Abstract
BackgroundBecause in vitro blood–brain barrier (BBB) models are important tools for studying brain diseases and drug development, we recently established a new line of conditionally immortalized human brain microvascular endothelial cells (HBMEC/ciβ) for use in such models. Since one of the most important functional features of the BBB is its strong intercellular adhesion, in this study, we aimed at improving HBMEC/ciβ barrier properties by means of culture media modifications, thus enhancing their use for future BBB studies. In addition, we simultaneously attempted to obtain insights on related mechanistic properties.MethodsSeveral types of culture media were prepared in an effort to identify the medium most suitable for culturing HBMEC/ciβ. The barrier properties of HBMEC/ciβ were examined by determining Na+-fluorescein permeability and transendothelial electric resistance (TEER). Endothelial marker mRNA expression levels were determined by quantitative real-time polymerase chain reaction. Adherens junction (AJ) formation was examined by immunocytochemistry. Cell migration ability was analyzed by scratch assay. Furthermore, cellular lipid composition was examined by liquid chromatography-time-of-flight mass spectrometry.ResultsOur initial screening tests showed that addition of hydrocortisone (HC) to the basal medium significantly reduced the Na+-fluorescein permeability and increased the TEER of HBMEC/ciβ monolayers. It was also found that, while AJ proteins were diffused in the cytoplasm of HBMEC/ciβ cultured without HC, those expressed in cells cultured with HC were primarily localized at the cell border. Furthermore, this facilitation of AJ formation by HC was in concert with increased endothelial marker mRNA levels and increased ether-type phosphatidylethanolamine levels, while cell migration was retarded in the presence of HC.ConclusionsOur results show that HC supplementation to the basal medium significantly enhances the barrier properties of HBMEC/ciβ. This was associated with a marked phenotypic alteration in HBMEC/ciβ through orchestration of various signaling pathways. Taken together, it appears that overall effects of HC on HBMEC/ciβ could be summarized as facilitating endothelial differentiation characteristics while concurrently retarding mesenchymal characteristics.Electronic supplementary materialThe online version of this article (doi:10.1186/s12987-015-0003-0) contains supplementary material, which is available to authorized users.
Highlights
Because in vitro blood–brain barrier (BBB) models are important tools for studying brain diseases and drug development, we recently established a new line of conditionally immortalized human brain microvascular endothelial cells (HBMEC/ciβ) for use in such models
The results are not shown here, it was determined that, among the media, the EBM basal 2 medium supplemented with SingleQuots (EBM2-SQs) provided differential effects on the morphology and gene expression profile of HBMEC/ciβ when compared to those cultured with our initially-used medium, which was a CSC-complete recombinant serum-containing medium supplemented with cbR (CSC-cbR). (Please note that medium is called “basal medium-supplement” throughout the manuscript.)
The results showed that neither EBM2cbR nor EBM2-SQs improved the junctional properties of HBMEC/ciβ over the levels obtained using CSC-cbR
Summary
Because in vitro blood–brain barrier (BBB) models are important tools for studying brain diseases and drug development, we recently established a new line of conditionally immortalized human brain microvascular endothelial cells (HBMEC/ciβ) for use in such models. One of the most important features of the BBB is its extremely strong intercellular adhesion, which is established by adherens junctions (AJs) and tight junctions (TJs) between the endothelial cells [1] This forceful adhesion seals the paracellular route and prevents entry of a variety of substances, both small and large, into brain from blood, while simultaneously creating a foundation that allows BMEC transporters to take up or expel molecules indispensable for, or harmful to, the physiological functions of the brain. The BBB is a pivotal research target for various brain diseases and CNS drug development studies
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