A silicon nanomembrane platform for the visualization of immune cell trafficking across the human blood–brain barrier under flow

Here we report on the development of a breakthrough microfluidic human in vitro cerebrovascular barrier (CVB) model featuring stem cell-derived brain-like endothelial cells (BLECs) and nanoporous silicon nitride (NPN) membranes (µSiM-CVB). The nanoscale thinness of NPN membranes combined with their high permeability and optical transparency makes them an ideal scaffold for the assembly of an in vitro microfluidic model of the blood–brain barrier (BBB) featuring cellular elements of the neurovascular unit (NVU). Dual-chamber devices divided by NPN membranes yield tight barrier properties in BLECs and allow an abluminal pericyte-co-culture to be replaced with pericyte-conditioned media. With the benefit of physiological flow and superior imaging quality, the µSiM-CVB platform captures each phase of the multi-step T-cell migration across the BBB in live cell imaging. The small volume of <100 µL of the µSiM-CVB will enable in vitro investigations of rare patient-derived immune cells with the human BBB. The µSiM-CVB is a breakthrough in vitro human BBB model to enable live and high-quality imaging of human immune cell interactions with the BBB under physiological flow. We expect it to become a valuable new tool for the study of cerebrovascular pathologies ranging from neuroinflammation to metastatic cancer.

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Abstract TP86: The Ischemic Blood-brain Barrier In A Dish: Use Of Patient-derived Stem Cells To Model The Response Of The Neurovascular Unit To Oxygen-glucose Deprivation Stress

Stroke ◽

10.1161/str.48.suppl_1.tp86 ◽

2017 ◽

Vol 48 (suppl_1) ◽

Author(s):

Shyanne Page ◽

Ronak Patel ◽

Abraham Alahmad

Keyword(s):

Cell Death ◽

Ischemic Stroke ◽

Blood Brain Barrier ◽

Barrier Function ◽

Neurovascular Unit ◽

Barrier Properties ◽

Cell Types ◽

Brain Barrier ◽

Blood Brain

The blood-brain barrier (BBB) constitutes a component of the neurovascular unit formed by specialized brain microvascular endothelial cells (BMECs) surrounded by astrocytes, pericytes and neurons. During ischemic stroke injury, the BBB constitutes the first responding element resulting in the opening of the BBB and eventually neural cell death by excitotoxicity. A better understanding of the cellular mechanisms underlying the opening of the BBB during ischemic stroke is essential to identify targets to restore such barrier function after injury. Current in vitro models of the human BBB, based on primary or immortalized BMECs monocultures, display poor barrier properties but also lack one or two cellular components of the neurovascular unit.In this study, we designed an integrative in vitro model of the BBB by generating BMECs, astrocytes and neurons using patient-derived BMECs from two iPSC lines (IMR90-c4 and CTR66M). We were able to obtain all three cell types from these two cell lines. iPSC-derived BMECs showed barrier properties similar or better barrier function than hCMEC/D3 monolayer (an immortalized adult somatic BMEC). Furthermore, iPSC—derived astrocytes were capable to induce barrier properties in BMECs upon co-cultures. whereas iPSC-derived neurons were capable to form extensive and branched neurites. Upon OGD stress, iPSC-derived BMECs showed a disruption of their barrier function as early as 6 hours of OGD stress and showed a complete disruption by 24 hours. Such disruption was reversed by reoxygenation. Interestingly such barrier disruption occurs through a VEGF-independent mechanism. In the other hand, iPSC-derived neurons showed a significant decrease in cell metabolic activity preceding neurites pruning. Finally, astrocytes showed the most robust phenotype, as we noted no cell death by 24 hours OGD.In this study, we demonstrated the ability to differentiate three cell types from the same patient in two iPSC lines. We also demonstrated the ability of these cells to respond to OGD/reoxygenation stress in agreement with the current literature. We are currently investigating the molecular mechanisms by which OGD/reoxygenation drive the cellular response in these cell types.

