Bioelectronic in vitro models for monitoring intestinal host-pathogen interactions

Abstract

The human gastrointestinal tract is a highly complex, dynamic interface where host-microbe interactions not only underpin digestion and immunity but also disease pathogenesis. Studying this in vitro requires models that not only sufficiently recapitulate the physiological complexity of the barrier being investigated but also offer sensitive, time-resolved, insight into the biochemical and biophysical processes occurring at that interface. This thesis presents the integration of organic bioelectronics, functional materials and tissue engineering principles with intestinal models at varying biological length scales and complexities to enable monitoring of interactions between pathogens, or their metabolites, and the host. By interfacing biology with organic electronics, I demonstrate how electrical impedance measurements can sensitively detect barrier disruption, pathogen-induced damage, and dynamic changes in physiology that are not accessible with conventional endpoint assays. To achieve this, the gut is viewed a series of biological barriers, or obstacles, in which a pathogen could face as it seeks to invade the host – including the colonic mucus layer, polarised epithelial cell membrane, and mucosa. Each barrier is modelled using a purpose-built platform that allows for the mechanistic dissection of how pathogens and their products interact with, and breach, these defences. Beginning at the innermost barrier of the gut, Chapter 2 introduces a mucus-on-a-chip model which enables the label-free electrochemical monitoring of mucus barrier disruption by mucolytic compounds and biofilms formation by E. coli LF82, a strain associated with Crohn’s disease. This model is validated using rheological and electrochemical techniques and is demonstrated to recapitulate key features of native mucus including viscoelasticity. Chapter 3 explores the interactions between a pathogen and the enterocyte cell membrane by developing a cell-membrane-on-a-chip model with membrane components derived directly from the apical and basal regions of epithelial cells which express native receptors. By isolating and interfacing native plasma membrane vesicles onto conducting polymer microelectrode arrays, a direct comparison of the susceptibility of apical and basal membranes to alpha hemolysin toxin is made, in addition to the investigation of pathogenic extracellular vesicles interacting with the host cell membrane. These supported lipid bilayers interfaced with conducting polymer microelectrode arrays provide a quantitative measure of membrane damage revealing membrane-specific responses. This tool enables the investigation of specific host factors that mediate toxin sensitivity and the identification of proteins that may represent therapeutic targets. Chapter 4 reports the development and viral infection of the 3D gut E-Transmembrane, a stratified triculture model of the epithelium and underlying lamina propria on a soft, conducting polymer, scaffold. This platform incorporates fibroblasts, epithelial cells, and goblet cells in a physiological architecture that is compatible with both electrical and optical monitoring of viral infection. The platform revealed distinct infection kinetics with altered viral titre and electrical barrier resistance compared to conventional systems. This, in addition to the distinct cytokine release is attributed to the architectural complexity of the E-Transmembrane highlighting the importance of cell-cell communication and physiological relevance. These findings suggest advanced tissue-engineered models are essential for accurately predicting host response to infection and thus could enable more generalisable preclinical evaluation of antiviral strategies. Across these platforms, electrochemical impedance spectroscopy (EIS) was employed to enable non-invasive, label-free, and quantitative monitoring of biological barriers and pathogen interactions. The use of EIS throughout this work allows for time resolved tracking of pathogen-induced barrier disruption thus providing a powerful alternative to destructive or low throughput biochemical assays. Together, this work establishes a toolkit for interrogating gut-associated pathogenesis across a spectrum of reductionist to physiologically complex tissue models. The platforms here are modular and adaptable, enabling rapid screening of pathogen interactions and pharmaceutical candidates and demonstrates compatibility with conventional imaging and molecular biology techniques. With the growing threat of emerging pathogens and the uncertainty surrounding the efficacy of existing therapeutics against novel agents, advanced bioelectronic models offer a powerful path forward for predictive, mechanistic studies of the invisible battles unfolding at the various biological interfaces of the gut. Crucially, by bridging engineering innovation with biological relevance, such tools can facilitate rapid, translatable, preclinical testing and enable personalised therapeutic strategies to ultimately improve patient outcomes.