Construction of 3D mucosal models to enhance the structure and function of epithelia

Several cell-based culture models have been developed to better understand epithelial function in humans. These primarily use cell lines grown as single layer monocultures on a two-dimensional (2D) substrate. 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.
The academic partner in this collaboration specializes in the development of technologies to improve the growth, structure and function of cell-based assays. Preliminary work has produced organotypic 3D tissue constructs consisting of a 3D stroma supporting a basement membrane and simple epithelium. We hypothesise that this construct will possess more realistic tissue-like structure and function compared to existing 2D epithelial models. Working with ECACC Culture Collections, we will develop a series of robust 3D mucosal models to evaluate the function of different human epithelial barriers. It is anticipated that the behaviour of existing cell lineages supplied by ECACC will be enhanced when part of a 3D organotypic model. Specifically, the student will: 1) (0-18 mths) develop and optimize models of human colon, kidney and respiratory mucosa. Stromal cells (e.g. tissue-specific fibroblasts, PBMCs, endothelial cells) will be seeded into a porous scaffold to re-constitute the sub-mucosal tissues. Specific extracellular matrix proteins will form a basement membrane onto which epithelial cells will be seeded to create a simple epithelium (e.g. A549, MDCK). The student will experiment with the type and ratio of the different cells, seeding density, period of growth, etc. to optimise the model and create a tissue-like mimetic; 2) (6-24 mths) Detailed analysis of structure will be performed comparing the morphology of the mucosal model with equivalent real tissue and the existing 2D model. Histology, electron microscopy, and immunofluorescence will be used to assess the structure of cells and expression of key proteins (e.g. ZO-1, Occludin) that are involved in barrier formation. This will be correlated Trans Epithelial Electrical Resistance (TEER) and passage of defined molecules under different growth conditions; 3) (18-30 mths) Assessment of the models will also include expression analysis of key transporters (e.g. P-gp, BCRP, MRPs (efflux); OATP1A2 (influx)) involved in epithelial function using real time PCR. Transport activity will be assessed directly by the addition of specific substrates and their movement across the mucosal construct. In addition, the permeability of the model to a series of model drugs will be assessed in relation to their known absorption fraction and permeability in vivo; 4) (24-36 mths) The model(s) will finally be tested under conditions to simulate an inflamed epithelial mucosa (such ischemic acute renal failure where defects in the kidney epithelium can occur). This will be achieved in combination with a pro-inflammatory stimuli followed by assessment of epithelial structure and function.