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Increased Transendothelial Transport of CCL3 Is Insufficient to Drive Immune Cell Transmigration through the Blood–Brain Barrier under Inflammatory Conditions In Vitro

Mediators of Inflammation ◽

10.1155/2017/6752756 ◽

2017 ◽

Vol 2017 ◽

pp. 1-11 ◽

Cited By ~ 4

Author(s):

Maxime De Laere ◽

Carmelita Sousa ◽

Megha Meena ◽

Roeland Buckinx ◽

Jean-Pierre Timmermans ◽

...

Keyword(s):

Blood Brain Barrier ◽

Immune Cell ◽

Brain Barrier ◽

Cell Infiltration ◽

Microvascular Endothelial Cell ◽

Immune Cell Infiltration ◽

Cell Trafficking ◽

Inflammatory Conditions ◽

Blood Brain

Many neuroinflammatory diseases are characterized by massive immune cell infiltration into the central nervous system. Identifying the underlying mechanisms could aid in the development of therapeutic strategies specifically interfering with inflammatory cell trafficking. To achieve this, we implemented and validated a blood–brain barrier (BBB) model to study chemokine secretion, chemokine transport, and leukocyte trafficking in vitro. In a coculture model consisting of a human cerebral microvascular endothelial cell line and human astrocytes, proinflammatory stimulation downregulated the expression of tight junction proteins, while the expression of adhesion molecules and chemokines was upregulated. Moreover, chemokine transport across BBB cocultures was upregulated, as evidenced by a significantly increased concentration of the inflammatory chemokine CCL3 at the luminal side following proinflammatory stimulation. CCL3 transport occurred independently of the chemokine receptors CCR1 and CCR5, albeit that migrated cells displayed increased expression of CCR1 and CCR5. However, overall leukocyte transmigration was reduced in inflammatory conditions, although higher numbers of leukocytes adhered to activated endothelial cells. Altogether, our findings demonstrate that prominent barrier activation following proinflammatory stimulation is insufficient to drive immune cell recruitment, suggesting that additional traffic cues are crucial to mediate the increased immune cell infiltration seen in vivo during neuroinflammation.

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Immune cell trafficking across the blood-brain barrier in the absence and presence of neuroinflammation

Vascular Biology ◽

10.1530/vb-19-0033 ◽

2020 ◽

Vol 2 (1) ◽

pp. H1-H18 ◽

Cited By ~ 6

Author(s):

Luca Marchetti ◽

Britta Engelhardt

Keyword(s):

Endothelial Cells ◽

Blood Brain Barrier ◽

Immune Cells ◽

Immune Cell ◽

Brain Barrier ◽

Proper Function ◽

Cell Trafficking ◽

Immune Cell Trafficking ◽

Blood Brain

To maintain the homeostatic environment required for proper function of CNS neurons the endothelial cells of CNS microvessels tightly regulate the movement of ions and molecules between the blood and the CNS. The unique properties of these blood vascular endothelial cells are termed blood-brain barrier (BBB) and extend to regulating immune cell trafficking into the immune privileged CNS during health and disease. In general, extravasation of circulating immune cells is a multi-step process regulated by the sequential interaction of adhesion and signalling molecules between the endothelial cells and the immune cells. Accounting for the unique barrier properties of CNS microvessels, immune cell migration across the BBB is distinct and characterized by several adaptations. Here we describe the mechanisms that regulate immune cell trafficking across the BBB during immune surveillance and neuroinflammation, with a focus on the current state-of-the-art in vitro and in vivo imaging observations.

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VLA-4 mediated adhesion of melanoma cells on the blood–brain barrier is the critical cue for melanoma cell intercalation and barrier disruption

Journal of Cerebral Blood Flow & Metabolism ◽

10.1177/0271678x18775887 ◽

2018 ◽

Vol 39 (10) ◽

pp. 1995-2010 ◽

Cited By ~ 5

Author(s):

Ana B García-Martín ◽

Pascale Zwicky ◽

Thomas Gruber ◽

Christoph Matti ◽

Federica Moalli ◽

...

