Periodic Reporting for period 1 - BiSCUIT (Biomimetic Sensorized Barriers-on-a-Chip: Unveiling a new Generation of Market-Ready Investigation Tools)
Período documentado: 2024-07-01 hasta 2025-12-31
Diseases of the central nervous system (CNS), including neurodegenerative disorders and brain cancers, represent one of the most pressing global health challenges, yet therapeutic development in this field remains remarkably inefficient. A major bottleneck is the blood–brain barrier (BBB), a highly selective and dynamic interface that tightly regulates molecular transport between the bloodstream and the brain parenchyma. Current preclinical models fail to reliably predict human BBB permeability and toxicity, leading to costly late-stage failures and extensive reliance on animal experimentation.
Traditional in vitro approaches lack 3D architecture, physiological flow, and real-time functional readouts. Conversely, in vivo animal models are expensive, ethically constrained, time-consuming, and often poorly predictive due to interspecies differences. As a result, there is a clear and unmet need for advanced human-relevant platforms that can faithfully reproduce BBB structure and function while enabling quantitative assessment of barrier integrity / model maturation and drug transport.
The main objective of the project BiSCUIT was to address this gap by developing sensorized, biomimetic 3D BBB-on-chip platform that combines real-scale vascular geometry, controlled microfluidic flow, and embedded with real-time monitoring of maturation and integrity through impedance-based measurements, such as trans-endothelial electrical resistance (TEER). This approach moves beyond endpoint assays, allowing continuous tracking of barrier dynamics under physiological and pathological conditions.
A further objective was to demonstrate the predictive power of the platform for drug delivery and nanomedicine, by assessing the transport and cellular interaction of therapeutic molecules and nanoparticles in conditions that closely mimic the human neurovascular unit. In the broader vision of the project, the BBB model is conceived as a modular component that can interface with advanced 3D disease models, including brain tumor cultures, enabling integrated studies of barrier crossing and downstream therapeutic efficacy.
The expected impact of the project is significant. Scientifically, it provided a robust and quantitative tool to investigate BBB physiology, dysfunction, and drug permeability with unprecedented spatiotemporal resolution. Technologically, it established a new generation of sensorized organ-on-chip systems that merge biomimetic microfabrication with real-time functional readouts. From a societal and economic perspective, the platform has the potential to reduce animal use in line with the 3Rs principle, lower preclinical development costs, and accelerate the translation of effective CNS therapies.
Traditional in vitro approaches lack 3D architecture, physiological flow, and real-time functional readouts. Conversely, in vivo animal models are expensive, ethically constrained, time-consuming, and often poorly predictive due to interspecies differences. As a result, there is a clear and unmet need for advanced human-relevant platforms that can faithfully reproduce BBB structure and function while enabling quantitative assessment of barrier integrity / model maturation and drug transport.
The main objective of the project BiSCUIT was to address this gap by developing sensorized, biomimetic 3D BBB-on-chip platform that combines real-scale vascular geometry, controlled microfluidic flow, and embedded with real-time monitoring of maturation and integrity through impedance-based measurements, such as trans-endothelial electrical resistance (TEER). This approach moves beyond endpoint assays, allowing continuous tracking of barrier dynamics under physiological and pathological conditions.
A further objective was to demonstrate the predictive power of the platform for drug delivery and nanomedicine, by assessing the transport and cellular interaction of therapeutic molecules and nanoparticles in conditions that closely mimic the human neurovascular unit. In the broader vision of the project, the BBB model is conceived as a modular component that can interface with advanced 3D disease models, including brain tumor cultures, enabling integrated studies of barrier crossing and downstream therapeutic efficacy.
The expected impact of the project is significant. Scientifically, it provided a robust and quantitative tool to investigate BBB physiology, dysfunction, and drug permeability with unprecedented spatiotemporal resolution. Technologically, it established a new generation of sensorized organ-on-chip systems that merge biomimetic microfabrication with real-time functional readouts. From a societal and economic perspective, the platform has the potential to reduce animal use in line with the 3Rs principle, lower preclinical development costs, and accelerate the translation of effective CNS therapies.
The core technical activity of BiSCUIT consisted of developing biomimetic BBB-on-chips system integrating microfluidics, high-resolution 3D microfabrication, and embedded electrical sensing for real-time functional monitoring.
The integration of impedance-based sensing represented a key technical milestone, and it has been validated though a soft-lithography-based microfluidic model. Using electrochemical impedance spectroscopy and equivalent-circuit modeling, the system enabled non-invasive monitoring of BBB formation and maturation. Quantitatively, BBB models exhibited TEER values of 17.8 ± 1.6 Ω·cm2, consistent with state-of-the-art dynamic BBB-on-chip platforms. Permeability assays using 70 kDa FITC–dextran showed an ≈80% reduction in apparent permeability in well-formed barriers compared to controls, confirming functional tight junction formation.
The platform was further validated for drug and nanomaterial testing. Dynamic permeability experiments demonstrated the ability to discriminate compounds with different BBB-crossing profiles: temozolomide showed rapid and extensive transport across the barrier (up to ≈96% normalized passage after 60 min), while doxorubicin exhibited limited permeability (≈33% after 60 min), in agreement with known clinical behavior. Real-scale models were tested as well, and validated though the testing of an antioxidant nanomedicine platform (Prussian Blue nanoparticles).
In parallel, complementary impedance-based platforms exploiting 3D microfabricated scaffolds were developed to demonstrate scalability and versatility for CNS-related drug screening. These systems enabled multiplexed, real-time monitoring of 3D tumor spheroids, detecting drug-induced cytotoxic effects that were not evident with conventional endpoint assays, and achieving >80% responsive sensing sites across multielectrode arrays.
