Modelling a Human Blood-Brain Barrier Co-Culture Using an Ultrathin Silicon Nitride Membrane-Based Microfluidic Device

Hudecz, Diána; McCloskey, Molly C.; Vergo, Sandra; Christensen, Søren; McGrath, James L.; Nielsen, Mogens

doi:10.3390/ijms24065624

Cited by 3 publications

(1 citation statement)

References 56 publications

(118 reference statements)

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“…In these experiments, the total antibody signal will be comprised of specific EV labeling events (true positives) as well as disassociated antibody complexes large enough to be captured by NPN pores (antibody molecules pass freely through the pores of NPN). 48 Just as counting becomes inaccurate at high capture densities, a false colocalization of pan EV and unassociated antibody signals will be increasingly likely at high concentrations. To develop guidelines to help avoid misinterpreting false colocalization, we performed CAD-LB with two nanoparticle species, each labeled with a distinct fluorescent dye (red or blue) and exclusively visualized in separate fluorescence microscope channels.…”

Section: Evaluating the Accuracy And Dynamic Range Of Cad-lbmentioning

confidence: 99%

Rapid Assessment of Biomarkers on Single Extracellular Vesicles Using ‘Catch and Display’ on Ultrathin Nanoporous Silicon Nitride Membranes

Walker,

Lucas,

Dewey

et al. 2024

Preprint

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Extracellular vesicles (EVs) are particles secreted by all cells which carry bioactive cargo and facilitate intercellular communication with roles in normal physiology and disease pathogenesis. EVs have tremendous diagnostic and therapeutic potential and accordingly, the EV field has grown exponentially in recent years. Bulk assays lack the sensitivity to detect rare EV subsets relevant to disease, and while single EV analysis techniques remedy this, they are undermined by complicated detection schemes often coupled with prohibitive instrumentation. To address these issues, we propose a microfluidic technique for EV characterization called 'catch anddisplay forliquidbiopsy (CAD-LB)'. CAD-LB rapidly captures fluorescently-labeled EVs in the similarly-sized pores of an ultrathin silicon nitride membrane. Minimally processed sample is introduced via pipette injection into a simple microfluidic device which is directly imaged using fluorescence microscopy for a rapid assessment of EV number and biomarker colocalization. In this work, nanoparticles were first used to define the accuracy and dynamic range for counting and colocalization by CAD-LB. Following this, the same assessments were made for purified EVs and for unpurified EVs in plasma. Biomarker detection was validated using CD9 in which Western blot analysis confirmed that CAD-LB faithfully recapitulated differing expression levels among samples. We further verified that CAD-LB captured the known increase in EV associated ICAM-1 following the cytokine stimulation of endothelial cells. Finally, to demonstrate CAD-LB's clinical potential, we show that EV biomarkers indicative of immunotherapy responsiveness are successfully detected in the plasma of bladder cancer patients undergoing immune checkpoint blockade.

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Section: Evaluating the Accuracy And Dynamic Range Of Cad-lbmentioning

confidence: 99%

Rapid Assessment of Biomarkers on Single Extracellular Vesicles Using ‘Catch and Display’ on Ultrathin Nanoporous Silicon Nitride Membranes

Walker,

Lucas,

Dewey

et al. 2024

Preprint

Self Cite

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Sided Stimulation of Endothelial Cells Modulates Neutrophil Trafficking in an In Vitro Sepsis Model