Keyword(s):

Brain Metastasis ◽

Blood Brain Barrier ◽

Barrier Properties ◽

Melanoma Cells ◽

Brain Barrier ◽

Adhesive Interaction ◽

Inflammatory Conditions ◽

Blood Brain ◽

In Vitro Bbb Model

Melanoma is the most aggressive skin cancer in humans. One severe complication is the formation of brain metastasis, which requires extravasation of melanoma cells across the tight blood–brain barrier (BBB). Previously, VLA-4 has been assigned a role for the adhesive interaction of melanoma cells with non-BBB endothelial cells. However, the role of melanoma VLA-4 for breaching the BBB remained unknown. In this study, we used a mouse in vitro BBB model and imaged the shear resistant arrest of melanoma cells on the BBB. Similar to effector T cells, inflammatory conditions of the BBB increased the arrest of melanoma cells followed by a unique post-arrest behavior lacking immediate crawling. However, over time, melanoma cells intercalated into the BBB and compromised its barrier properties. Most importantly, antibody ablation of VLA-4 abrogated melanoma shear resistant arrest on and intercalation into the BBB and protected the BBB from barrier breakdown. A tissue microarray established from human brain metastasis revealed that indeed a majority of 92% of all human melanoma brain metastases stained VLA-4 positive. We propose VLA-4 as a target for the inhibition of brain metastasis formation in the context of personalized medicine identifying metastasizing VLA-4 positive melanoma.

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Human pluripotent stem cell–derived brain pericyte–like cells induce blood-brain barrier properties

Science Advances ◽

10.1126/sciadv.aau7375 ◽

2019 ◽

Vol 5 (3) ◽

pp. eaau7375 ◽

Cited By ~ 40

Author(s):

Matthew J. Stebbins ◽

Benjamin D. Gastfriend ◽

Scott G. Canfield ◽

Ming-Song Lee ◽

Drew Richards ◽

...

Keyword(s):

Stem Cells ◽

Endothelial Cells ◽

Blood Brain Barrier ◽

Neurovascular Unit ◽

Barrier Properties ◽

Cell Types ◽

Brain Barrier ◽

Human Model ◽

Blood Brain ◽

Brain Pericytes

Brain pericytes play important roles in the formation and maintenance of the neurovascular unit (NVU), and their dysfunction has been implicated in central nervous system disorders. While human pluripotent stem cells (hPSCs) have been used to model other NVU cell types, including brain microvascular endothelial cells (BMECs), astrocytes, and neurons, hPSC-derived brain pericyte–like cells have not been integrated into these models. In this study, we generated neural crest stem cells (NCSCs), the embryonic precursor to forebrain pericytes, from hPSCs and subsequently differentiated NCSCs to brain pericyte–like cells. These cells closely resembled primary human brain pericytes and self-assembled with endothelial cells. The brain pericyte–like cells induced blood-brain barrier properties in BMECs, including barrier enhancement and reduced transcytosis. Last, brain pericyte–like cells were incorporated with iPSC-derived BMECs, astrocytes, and neurons to form an isogenic human model that should prove useful for the study of the NVU.

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Serum-derived factors in breast cancer patients change barrier properties of the human in vitro blood-brain barrier model

10.1055/s-0040-1710613 ◽

2020 ◽

Author(s):

C Curtaz ◽

C Schmitt ◽

S-L Herbert ◽

A Quenzer ◽

J Feldheim ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Patients ◽

Blood Brain Barrier ◽

Barrier Properties ◽

Brain Barrier ◽

Breast Cancer Patients ◽

Blood Brain ◽

Blood Brain Barrier Model ◽

Barrier Model

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Effects of Chlorpyrifos Exposure on Acetylcholine Metabolism across a Model Blood-Brain Barrier

ECS Meeting Abstracts ◽

10.1149/ma2019-02/55/2426 ◽

2019 ◽

Vol MA2019-02 (55) ◽

pp. 2426-2426

Author(s):

