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

对肠道吸收和分泌功能的研究，大多涉及使用体内试验或体外动物组织转运模型。然而，由于体内环境的复杂性和标准化的难度，在这样的动物系统中很难解释详细的机制研究。为了更好地了解药物在人体内的肠道通透性，已经开发了几个简单的基于细胞的培养模型。然而，这样的培养还不发达，只能部分概括上皮粘膜的结构。这些类型的模型有许多缺陷，包括：1)缺乏其他类型的细胞；2)缺乏有组织的三维(3D)结构；3)上皮和底层间质组织之间缺乏必要的细胞间信号。这些限制导致了通透性、受体复合体和参与吸收和分泌的转运蛋白的功能差异。有很大的需求创造出结构和功能都得到改善的高级肠道模型。我们假设，与现有的2D上皮模型相比，3D肠粘膜结构将具有更真实的组织样结构和功能。本研究的目的是展示人体三维肠粘膜模型在评估健康和疾病中上皮屏障功能方面的优越性。学生将：1)构建人体肠粘膜的三维培养模型。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。不同类型的肠道细胞将被种植到支架上，以产生由上皮层和粘膜下层组成的独特的3D人体肠道结构，以在体外重建肠粘膜的解剖结构；2)展示该肠道结构的优越结构和功能。3D模型的细胞结构将与真实的肠道组织样本和现有的2D体外模型进行比较。这将通过检测紧密连接蛋白和测量上皮细胞阻力来关注上皮细胞的极化和物理屏障的形成。为了评估该结构的功能特征，将使用Ussing小室和其他功能分析来测量离子通量，并确定转运蛋白分子的表达并将其与生理活性相关联；3)模拟已知病理条件的各个方面，其中模型部分模拟异常的肠道功能。促炎刺激将被用来诱导病理反应，以模拟发炎的肠粘膜，并在3D结构中包含共同培养的炎症细胞(如结肠炎和克罗恩病)。这将随后被描述为结构和功能缺陷的特征；4)基于上述3D构建的先进系统，使用人类诱导多能干细胞(IPSC)来源的上皮细胞来模拟涉及正常肠上皮离子转运失败的病理条件。肠道3D构建将结合最初从囊性纤维化患者产生的IPSCs的上皮细胞衍生品。该结构的功能缺陷将被评估以模拟囊性纤维化的特征。

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.