A 3D in vitro model of the human gut-microbiome : a bioelectronics approach

Over the past decades, the human gut microbiota has emerged as a key player in the bidirectional communication of the gut-brain axis, affecting various aspects of health and disease. Although a vast array of studies have revealed the specific role of gut microbiota in various physiological and pathophysiological conditions, the mechanisms underlying the intricate cross-talk between the host and the intestinal flora remain a mystery. Until recently, the majority of studies that seek to explore these mechanisms relied almost exclusively on animal models, and particularly gnotobiotic mice. Despite the great progress made with these models, various limitations, including ethical considerations and interspecies differences, which limit the translatability of data to human systems, pushed researchers to seek for alternatives. The field of in vitro models has experienced tremendous growth recently, thanks to advances in 3D cell biology, materials science and bioengineering. These advances have enabled the development of novel in vitro models of the human gut, among other tissues, that more faithfully emulate the native tissue structure and function, including organotypic cultures and bioengineered tissues. In parallel, organs-on-chips technology has emerged as a promising alternative to traditional approaches, and especially animal models, for the development of more predictive in vitro models that can successfully replicate parts of human physiology and organ function. Successful organs-on-chips integrate human cells within tissues surrounded by biomolecular components, and have benefited from the use of inert 3D gels and scaffolds as templates to prompt tissue formation. However, the fidelity of the organs-on-chips cellular models that, in most cases, still rely on simplistic two-dimensional cellular architectures, and the lack of in-line quantitative assessment of the tissue physiology, hamper their clinical translation. Thanks to recent advancements in the field, bioelectronics now offer a broad toolbox at the service of various biomedical applications. Integration of bioelectronic devices with bioengineering and organs-on-chips models can be particularly favourable for delivering more sophisticated biomimetic models with in-line monitoring capabilities, apt for studies looking at host-microbiome interactions. The aim of this dissertation was to develop a bioelectronic platform to host and monitor a 3D model of the human intestine and microbiome. To achieve this goal, a multidisciplinary approach was employed, combining principles from 3D cell biology and tissue engineering with organic electronics and organs-on-chips approaches. The first stage was to identify the most appropriate constituents of the intestinal model, both in terms of biology and electronics. Different co-culture systems, comprising intestinal and immune cells, were established and interfaced either with hydrogels or conducting polymer scaffolds, fabricated to mimic the native extracellular niche. In the next stage, the intestinal model was further enhanced to incorporate components of the intestinal wall structure and was successfully integrated in two different bioelectronic platforms, the Bioelectronic Transmembranes and the L-Tubistor. In these platforms, the electroactive scaffolds served as a template for the 3D human intestine while facilitating dynamic electrical monitoring of tissue formation for about 3-4 weeks. While in both cases a fully differentiated intestinal epithelium was established, exhibiting typical features of its native counterpart, such as polarisation and stratification, progressing from the Bioelectronic Transmembranes to the hollow tubular scaffolds of the L-Tubistor resulted in a more biomimetic in vitro platform. The intrinsic electrical conductivity of the scaffolds allowed for non-invasive, in-line functional assessment of the tissue, alleviating the need for post hoc placement of invasive sensory probes or electrodes. Finally, stable co-cultures of the in vitro intestinal models and simple bacterial strains - commensal to the human gut - were established and characterised optically and electrically, showing great promise towards building a complete human gut-microbiome in vitro platform. In conclusion, simple prototype in vitro platforms were engineered in this project that combine 3D intestinal tissues with integrated sensing capabilities, constituting valuable tools to study intestinal (patho-) physiology, host-pathogen and host-microbe interactions. Most importantly, this work provides a framework for interfacing complex biological systems with highly sensitive in-line electronic assays, representing a paradigm shift in modelling human (patho-) physiology.