The neurovascular unit (NVU) is a complex structure comprising endothelial cells, pericytes, astrocytes, neurons, and extracellular matrix (ECM) elements. It plays an essential role in regulating cerebral blood flow and maintaining the integrity of the blood-brain barrier (BBB). Over the years, several tissue engineering approaches modelling the NVU in vitro have gained popularity as they offer a platform to investigate the cellular cross-talk, unravel disease mechanisms as well as brain targeted therapeutics effects. While in vivo models have been invaluable in advancing our understanding of the brain functions, they also have significant limitations when it comes to tissue engineering purposes. Besides being expensive to establish and maintain, in vivo models are often species-specific and may not accurately reflect human pathophysiology. Additionally, ethical concerns around the use of animals in research can limit the types of studies that can be performed.

These limitations highlight the need for alternative in vitro models that can more accurately mimic the NVU structure and functions in health and disease. Two-dimensional (2D) cell culture models, such as Transwell systems, have been widely used for in vitro NVU modelling allowing the investigation of cell-cell interactions and cellular influence on the BBB properties and integrity. However, one setback of such 2D models is that they do not fully recapitulate the complex 3D microenvironment of the NVU, which can negatively impact cell behaviour and signalling pathways. Additionally, in 2D models, shear stress and other mechanical forces experienced by endothelial cells are not representative of physiological conditions in vivo. Organ-on-chips technology, on the other hand, has emerged as a promising and more robust alternative platform to study the dynamic interactions between neuronal and vascular components under controlled conditions. Nevertheless, some challenges, such as difficulty in accurately replicating the complex 3D architecture and physiology of the native NVU still remain.

Tissue engineering integrating bioelectronics has emerged as a promising strategy for in vitro modelling and monitoring of complex biological systems such as the NVU. This approach entails combining tissue-engineered equivalents (e.g., scaffolds, hydrogels) with integrated electronic sensors and devices, enabling real-time monitoring of cellular and molecular processes in situ. In addition, the seamless incorporation of bioelectronics within tissue-engineered systems enables the real-time and continuous recording of experimental conditions, hence avoiding end-point assay experiments. Furthermore, tissue engineering integrating bioelectronics has the remarkable potential to revolutionize disease modelling and drug screening by allowing the development of high-throughput platforms for screening drug candidates and personalized medicine. While there are still challenges to overcome, such as the biocompatibility of the electronic components, device design optimisation and the need for better integration methods, this approach holds great promise for advancing our understanding of complex biological systems and improving the design/development of therapeutics.

The objective of this dissertation was to create a novel bioelectronic model of the neurovascular unit (NVU) that could continuously monitor the blood-brain barrier (BBB) in vitro. To achieve this goal, the research combined fundamental principles from 3D cell biology, material science, and tissue engineering.

The first approach was to establish a 2D Transwell system as a reductionist and informative support for multiple NVU model configurations. The 2D system was used to determine the optimal cell culture conditions for endothelial cells, astrocytes, and neuronal cells. The resulting NVU in vitro platform was then characterised using optical and electrical methods to evaluate BBB integrity under different conditions. To improve and advance such biological model, the next step was to create a 3D biomimetic scaffold that resembled the brain extracellular matrix (ECM). The scaffold was designed to be a more physiologically relevant substrate than the 2D Transwell systems. A composite electroactive scaffold integrating ECM elements was generated to test its suitability to support neuronal cell culture and differentiation in situ. The biomimetic 3D scaffold was then used to host the multicellular NVU model, which demonstrated the formation of an apical endothelial barrier. Finally, the 3D NVU system was successfully incorporated into an in-house engineered bioelectronic platform called "e-Transmembrane." This platform enabled in-line, non-invasive, and dynamic monitoring of the NVU cell types and BBB development. The integration of the 3D NVU system into the e-Transmembrane platform represented a significant leap towards a more sophisticated and realistic model of the NVU and its BBB.

In conclusion, this study described above has significant potential for advancing our understanding of the blood-brain barrier (BBB) and the neurovascular unit (NVU) in vitro. Furthermore, such bioelectronic NVU model could represent a reliable test bed for drug discovery and screening by providing a more reliable and predictive platform for testing the permeability of drugs across the BBB.