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Patient, J.D. (2016) Development of an in-vitro epithelial-myofibroblast intestinal model. PhD thesis, University of Nottingham.

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Abstract

In vitro studies of drug permeability are traditionally carried out using cultured monolayers of epithelial cell lines grown on semi permeable membranes. Caco-2 cells, which are colon-derived, spontaneously form polarised cell layers when cultured in vitro which are akin to an epithelium of enterocyte-like cells. The Caco-2 model has been developed as a powerful in vitro tool in the early assessment of human drug permeability and is even approved by regulatory agencies for biowaver applications (i.e. in vitro tests in lieu of in vivo animal experiments).

As Caco-2 cells are derived from colon tissue they represent a more formidable barrier to drug absorption than the upper regions of the intestine which is where the majority of oral drugs permeate into the body. Whilst the Caco-2 model, alongside other in vitro methods, has provided a significant means to understand the mechanics of drug permeability. Many researchers have sought to improve upon the existing unicellular model; it is hoped that this will result in a more relevant and predictive model for researchers to test new drugs but also to dissect cellular cross talk and to probe cell-matrix interactions.

Myofibroblasts are a niche cell type located subjacent to epithelial tissues which regulate the integrity, growth and differentiation of the overlying epithelium. In this study the co-culture of human epithelial cell lines with a myofibroblast cell line, CCD-18co, were investigated to study how myofibroblasts influence the barrier integrity of epithelia in vivo. Additionally, nanofibre scaffolds produced by electrospinning were explored as 3-dimensional and topographically relevant cell scaffolds to support the growth of intestinal cells in vitro.

In a traditional transwell format, cultured epithelial lines Caco-2 (intestinal), HT-29 (intestinal) and Calu-3 (airway) in co-cultures with CCD-18co revealed cell-line specific response with respect to the modulation of barrier integrity. The mechanism of the modulation was confirmed to be mediated through paracrine signalling by using myofibroblast conditioned media.

Fibre scaffolds which mimic the fibrillar nature of the extra cellular matrix and basement membrane were produced by electrospinning using the polymer, poly (ethylene terephthalate). Nanofibre scaffolds were characterised and further optimised for cell culture with surface coating with collagen to achieve adequate cell attachment and confluence. Work was also conducted to incorporate villi architectures into the fibre scaffolds; the potential of this ambition was investigated by using models produced by rapid prototyping. These models, which demonstrated good fidelity with the actual villi dimensions found in vivo, were used during the electrospinning process to shape the polymer scaffolds towards the geometry found in intestinal tissue.

A number of molecular tracers and model drug compounds were used to evaluate the permeability profiles of Caco-2 monocultures, Caco-2/CCD-18co co-cultures cultured on the two different culture substrates in addition to the assessment of resected porcine intestinal tissue sections. Caco-2 cells and Caco-2/CCD-18co co-cultures grown on nanofibre substrates were found to have lower electrical resistance and higher permeability properties than their transwell equivalents. Caco-2/CCD-18co co-cultures on conventional transwell inserts demonstrated a permeability profile closer to the resected porcine tissue and reported human tissue values than the conventional Caco-2 model whilst maintaining p-glycoprotein assay sensitivity.

This work forms a solid foundation for further research into the role of myofibroblasts in epithelial cell function and in the development of more predictive in vitro cell models for widespread scientific research.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Rose, F.R.A.J. Ghaem-Maghami, A. Roberts, C.J. Williams, P.M. White, L.
Subjects:	Q Science > QP Physiology > QP1 Physiology (General) including influence of the environment
Faculties/Schools:	UK Campuses > Faculty of Science > School of Pharmacy
Item ID:	33617
Depositing User:	Patient, Jamie
Date Deposited:	21 Jul 2016 12:44
Last Modified:	12 Oct 2017 20:44
URI:	https://eprints.nottingham.ac.uk/id/eprint/33617

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