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

Lead Research Organisation: Durham University
Department Name: Biosciences

Abstract

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.

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
NC/N00289X/1 01/10/2016 30/09/2019
1804540 Studentship NC/N00289X/1 01/10/2016 30/09/2019 Nicole Darling