Ahmad,

Linares,

Pietropaoli

et al. 2024

Adv Healthcare Materials

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Sepsis pathophysiology involves complex interactions between vascular endothelium and circulating immune cells. Importantly, while the role of dysregulated polymorphonuclear leukocyte (PMN) transmigration in septic mediated tissue damage is well documented, strategies to mitigate aberrant transmigration across endothelium have yet to yield viable therapeutics. Much of this can be attributed to the usage of animal models in preclinical trials that lack translational relevance. Recently, however, microphysiological systems (MPS) have emerged as novel in vitro mimetics that facilitate the development of human models of disease. With this advancement, we can now directly probe aspects of endothelial physiology that are difficult to assess with other models. Here, we study the role of endothelial cell (EC) apicobasal polarity and how sided presentation of inflammatory cytokines modulates subsequent leukocyte trafficking response by utilizing the µSiM‐MVM (microphysiological system enabled by a silicon membrane – microvascular mimetic). The apicobasal polarity of ECs with respect to inflammation and leukocyte trafficking is a scarcely studied phenomenon that may be critical to understand the dynamic interplay between ECs and PMNs in sepsis and other causes of systemic inflammation. Here we stimulated ECs either apically or basally with a cytokine cocktail to model a septic‐like challenge before introducing healthy donor PMNs into the device. At low concentrations we found that a tissue sided (basally oriented) stimulation generated a stronger PMN transmigratory response versus apical stimulation. Importantly, healthy PMNs were unable to migrate towards a bacterial peptide chemoattractant when ECs were apically stimulated at low doses. This mimics the impaired ability of PMNs to identify a source of infection seen in sepsis, but identifies an EC role in the impairment for the first time, to our knowledge. Only by escalating the apical inflammatory stimulus by a factor of five were we able to overcome the EC barrier and solicit high PMN transmigration levels from the blood to the tissue side of the chip. These results, using the unique capabilities of the µSiM‐MVM, demonstrate that EC apicobasal polarity modulates differential PMN transmigratory behavior in a way that provides insight into the mechanisms underlying sepsis.This article is protected by copyright. All rights reserved

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Microfluidic models of the neurovascular unit: a translational view

Wevers,

De Vries

2023

Fluids Barriers CNS

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The vasculature of the brain consists of specialized endothelial cells that form a blood-brain barrier (BBB). This barrier, in conjunction with supporting cell types, forms the neurovascular unit (NVU). The NVU restricts the passage of certain substances from the bloodstream while selectively permitting essential nutrients and molecules to enter the brain. This protective role is crucial for optimal brain function, but presents a significant obstacle in treating neurological conditions, necessitating chemical modifications or advanced drug delivery methods for most drugs to cross the NVU. A deeper understanding of NVU in health and disease will aid in the identification of new therapeutic targets and drug delivery strategies for improved treatment of neurological disorders.To achieve this goal, we need models that reflect the human BBB and NVU in health and disease. Although animal models of the brain’s vasculature have proven valuable, they are often of limited translational relevance due to interspecies differences or inability to faithfully mimic human disease conditions. For this reason, human in vitro models are essential to improve our understanding of the brain’s vasculature under healthy and diseased conditions. This review delves into the advancements in in vitro modeling of the BBB and NVU, with a particular focus on microfluidic models. After providing a historical overview of the field, we shift our focus to recent developments, offering insights into the latest achievements and their associated constraints. We briefly examine the importance of chip materials and methods to facilitate fluid flow, emphasizing their critical roles in achieving the necessary throughput for the integration of microfluidic models into routine experimentation. Subsequently, we highlight the recent strides made in enhancing the biological complexity of microfluidic NVU models and propose recommendations for elevating the biological relevance of future iterations.Importantly, the NVU is an intricate structure and it is improbable that any model will fully encompass all its aspects. Fit-for-purpose models offer a valuable compromise between physiological relevance and ease-of-use and hold the future of NVU modeling: as simple as possible, as complex as needed.

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Modelling a Human Blood-Brain Barrier Co-Culture Using an Ultrathin Silicon Nitride Membrane-Based Microfluidic Device

Cited by 3 publications

References 56 publications

Rapid Assessment of Biomarkers on Single Extracellular Vesicles Using ‘Catch and Display’ on Ultrathin Nanoporous Silicon Nitride Membranes

Rapid Assessment of Biomarkers on Single Extracellular Vesicles Using ‘Catch and Display’ on Ultrathin Nanoporous Silicon Nitride Membranes

Sided Stimulation of Endothelial Cells Modulates Neutrophil Trafficking in an In Vitro Sepsis Model

Microfluidic models of the neurovascular unit: a translational view

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