Ethan S. McClain ◽

Dusty R. Miller ◽

Jacquelyn A Brown ◽

John P Wikswo ◽

David E. Cliffel

Keyword(s):

Blood Brain Barrier ◽

Neurovascular Unit ◽

Acetylcholinesterase Activity ◽

Cell Types ◽

Electrochemical Analysis ◽

Brain Barrier ◽

Agricultural Industry ◽

Tight Junction Formation ◽

Blood Brain

Organophosphate (OP) compounds, used throughout the agricultural industry as insecticides, are known to directly and irreparably alter brain function in humans. Exposure to OPs decreases acetylcholinesterase activity and leads to a buildup of acetylcholine, with chronic exposure to sub-lethal levels inducing neuropathy. This buildup of acetylcholine can be monitored through electrochemical methods to study the effects of OP toxicity. The microclinical analyzer (µCA), an in vitro microfluidic device allowing for electrochemical analysis using a screen-printed electrode, can be modified with enzymes to detect acetylcholine. Using the µCA in combination with the neurovascular unit (NVU), an organotypic model of the blood-brain barrier (BBB), can provide a better understanding of the BBB forms, functions, and responds to insults. The NVU supports all the cell types necessary for proper BBB formation (endothelial cells, astrocytes, pericytes, and neurons) and provides the flow-created shear forces for mature tight junction formation. The µCA and NVU were used study the effects of chlorpyrifos on acetylcholine concentrations present across the BBB. Understanding the effects of OP like chlorpyrifos on neurotoxicity can contributes to the assessment and treatment of chronic and acute exposure and inform policy decisions around the uses of OP pesticides in the agricultural industry.

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Nitric Oxide Isoenzymes Regulate Lipopolysaccharide-Enhanced Insulin Transport across the Blood-Brain Barrier

Endocrinology ◽

10.1210/en.2007-1091 ◽

2008 ◽

Vol 149 (4) ◽

pp. 1514-1523 ◽

Cited By ~ 36

Author(s):

William A. Banks ◽

Shinya Dohgu ◽

Jessica L. Lynch ◽

Melissa A. Fleegal-DeMotta ◽

Michelle A. Erickson ◽

...

Keyword(s):

Nitric Oxide ◽

Blood Brain Barrier ◽

Neurovascular Unit ◽

Brain Barrier ◽

Mrna Levels ◽

Insulin Transport ◽

Blood Brain ◽

Inducible Nos

Insulin transported across the blood-brain barrier (BBB) has many effects within the central nervous system. Insulin transport is not static but altered by obesity and inflammation. Lipopolysaccharide (LPS), derived from the cell walls of Gram-negative bacteria, enhances insulin transport across the BBB but also releases nitric oxide (NO), which opposes LPS-enhanced insulin transport. Here we determined the role of NO synthase (NOS) in mediating the effects of LPS on insulin BBB transport. The activity of all three NOS isoenzymes was stimulated in vivo by LPS. Endothelial NOS and inducible NOS together mediated the LPS-enhanced transport of insulin, whereas neuronal NOS (nNOS) opposed LPS-enhanced insulin transport. This dual pattern of NOS action was found in most brain regions with the exception of the striatum, which did not respond to LPS, and the parietal cortex, hippocampus, and pons medulla, which did not respond to nNOS inhibition. In vitro studies of a brain endothelial cell (BEC) monolayer BBB model showed that LPS did not directly affect insulin transport, whereas NO inhibited insulin transport. This suggests that the stimulatory effect of LPS and NOS on insulin transport is mediated through cells of the neurovascular unit other than BECs. Protein and mRNA levels of the isoenzymes indicated that the effects of LPS are mainly posttranslational. In conclusion, LPS affects insulin transport across the BBB by modulating NOS isoenzyme activity. NO released by endothelial NOS and inducible NOS acts indirectly to stimulate insulin transport, whereas NO released by nNOS acts directly on BECs to inhibit insulin transport.

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