Primary Porcine Brain Microvessel Endothelial Cell Isolation and Culture

Patabendige, Adjanie; Abbott, N. Joan

doi:10.1002/0471142301.ns0327s69

Cited by 27 publications

(17 citation statements)

References 23 publications

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“…Here we describe a simple "white matter in vitro BBB model" consisting of CECs of porcine origin. Porcine in vitro BBB models are widely used in the literature (12,31,38) because of their robustness and smaller interspecies differences to human. The porcine brain is large enough for clear macroscopic delimitation of cortical grey matter and subcortical white matter.…”

Section: Resultsmentioning

confidence: 99%

Differences in the molecular structure of the blood-brain barrier in the cerebral cortex and white matter: an in silico, in vitro, and ex vivo study

Nyúl‐Tóth

Suciu

Molnár

et al. 2016

American Journal of Physiology-Heart and Circulatory Physiology

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The blood-brain barrier (BBB) is the main interface controlling molecular and cellular traffic between the central nervous system (CNS) and the periphery. It consists of cerebral endothelial cells (CECs) interconnected by continuous tight junctions, and closely associated pericytes and astrocytes. Different parts of the CNS have diverse functions and structures and may be subject of different pathologies, in which the BBB is actively involved. It is largely unknown, however, what are the cellular and molecular differences of the BBB in different regions of the brain. Using in silico, in vitro, and ex vivo techniques we compared the expression of BBB-associated genes and proteins (i.e., markers of CECs, brain pericytes, and astrocytes) in the cortical grey matter and white matter. In silico human database analysis (obtained from recalculated data of the Allen Brain Atlas), qPCR, Western blot, and immunofluorescence studies on porcine and mouse brain tissue indicated an increased expression of glial fibrillary acidic protein in astrocytes in the white matter compared with the grey matter. We have also found increased expression of genes of the junctional complex of CECs (occludin, claudin-5, and α-catenin) in the white matter compared with the cerebral cortex. Accordingly, occludin, claudin-5, and α-catenin proteins showed increased expression in CECs of the white matter compared with endothelial cells of the cortical grey matter. In parallel, barrier properties of white matter CECs were superior as well. These differences might be important in the pathogenesis of diseases differently affecting distinct regions of the brain.

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Section: Resultsmentioning

confidence: 99%

Differences in the molecular structure of the blood-brain barrier in the cerebral cortex and white matter: an in silico, in vitro, and ex vivo study

Nyúl‐Tóth

Suciu

Molnár

et al. 2016

American Journal of Physiology-Heart and Circulatory Physiology

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“…TEER across the BBB model was determined using STX100C electrodes connected to an EVOM2 epithelial voltohmmeter (World Precision Instruments, UK) as described previously ( Patabendige & Abbott, 2014 ; Patabendige et al, 2013a ). The resistance of cells grown on Transwell filter inserts was corrected for resistance across a cell-free insert, and multiplied by surface area, to give TEER in ohms x cm 2 (Ω·cm 2 ).…”

Section: Methodsmentioning

confidence: 99%

Brain microvascular endothelial-astrocyte cell responses following Japanese encephalitis virus infection in an in vitro human blood-brain barrier model

Patabendige

Michael

Craig

et al. 2018

Molecular and Cellular Neuroscience

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Japanese encephalitis virus (JEV) remains a leading cause of encephalitis, globally, which continues to grow in importance despite the availability of vaccines. Viral entry into the brain can occur via the blood-brain barrier (BBB), and inflammation at the BBB is a common final pathway in many brain infections. However, the role of the BBB during JEV infection and the contribution of the endothelial and astrocytic cell inflammation in facilitating virus entry into the brain are incompletely understood. We established a BBB model using human brain endothelial cells (HBECs) and human astrocytes. HBECs are polarised, and therefore the model was inoculated by JEV from the apical side to simulate the in vivo situation. The effects of JEV on the BBB permeability and release of inflammatory mediators from both apical and basolateral sides, representing the blood and the brain side respectively were investigated. JEV infected HBECs with limited active virus production, before crossing the BBB and infecting astrocytes. Control of JEV production by HBECs was associated with a significant increase in permeability, and with elevation of many host mediators, including cytokines, chemokines, cellular adhesion molecules, and matrix metalloproteases. When compared to the controls, significantly higher amounts of mediators were released from the apical side as opposed to the basolateral side. The increased release of mediators over time also correlated with increased BBB permeability. Treatment with dexamethasone led to a significant reduction in the release of interleukin 6 (IL6), C-C motif chemokine ligand 5 (CCL5) and C-X-C motif chemokine ligand 10 (CXCL10) from the apical side with a reduction in BBB disruption and no change in JEV production. The results are consistent with the hypothesis that JEV infection of the BBB triggers the production of a range of host mediators from both endothelial cells and astrocytes, which control JEV production but disrupt BBB integrity thus allowing virus entry into the brain. Dexamethasone treatment controlled the host response and limited BBB disruption in the model without increasing JEV production, supporting a re-investigation of its use therapeutically.

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“…However, these models are time‐consuming, expensive and have low‐throughput compared with cell models . Primary porcine and bovine cells have high barrier integrity and low permeability . However, primary cells require resource‐demanding isolation procedures, have limited availability, and suffer from batch‐to‐batch variation.…”

Section: Introductionmentioning

confidence: 99%

Barrier Properties and Transcriptome Expression in Human iPSC-Derived Models of the Blood–Brain Barrier

Delsing

Dönnes²,

Sánchez

et al. 2018

Stem Cells

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Cell-based models of the blood-brain barrier (BBB) are important for increasing the knowledge of BBB formation, degradation and brain exposure of drug substances. Human models are preferred over animal models because of interspecies differences in BBB structure and function. However, access to human primary BBB tissue is limited and has shown degeneration of BBB functions in vitro. Human induced pluripotent stem cells (iPSCs) can be used to generate relevant cell types to model the BBB with human tissue. We generated a human iPSC-derived model of the BBB that includes endothelial cells in coculture with pericytes, astrocytes and neurons. Evaluation of barrier properties showed that the endothelial cells in our coculture model have high transendothelial electrical resistance, functional efflux and ability to discriminate between CNS permeable and non-permeable substances. Whole genome expression profiling revealed transcriptional changes that occur in coculture, including upregulation of tight junction proteins, such as claudins and neurotransmitter transporters. Pathway analysis implicated changes in the WNT, TNF, and PI3K-Akt pathways upon coculture. Our data suggest that coculture of iPSC-derived endothelial cells promotes barrier formation on a functional and transcriptional level. The information about gene expression changes in coculture can be used to further improve iPSC-derived BBB models through selective pathway manipulation. Stem Cells 2018.

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Primary Porcine Brain Microvessel Endothelial Cell Isolation and Culture

Cited by 27 publications

References 23 publications

Differences in the molecular structure of the blood-brain barrier in the cerebral cortex and white matter: an in silico, in vitro, and ex vivo study

Differences in the molecular structure of the blood-brain barrier in the cerebral cortex and white matter: an in silico, in vitro, and ex vivo study

Brain microvascular endothelial-astrocyte cell responses following Japanese encephalitis virus infection in an in vitro human blood-brain barrier model

Barrier Properties and Transcriptome Expression in Human iPSC-Derived Models of the Blood–Brain Barrier

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