; intestinal

Investigations of intestinal absorption and secretory function, most often involve the use in vivo assays or ex vivo animal tissue transport models. However, due to the complexities of the in vivo environment and difficulty to standardize, detailed mechanistic studies are hard to interpret in such animal systems. Several simplistic cell-based culture models have been developed to better understand drug intestinal permeability in humans. However, such cultures are under developed and only partly recapitulate the structure of the epithelial mucosa. There are many deficits with these types of model including: 1) lack of other cell types; 2) lack of organized three dimensional (3D) structure; 3) the absence of essential inter-cellular signalling between the epithelium and underlying stromal tissues. Such limitations result in functional differences in permeability, receptor complexes, and transporters involved in absorption and secretion. There is significant demand to create superior intestinal models with improved structure and function. We hypothesise that a 3D intestinal mucosal construct will possess more realistic tissue-like structure and function compared to existing 2D epithelial models. The aim of this study is to demonstrate the superiority of a human 3D intestinal mucosa model to evaluate the function of the epithelial barrier in health and disease.The student will:1) Construct a 3D culture model of the human intestinal mucosa. Cultured cells will be adapted to 3D growth prior to construction of the model to further enhance its structure and functional performance. Different intestinal cell types will be seeded onto a scaffold to produce a unique 3D human intestinal construct comprised of epithelial and sub-mucosal layers to reconstitute the anatomy of the intestinal mucosa in vitro;2) Demonstrate the superior structure and function of the intestinal construct. The cellular architecture of the 3D model will compared with samples of real intestinal tissue and the existing 2D in vitro model. This will focus on polarization of epithelial cells and the formation of the physical barrier by examining tight junction proteins and measurement of epithelial resistance. To assess the functional characteristics of the construct, ion fluxes will be measured and the expression of transporter molecules will be determined and correlated with physiological activity using Ussing chambers and other functional assays;3) Simulate aspects of a known pathological condition where the model partially mimics abnormal intestinal function. A pro-inflammatory stimulus will be used induce a pathological response to mimic an inflamed intestinal mucosa together with the inclusion of inflammatory cells co-cultured within the 3D construct (as in Colitis and Crohn's disease). This will be subsequently characterized for structural and functional deficits;4) Generation an advanced system based on the 3D construct above using human induced Pluripotent Stem Cell (iPSC)-derived epithelial cells to simulate a pathological condition involving failure of normal intestinal epithelial ion transport. Intestinal 3D constructs will be made incorporating epithelial cell derivatives of iPSCs originally produced from cystic fibrosis patients. The functional deficits of the construct will be assessed to model features of cystic fibrosis.

研究肠道吸收和分泌功能，通常涉及体内测定或体内动物组织传输模型的使用。但是，由于体内环境的复杂性和标准化的难度，在此类动物系统中很难解释详细的机械研究。已经开发出几种简单的基于细胞的培养模型，以更好地了解人类的药物肠道通透性。但是，这种培养物正在开发中，仅部分概括了上皮粘膜的结构。这些类型的模型有许多缺陷，包括：1）缺乏其他细胞类型； 2）缺乏有组织的三维（3D）结构； 3）上皮和基础基质组织之间没有必需的细胞间信号。这种局限性导致渗透性，受体复合物和参与吸收和分泌的转运蛋白的功能差异。有很大的需求以创建具有改进的结构和功能的上层肠模型。我们假设与现有的2D上皮模型相比，3D肠粘膜构建体将具有更现实的组织样结构和功能。这项研究的目的是证明人类3D肠粘膜模型的优越性，以评估上皮屏障在健康和疾病中的功能。学生将：1）构建人类肠粘膜的3D培养模型。在构建模型之前，培养的细胞将适应3D生长，以进一步增强其结构和功能性能。不同的肠细胞类型将播种到支架上，以产生由上皮和亚粘膜层组成的独特的3D人类肠结构，以重建体外肠道粘膜的解剖结构； 2）证明了肠结构的优质结构和功能。 3D模型的细胞结构将与真实肠道组织和现有2D体外模型的样品进行比较。这将着重于上皮细胞的极化和通过检查紧密连接蛋白和上皮耐药性测量的物理屏障的形成。为了评估构建体的功能特征，将测量离子通量，并使用USSING室和其他功能测定法确定转运蛋白分子的表达并与生理活性相关； 3）模拟已知的病理状况的各个方面，其中该模型部分模拟了模拟异常的肠胃正常肠功能。促炎性刺激将引起对模仿发炎的肠粘膜的病理反应，以及在3D构建体中共培养的炎症细胞（如结肠炎和克罗恩病）。随后将以结构和功能性缺陷来表征； 4）使用人类诱导的多能干细胞（IPSC）衍生的上皮细胞基于上述3D构建体的生成高级系统，以模拟涉及正常肠内肠上皮离子转运的病理状况。将制作肠3D构建体，结合最初由囊性纤维化患者产生的IPSC的上皮细胞衍生物。该构建体的功能缺陷将被评估为模拟囊性纤维化的特征。