Development of a physiologically complex model of the human intestine for the study of absorption and detoxification

Abstract

The attrition rates of pharmaceutical therapeutics are extremely high, with only 5% of drug candidates entering human trials eventually gaining approval. One of the main reasons attributed to such failures is a lack of suitable preclinical models that can accurately predict in vivo pharmacokinetics. As the primary site of absorption for oral therapeutics, the intestine is a dominant organ determining the final bioavailability of drug candidates. Yet the standard model, a monolayer of epithelial cells cultured on a semipermeable plastic membrane, lacks the physiologically relevant architecture, extracellular matrix (ECM) components and underlying stromal cells of intestinal tissue. Consequently, a number of limitations still persist in its utility to accurately predict in vivo compound permeability. This thesis has evaluated electrospun poly(L-lactic acid) (PLLA) fibrous membranes as an alternative cell culture surface with which to develop a more physiologically relevant model of the human intestine. Characterisation of these fibrous membranes revealed a topographic architecture highly reminiscent of the fibrillous ECM structure of the intestinal basement membrane, which promoted the adhesion and growth of Caco-2 cells into contiguous monolayer. Immunofluorescence imaging, gene and protein expression techniques validated the PLLA fibre monolayer as equivalent in its differentiation and tight junction formation to the standard model, yet demonstrated notable morphological differences and improved barrier properties. Following this, the physiological complexity of the PLLA fibre monolayer was further developed to include additional stromal elements, namely adjacent subepithelial fibroblast cells. A series of co-culture models with differing proximities of fibroblast cells revealed that intestinal permeability, enzyme and transporter protein activity had different sensitivities to fibroblast signalling. Comparison of fibroblasts co-cultures and monolayers in both standard and PLLA fibrous inserts, demonstrated that intestinal activity was most improved by both topographic cell culture surface and by fibroblasts incorporation. Taken together, the PLLA fibrous epithelial/fibroblast bilayer fabricated in this thesis provided a model of the intestine with the most physiological relevant function, and an improved preclinical tool for the study of absorption and detoxification.

Date of Award	1 Aug 2021
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Jian Lu (Supervisor) & Jeffrey Penny (Supervisor)