The human Blood Brain Barrier (hBBB) is a complex cellular architecture separating the blood from the brain parenchyma. Its integrity and perfect functionality are essential for preventing neurotoxic plasma components and pathogens enter the brain. Although vital for preserving the correct brain activity, the low permeability of hBBB represents a huge impediment to treat mental and neurological disorders or to address brain tumors. Indeed, the vast majority of potential drug treatments are unable to reach the brain crossing the hBBB. On the other hand, hBBB integrity can be damaged or its permeability increase as a result of infections or in presence of neurodegenerative diseases. Current in vitro systems and in vivo animal models used to study the molecular/drug transport mechanism through the hBBB have several intrinsic limitations that are difficult to overcome. In this scenario, Organ-on-Chip (OoC) models based on microfluidic technologies are considered promising innovative platforms that combine the handiness of an in vitro model with the complexity of a living organ, while reducing time and costs. In this review, we focus on recent advances in OoCs for developing hBBB models, with the aim of providing the reader with a critical overview of the main guidelines to design and manufacture a hBBB-on-chip, whose compartments need to mimic the ‘blood side’ and ‘brain side’ of the barrier, to choose the cells types that are both representative and convenient, and to adequately evaluate the barrier integrity, stability, and functionality.

Organ‐on‐Chip (OoC) technology provides a powerful platform for neurovascular research, enabling the precise replication of the blood‐brain barrier (BBB) microenvironment, including its 3D architecture and the influence of dynamic blood flow. This study introduces a novel microfluidic device designed to investigate the morphological and structural adaptations of human brain endothelial cells (ECs) within narrow, square‐shaped microchannels that closely mimic the microvessels of the brain's microcirculation. The endothelial microchannels are layered above a microchamber filled with Matrigel and abluminal vascular cells, enhancing cell‐cell interactions across the BBB interface. The system integrates co‐culture with pericytes and astrocytes while subjecting brain ECs to physiologically relevant pulsatile flow. The findings reveal that the morphology and cytoskeletal organization of brain ECs are distinctly influenced by pulsatile flow depending on the presence of pericytes and astrocytes. Specifically, in the absence of perivascular support, brain ECs exhibit a stretched morphology with prominent actin stress fibers, while co‐culture with pericytes and astrocytes promotes endothelial rearrangement, leading to lumen formation and enhanced barrier properties. This study highlights the essential role of perivascular cells in modulating endothelial responses under microvascular confinement and physiologically relevant flow. These insights advance in vitro models of the neurovascular unit and BBB mechanobiology.

Cell migration assays provide valuable insights into pathological conditions, such as tumor metastasis and immune cell infiltration, and the regenerative capacity of tissues. In vitro tools commonly used for cell migration studies exploit commercial transwell systems, whose functionalities can be improved through engineering of the pore pattern. In this context, we propose the fabrication of a transwell-like device pursued by combining the proton beam writing (PBW) technique with wet etching onto thin layers of polydimethylsiloxane (PDMS). The resulting transwell-like device incorporates a PDMS membrane with finely controllable pore patterning that was used to study the arrangement and migration behavior of HCMEC/D3 cells, a well-established human brain microvascular endothelial cell model widely used to study vascular maturation in the brain. A comparison between commercial polycarbonate membranes and the PBW-holed membranes highlights the impact of the ordering of the pattern and porosity on cellular growth, self-organization, and transmigration by combining fluorescent microscopy and advanced digital processing. Endothelial cells were found to exhibit distinctive clustering, alignment, and migratory behavior close to the pores of the designed PBW-holed membrane. This is indicative of activation patterns associated with cytoskeletal remodeling, a critical element in the angiogenic process. This study stands up as a novel approach toward the development of more biomimetic barrier models (such as organ-on-chips).

Lipopolysaccharides (LPS) from Gram-negative bacteria represent a significant challenge across various industries due to their prevalence and pathogenicity and the limitations of existing detection methods. Traditional approaches, such as the rabbit pyrogen test (RPT) and the Limulus Amebocyte Lysate (LAL) assay, have served as gold standards for endotoxin detection. However, these methods are constrained by high costs, lengthy processing times, environmental concerns, and the need for significant reagent volumes, which limit their scalability and application in resource-limited settings. In this study, we introduce an innovative microfluidic platform that integrates the LAL assay within microdroplets, addressing the critical limitations of traditional techniques. By leveraging the precise fluid control and reaction isolation offered by microdroplet technology, the system reduces reagent consumption, enhances sensitivity, and enables high-throughput analysis. Calibration tests were performed to validate the platform’s ability to detect LPS, using colorimetric measurements. Results demonstrated comparable or improved performance relative to traditional systems, achieving lower detection limits and greater accuracy. This work demonstrates a proof-of-concept miniaturisation of the pharmacopoeial LAL assay. The method yielded low intra-assay variability (σ ≈ 0.002 OD; CV ≈ 0.9% over n = 50 droplets per point) and a LOD estimated from calibration statistics after path-length normalisation. Broader adoption will require additional comparative validation and standardisation. This scalable, cost-effective, and environmentally sustainable approach offers a practical solution for endotoxin detection in clinical diagnostics, biopharmaceutical production, and environmental monitoring. The proposed technology paves the way for advanced LPS detection methods that meet stringent safety standards while improving efficiency, affordability, and adaptability for diverse applications.

Barriers in the human body play a crucial role in regulating the exchange of substances between compartments, with permeability alterations occurring under both physiological and pathological conditions. In vitro barrier models are essential tools for studying the mechanisms of molecular diffusion across these barriers. Traditional coculture systems or advanced organ‐on‐chip (OoC) platforms mostly utilize permeable membranes based on artificial, nonbiodegradable materials. In this study, we introduced cellulose nanofibrils (CNFs)‐based membranes to develop a new class of in vitro barrier systems. CNFs, derived from natural sources, are nontoxic, biodegradable, optically transparent, and feature a 3D fibrillar structure that mimics the cellular basement membrane. We successfully modulated the permeability of CNF‐based membranes, interposed in dual‐chamber polydimethylsiloxane devices, to small molecules through chemical and enzymatic treatments, while preserving their ability to allow cell adhesion and growth. This technology holds potential for its integration in next‐generation OoC devices, offering more realistic and complex models that closely mimic the physiological behavior of human barriers.