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A Hybrid Nanofiber/Paper Cell Culture Platform for Building a 3D Blood-brain Barrier Model

ABSTRACT: The blood brain barrier (BBB) protects the central nervous system from toxins and pathogens in the blood by regulating permeation of molecules through the barrier interface. In vitro BBB models described to date reproduce some aspects of BBB functionality, but also suffer from incomplete phenotypic expression of brain endothelial traits, difficulty in reproducibility and fabrication, or overall cost. To address these limitations, we describe a three-dimensional (3D) BBB model based on a hybrid paper/nanofiber scaffold. The cell culture platform utilizes lens paper as a framework to accommodate 3D culture of astrocytes. An electrospun nanofiber layer is coated onto one face of the paper to mimic the basement membrane and support growth of an organized two-dimensional layer of endothelial cells (ECs). Human induced pluripotent stem cell-derived ECs and astrocytes are co-cultured to develop a human BBB model. Morphological and spatial organization of model are validated with confocal microscopy. Measurements of transendothelial resistance and permeability demonstrate the BBB model develops a high-quality barrier and responds to hyperosmolar treatments. RNA-sequencing shows introduction of astrocytes both regulates EC tight junction proteins and improves endothelial phenotypes related to vasculogenesis. This model shows promise as a model platform for future in vitro studies of the BBB.

ORGANISM(S): Homo sapiens

PROVIDER: GSE181332 | GEO | 2021/11/01

REPOSITORIES: GEO

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Project description:The blood-brain barrier (BBB) consists of specific physical barriers, enzymes and transporters, which together maintain the necessary extracellular environment of the central nervous system (CNS). The main physical barrier is found in the CNS endothelial cell, and depends on continuous complexes of tight junctions combined with reduced vesicular transport. Other possible constituents of the BBB include extracellular matrix, astrocytes and pericytes, but the relative contribution of these different components to the BBB remains largely unknown. Here we demonstrate a direct role of pericytes at the BBB in vivo. Using a set of adult viable pericyte-deficient mouse mutants we show that pericyte deficiency increases the permeability of the BBB to water and a range of low-molecular-mass and high-molecular-mass tracers. The increased permeability occurs by endothelial transcytosis, a process that is rapidly arrested by the drug imatinib. Furthermore, we show that pericytes function at the BBB in at least two ways: by regulating BBB-specific gene expression patterns in endothelial cells, and by inducing polarization of astrocyte end-feet surrounding CNS blood vessels. Our results indicate a novel and critical role for pericytes in the integration of endothelial and astrocyte functions at the neurovascular unit, and in the regulation of the BBB. The brain microvascular fragments were isolated from mice with different genotypes, each represented by 3-4 biological replicates. Genotypes 1-2: Platelet derived growth factor-B (PDGF-B) retention-motif knockout (pdgfbret/ret) represent the pericyte-deficient situation, and the heterozygous mice (pdgfbret/+) are used as controls. Genotypes 3-4: Hypomorphic PDGF-B mutants that rescue pdgfb-/- null mice, in which a one copy of a conditionally silent human PDGF-B transgene targeted to the Rosa 26 locus (R26P) is turned on by endothelial-specific expression of Cre recombinase. In this data set these mice are named as Tie2Cre, R26P+/0, pdgfb-/- (representing the pericyte-deficient situation). Mice wt for pdgfb (pdgfb+/+) and carrying one silent copy of R26P (R26P+/0), are used as controls. Genotype 5: Adult Notch3+/+ wildtype (WT).

