Developing bioelectronic sensors for 3D in vitro models at the air liquid interface: Innovative technology for biomedical and respiratory research.
Respiratory diseases and airborne infections are significant global health issues and a major cause of morbidity and mortality worldwide. According to the World Health Organization (WHO), respiratory diseases rank as the third leading cause of death globally, with over 10 million deaths per year attributed to respiratory illnesses. Chronic respiratory conditions are characterized by aberrant immune and epithelial barrier function, leading to respiratory inflammation and tissue damage. Despite substantial progress in identifying therapeutic targets and drug candidates, the efficacy of novel treatments in clinical trials remains low. One of the reasons for this, is that the current gold standard models of human disease used for drug screening rely on two-dimensional (2D) cell culture and/or animal testing. However, neither of these models accurately reflects the native microenvironment of human tissues. Recent advancements in bioengineering have resulted in the development of *in vitro* tissue models that more closely mimic the native tissue microenvironment. These frameworks include the development of extracellular matrix (ECM) materials, microfluidics, and three dimensional (3D) organoids. However, the clinical translatability of these models is limited by the lack of sensor technologies capable of monitoring them non-invasively and in real time. Gold standard analysis techniques rely on sample labelling or destructive end-point analysis, while electronic methods require large, rigid electrode materials that are ill adapted to soft 3D microarchitecture. Additionally, these electronics require submersion in an electrolyte for operation, which is inadequate for native air-liquid interfaced tissue such as the lungs. In this context, the goal of this thesis project was to address these challenges from multiple angles and in a multidisciplinary manner. The first goal was biology-focused and aimed at transitioning from 2D monocultures to a multicellular 3D model. The development of the biological respiratory model was based on a co-culture of primary human bronchial epithelial cells and lung fibroblasts in collagen coated cell culture inserts. This model highlighted the impact that multicellular cross-talk can have on the development/differentiation of *in vitro* models and their differing response to drug stimulation. The second goal was materials-focused and aimed at creating a biomimetic ECM environment of the lung. A composite electroactive scaffold was developed using an organic conducting polymer blended with human Fibrinogen protein, which replicated the mechanical and morphological properties of the human airway. The platform was capable of monitoring barrier formation, in real-time over 32 days, and, permits continuous, simultaneous electrical readouts. The third goal was technology-focused and resulted in the design and fabrication of a novel microelectronic device that was capable of monitoring epithelial barrier function and perturbations of primary *in vitro* models at the air liquid interface (ALI). By developing integrated bioelectronic sensors, this work demonstrates the ability to monitor advanced 3D tissue-like systems non-invasively and in real time. Additionally, and for the first time, patient-specific electrical signatures of respiratory epithelial dysfunction were measured at ALI. This technology will also have the potential for use in other air interfaced model systems, including the gut, skin, eye and brain, for diagnostic, drug screening and personalised medicine applications. Overall, it is hoped the versatile biotechnology platforms developed here will contribute to the field of biomedical and respiratory research.
3D in vitro models, Air liquid interface, Bioelectronics, Epithelial barriers, Organic electronics, Respiratory disease
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