Real-time monitoring of a 3D blood–brain barrier model maturation and integrity with a sensorized microfluidic device†

Abstract

A significant challenge in the treatment of central nervous system (CNS) disorders is represented by the presence of the blood–brain barrier (BBB), a highly selective membrane that regulates molecular transport and restricts the passage of pathogens and therapeutic compounds. Traditional in vivo models are constrained by high costs, lengthy experimental timelines, ethical concerns, and interspecies variations. In vitro models, particularly microfluidic BBB-on-a-chip devices, have been developed to address these limitations. These advanced models aim to more accurately replicate human BBB conditions by incorporating human cells and physiological flow dynamics. In this framework, here we developed an innovative microfluidic system that integrates thin-film electrodes for non-invasive, real-time monitoring of BBB integrity using electrochemical impedance spectroscopy (EIS). EIS measurements showed frequency-dependent impedance changes, indicating BBB integrity and distinguishing well-formed from non-mature barriers. The data from EIS monitoring was confirmed by permeability assays performed with a fluorescence tracer. The model incorporates human endothelial cells in a vessel-like arrangement to mimic the vascular component and three-dimensional cell distribution of human astrocytes and microglia to simulate the parenchymal compartment. By modeling the BBB-on-a-chip with an equivalent circuit, a more accurate trans-endothelial electrical resistance (TEER) value was extracted. The device demonstrated successful BBB formation and maturation, confirmed through live/dead assays, immunofluorescence and permeability assays. Computational fluid dynamics (CFD) simulations confirmed that the device mimics in vivo shear stress conditions. Drug crossing assessment was performed with two chemotherapy drugs: doxorubicin, with a known poor BBB penetration, and temozolomide, conversely a specific drug for CNS disorders and able to cross the BBB, to validate the model predictive capability for drug crossing behavior. The proposed sensorized microfluidic device represents a significant advancement in BBB modeling, offering a versatile platform for CNS drug development, disease modeling, and personalized medicine.