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.

肠道吸收和分泌功能的研究最常涉及使用体内测定或离体动物组织运输模型。然而，由于体内环境的复杂性和标准化的难度，在此类动物系统中进行详细的机制研究很难解释。已经开发了几种基于细胞的简单培养模型，以更好地了解药物在人体中的肠道渗透性。然而，此类培养物尚不发达，并且仅部分概括了上皮粘膜的结构。这些类型的模型存在许多缺陷，包括：1）缺乏其他细胞类型； 2）缺乏有组织的三维（3D）结构； 3）上皮和下面的基质组织之间缺乏必要的细胞间信号传导。这些限制导致渗透性、受体复合物以及参与吸收和分泌的转运蛋白的功能差异。人们迫切需要创建结构和功能得到改善的优质肠道模型。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真实的组织样结构和功能。本研究的目的是证明人体 3D 肠粘膜模型在评估上皮屏障在健康和疾病中的功能方面的优越性。学生将：1) 构建人体肠粘膜 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。将不同类型的肠道细胞接种到支架上，产生由上皮层和粘膜下层组成的独特的3D人体肠道构建体，以在体外重建肠粘膜的解剖结构；2）展示肠道构建体的优越结构和功能。 3D 模型的细胞结构将与真实肠道组织样本和现有 2D 体外模型进行比较。这将通过检查紧密连接蛋白和测量上皮电阻来重点关注上皮细胞的极化和物理屏障的形成。为了评估构建体的功能特征，将测量离子通量，并使用 Ussing 室和其他功能测定确定转运蛋白分子的表达并与生理活性相关联；3) 模拟已知病理状况的各个方面，其中模型部分模拟肠道功能异常。将使用促炎刺激物诱导病理反应以模拟发炎的肠粘膜以及在 3D 构建体中共培养的炎症细胞（如结肠炎和克罗恩病）。随后将对其结构和功能缺陷进行表征；4) 使用人诱导多能干细胞 (iPSC) 衍生的上皮细胞，基于上述 3D 构建体生成先进系统，以模拟涉及正常肠上皮离子运输失败的病理状况。肠道 3D 构建体将包含最初从囊性纤维化患者产生的 iPSC 的上皮细胞衍生物。将评估构建体的功能缺陷以模拟囊性纤维化的特征。