A final major achievement was the successful fabrication of real-scale porous microtubular scaffolds (10–50 µm diameter) by two-photon lithography, precisely aligned with thin-film platinum microelectrodes (Figure 1). This architecture enabled endothelial cells to organize into a vessel-like tubular structure under controlled flow conditions, closely mimicking brain capillaries.
The project delivered fully validated, sensorized 3D BBB and CNS-relevant in vitro platforms capable of real-time functional readouts, quantitative permeability assessment, and predictive drug screening. These outcomes establish a robust technological foundation for advanced CNS research, with clear potential to reduce animal testing, improve preclinical predictivity, and support future translational and personalized medicine applications.
The integration of impedance-based sensing represented a key technical milestone, and it has been validated though a soft-lithography-based microfluidic model. Using electrochemical impedance spectroscopy and equivalent-circuit modeling, the system enabled non-invasive monitoring of BBB formation and maturation. Quantitatively, BBB models exhibited TEER values of 17.8 ± 1.6 Ω·cm2, consistent with state-of-the-art dynamic BBB-on-chip platforms. Permeability assays using 70 kDa FITC–dextran showed an ≈80% reduction in apparent permeability in well-formed barriers compared to controls, confirming functional tight junction formation.
The platform was further validated for drug and nanomaterial testing. Dynamic permeability experiments demonstrated the ability to discriminate compounds with different BBB-crossing profiles: temozolomide showed rapid and extensive transport across the barrier (up to ≈96% normalized passage after 60 min), while doxorubicin exhibited limited permeability (≈33% after 60 min), in agreement with known clinical behavior. Real-scale models were tested as well, and validated though the testing of an antioxidant nanomedicine platform (Prussian Blue nanoparticles).
In parallel, complementary impedance-based platforms exploiting 3D microfabricated scaffolds were developed to demonstrate scalability and versatility for CNS-related drug screening. These systems enabled multiplexed, real-time monitoring of 3D tumor spheroids, detecting drug-induced cytotoxic effects that were not evident with conventional endpoint assays, and achieving >80% responsive sensing sites across multielectrode arrays.
A final major achievement was the successful fabrication of real-scale porous microtubular scaffolds (10–50 µm diameter) by two-photon lithography, precisely aligned with thin-film platinum microelectrodes (Figure 1). This architecture enabled endothelial cells to organize into a vessel-like tubular structure under controlled flow conditions, closely mimicking brain capillaries.
The project delivered fully validated, sensorized 3D BBB and CNS-relevant in vitro platforms capable of real-time functional readouts, quantitative permeability assessment, and predictive drug screening. These outcomes establish a robust technological foundation for advanced CNS research, with clear potential to reduce animal testing, improve preclinical predictivity, and support future translational and personalized medicine applications.
BiSCUIT generated advanced sensorized 3D in vitro platforms that realistically reproduce key structural, mechanical and functional features of the BBB and of related CNS microenvironments (patent-protected; Figure 2). By combining biomimetic microfabrication, controlled microfluidics, and real-time impedance sensing, the developed systems enable quantitative assessment of cellular responses under physiologically relevant conditions. These results address a critical limitation of current preclinical models, namely the lack of predictive, human-relevant tools for CNS drug development.
The potential impact is multifold. Scientifically, the platforms provide new insights into BBB maturation, dysfunction, and drug transport dynamics, supporting more reliable screening of therapeutics and nanomedicines. Technologically, they establish a new class of integrated, sensorized organ-on-chip systems capable of real-time functional readouts, reducing reliance on endpoint assays. Societally and economically, the results support the reduction of animal experimentation in line with the 3Rs principle and have the potential to lower costs and failure rates in CNS drug development pipelines.
To ensure further uptake and long-term success, several key needs can be identified. Further research and demonstration activities are required to validate the platforms across a broader range of compounds, disease models and patient-derived cells. Scale-up, standardization of fabrication and measurement protocols, and benchmarking against regulatory reference methods will be essential for industrial adoption. Access to translational funding and engagement with pharmaceutical and biotech stakeholders will facilitate commercialization. Finally, alignment with emerging regulatory and standardization frameworks will be crucial to support acceptance in preclinical and regulatory contexts; in this regard, at the end of the project, a CEN Workshop Agreement has been published (Guidelines for Blood-Brain Barrier on-Chip Models for Drug Delivery Testing).
The potential impact is multifold. Scientifically, the platforms provide new insights into BBB maturation, dysfunction, and drug transport dynamics, supporting more reliable screening of therapeutics and nanomedicines. Technologically, they establish a new class of integrated, sensorized organ-on-chip systems capable of real-time functional readouts, reducing reliance on endpoint assays. Societally and economically, the results support the reduction of animal experimentation in line with the 3Rs principle and have the potential to lower costs and failure rates in CNS drug development pipelines.
To ensure further uptake and long-term success, several key needs can be identified. Further research and demonstration activities are required to validate the platforms across a broader range of compounds, disease models and patient-derived cells. Scale-up, standardization of fabrication and measurement protocols, and benchmarking against regulatory reference methods will be essential for industrial adoption. Access to translational funding and engagement with pharmaceutical and biotech stakeholders will facilitate commercialization. Finally, alignment with emerging regulatory and standardization frameworks will be crucial to support acceptance in preclinical and regulatory contexts; in this regard, at the end of the project, a CEN Workshop Agreement has been published (Guidelines for Blood-Brain Barrier on-Chip Models for Drug Delivery Testing).