Multi-Scale Modelling of Host-Microbe Interactions: From Cell Membranes to Multi-Cellular Gut Models
Recently, the role of the intestinal tract in health and disease has become of increasing interest. Translation of conventional 2D in vitro and animal models, which have been the cornerstone for intestinal research, is however limited; while human studies are expensive and preclude individual mechanisms from being investigated. More tailored in vitro models therefore offer an attractive avenue to study intestinal health and disease including microbiome-gut-immune interactions. Recently, there has been much progress in the development of in vitro intestinal models with studies incorporating mechanical and biophysical cues, immune cells and supporting longer-term co-culture of microbiota, amongst others. Nevertheless, platforms which combine all of these and more faithfully represent the intestinal tract cells are still lacking. Given the complexity of the intestinal tract, however, reductionist models tailored for a specific research question still provide much utility and opportunity. To this end, this thesis provides a brief overview of the structure of the gut and associated immune tissue to introduce the complexities involved with the gut-immune axis and how these may be challenging to recapitulate in vitro. Further, considerations for in vitro intestinal model development are introduced, together with electrochemical techniques which have been employed to monitor these models. Three different scales of in vitro models are then presented, namely from cell-free cell membrane-based models, to traditional cell monolayers and stratified multi-cell type approaches, to address three microbe-related applications. Different electrochemical devices are used to monitor each for applications in host-pathogen interaction, microbiome differences in irritable bowel syndrome and microbe metabolite effects. To this end, cell membrane models are first presented for the detection of whole bacteria, namely the opportunistic pathogen Vibrio vulnificus (V. vulnificus) known for its severe disease including gastroenteritis, wound infection and sepsis. Focus is given to establishing intestinal cell-derived membrane models, studying cell line susceptibility by monitoring barrier changes due to cell monolayer-V. vulnificus interactions, with results showing increased disruption of enterocytes compared to goblet cells. Enterocyte-derived membrane models are then used to validate the cell membrane models for detecting live V. vulnificus, first in a simple buffer, and then, after validating the platform’s compatibility with human blood for the first time, in human blood; demonstrating the potential of the platform for pathogen detection and point-of-care applications. A gut-immune model comprising of an epithelial cell monolayer and an immune cell (macrophage)-laden collagen hydrogel, is next presented together with a novel conformable device which contacts cells directly. Improved sensitivity of the device on this more complex model, as well as a simple monolayer model, is confirmed compared to the gold standard EVOMTM commonly used to monitor barrier resistance. The models and devices, supported by cytokine and metabolomics analysis, are used to study how faecal matter supernatants from patients with irritable bowel syndrome affect intestinal barrier integrity, compared to faecal matter supernatants from their household controls. Not only is this studied more broadly, but subsets of IBS patients identified to harbour differing intestinal microbiota are also considered in more detail, showing how in vitro models could be used in future to identify IBS subtypes and potentially to inform treatment approaches. Finally, a gut-immune-vasculature model is presented and utilised to investigate the potential for the conformable device to, for the first time, monitor two cell barriers within the same model independently. The model-device combination, supported by cytokine analysis, is first used to study known isolated bacterial metabolites or dietary compounds, showing changes in epithelial and endothelial barrier resistance; before being used to test more complex bacterial metabolites generated through the growth of four intestinal bacteria individually as well as a community of 25 different bacteria. Throughout this dissertation, the potential for employing tailored in vitro intestinal models coupled with electrochemical monitoring techniques for host-microbe interactions studies is demonstrated, highlighting the potential for these platforms in research, diagnostics and clinical applications.
bioelectronics, in vitro models, intestinal models, in vitro monitoring
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