Shear Stress and Neuronal Pathologies: Organ-on-a-Chip Model to Study Neurovascular Interactions

The neurovascular unit (NVU) is an intricate structure that acts as the brain’s main gatekeeper. It is composed of several specialized cell types, including endothelial cells that line the blood vessels and form the first barrier, together with pericytes, astrocytes, neurons, and microglia. Their continuous communication, known as neurovascular coupling, is essential for maintaining brain function. When this delicate balance is disturbed, it can lead to various forms of neuronal dysfunction. A crucial factor in this communication is fluid shear stress (FSS), the frictional force created by blood flow along vessel walls. Physiological levels of FSS help endothelial cells acquire a Blood–Brain-Barrier (BBB)-like phenotype, strengthening the barrier and maintaining brain homeostasis. However, changes in blood flow can alter tight junctions’ organization and transporter activity, contributing to BBB disruption. Recent findings have linked such vascular alterations to neurodegenerative diseases, although the underlying molecular mechanisms remain largely unclear. The regulation of cerebral blood flow depends on feedback and feedforward mechanisms that adjust vascular tone in response to neuronal activity, a process called functional hyperemia. When this mechanism fails, it can result in insufficient energy delivery and impaired clearance of harmful molecules. Despite the clear connection between blood flow alterations and brain dysfunction, progress in understanding the link has been limited by the absence of realistic human models. Traditional 2D in-vitro systems do not recapitulate the complexity and dynamicity of the NVU, while in-vivo studies, mostly performed in animals, face important limitations due to species differences. Over the past decade, Organ-on-a-Chip (OoC) technology has emerged as a promising approach to bridge this gap. These microfluidic devices recreate key features of human tissues under controlled conditions, enabling more physiologically relevant studies. However, most NVU-on-a-chip models are made from PDMS (polydimethylsiloxane), a widely used but with some limitations material that absorbs small molecules and require complex fabrication procedures. To overcome these issues, this work employs 3D printing via stereolithography to create accessible and customizable NVU-on-a-chip platforms. This technique allows rapid prototyping, flexible design through CAD modeling, and the use of biocompatible materials suitable for biological experiments. Three main chip models were developed: • an open-top platform to study the effect of shear stress on endothelial cells; • an NVU-on-a-chip for investigating molecular cross-talk; • and a gut–brain axis model to explore inter-organ communication. In parallel, a low-cost Arduino-based TEER (Transendothelial/Transepithelial Electrical Resistance) sensor was designed to measure barrier integrity in real time, without labels or invasive methods. Using these modular systems, we demonstrated that 3D-printed microdevices can support the growth of endothelial and neuroglia cells while preserving their morphology and functionality. Live calcium imaging confirmed the responsiveness of both endothelial and neuronal cells to physiological stimuli. The gut–brain model further showed that inflammation in the gut compartment, induced by lipopolysaccharide (LPS), can affect neuronal activity, providing a useful tool to study systemic influences on the brain. Finally, integrating the Arduino-based TEER system into the NVU-chip allowed continuous monitoring of barrier properties. The combination of 3D printing and microfluidic design offers a cost-effective, flexible, and human-relevant approach for studying neurovascular physiology, disease mechanisms, and drug permeability in a dynamic environment.