Project description:The blood-brain barrier (BBB) dynamically controls and maintains a precisely balanced brain microenvironment necessary for reliable functioning. It is made up of highly specialized endothelial cells (ECs) in the lining of the vascular wall. These ECs are surrounded by the basal lamina, astrocytic perivascular endfeet, pericytes, microglia and neuronal processes that have been shown to contribute to barrier function1. In essence, the brain endothelium limits both transcellular and paracellular passage of cells and molecules into the central nervous system (CNS). Transcellular passage of hydrophilic molecules is limited due to a low rate of transcytotic vesicles, an extremely low pinocytotic activity, expression of active efflux transporters, and high metabolic activity. Paracellular diffusion of hydrophilic molecules and trafficking of immune cells is restricted by a network of tight junctions (TJs)2-5. Due to these characteristics, the BBB is able to protect the CNS from sudden changes in blood composition and uncontrolled influx of immune cells. In a large number of neuro-inflammatory diseases, an impaired function and an opening of the BBB are observed. In diseases of the CNS like multiple sclerosis or stroke or after brain trauma, the BBB becomes inflamed (mediated by for example reactive oxygen species (ROS) and hypoxia) and its function severely impaired which gives rise to enhanced cellular infiltration, thus contributing to tissue damage and neurological deficits. Due to the specialized nature of brain ECs, transmigration of leukocytes through cerebrovascular endothelium is likely to differ from that in other vascular beds. Generally, the transmigration process is closely controlled by the interaction of leukocytes with the endothelial surface followed by low velocity rolling, arrest, firm adhesion, and finally transmigration. For the diapedesis of immune cells across the cerebral vasculature a re-arrangement of the cytoskeleton and opening of the TJ complexes is required to allow cells to pass into the sub endothelial space. In the initial step of the adhesive cascade leukocytes adhere to the endothelium with low affinity. This adhesion is mediated by several members of the selectin family and their corresponding ligands. Despite the low affinity of these interactions, resulting contact of leukocytes with the endothelium leads to further activation of both cell types and finally transmigration of the leukocytes through the BBB6,7. The glycosylation of endothelial cells of different vascular bed origins To date it is unknown which specific carbohydrate structures on the BBB endothelium mediate leukocyte capture and rolling, and whether these structures are differentially expressed onto inflamed brain EC compared to normal brain ECs and vascular beds of other organs. Initial studies using qPCR and FACS analysis within our group reveal that brain endothelial cells have a different profile in their glycosylation-related genes compared to microvascular ECs upon inflammation, which may result in a different glycosylation profile of adhesive structures and may underlie rolling, adhesion and diapedesis of leukocytes. In this project we wish to identify specific, glycosylated structures on brain endothelial cells that mediate capture, rolling and diapedesis of leukocytes in the brain. To investigate the expression of glycosyltransferases in dendritic cells and the changes in expression associated with maturation. RNA preparations from stimulated and non-stimulated hcMEC/D3 (human brain endothelial cells line) and FMVEC (human promary microvascular endothelial cells isolated from foreskins) were sent to both Microarray Core (E) and Core(C). The RNA was put on an RNeasy Column, amplified, labeled, and hybridized to the Glycov3 microarrays. Data was sent to Dr. van Kooyk's lab for analysis.

Project description:The blood brain barrier (BBB) acts both a physical as well as a metabolic barrier isolating the Central Nervous System (CNS) from systemic circulation, blocking intravascular toxin entry and promoting toxic substance elimination to blood stream. The BBB is formed by four main cellular elements: endothelial cells, astrocyte end-feet, microglial cells, and pericytes. There is strong evidence that astrocytes are able to regulate the phenotypes of endothelial cells by directly contacting or secreting a range of chemical agents, resulting in BBB permeability changes and so on. Conversely, endothelium-derived leukemia inhibitory factor (LIF) has been shown to induce astrocytic differentiation, from studies in normal condition of cell culture. However, in pathological conditions, the specific regulatory mechanism that endothelial cells exert on astrocytes is still less known. In our recently study, a transwell-based, endothelial-astrocyte co-culture model was established to investigate this regulatory relationship in bacterial infection. An extraintestinal pathogenic Escherichia. coli strain was used to infect human brain microvascular endothelial cells (hBMEC) planted in the upper chamber, astrocytes co-cultured in the bottom well were actived without direct contact with bacteria or endothelial cell. The transcription levels of some chemokines and cytokines such as TNF-α and CXCL10 were significantly upregulated. We hypothesized that the secreted proteins from endothelial cells is the main cause of astrocyte activation and the culture supernatant of hBMEC infected by E.coli was obtained to perform the mass spectrometry analysis. In summary, the proteomic identification results showed that 2,692 common proteins were identified in the two groups of control and infected hBMEC, including 140 differentially expressed proteins. The bioinformatics analysis indicated that differentially expressed proteins are mainly classified into structural molecular activity and constituent of ribosome. KEGG pathway enrichment analysis showed that many proteins are involved in focal adhesion as well as a bunch of signaling pathways, PI3K-Akt, Rap1, Ras, MAPK and NF-kappaB signaling pathways, for example. The present study laid an important foundation for the subsequent study of secreted proteins of hBMEC in bacterial infections.

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A Hybrid Nanofiber/Paper Cell Culture Platform for Building a 3D Blood-brain Barrier Model

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