Abstract
BACKGROUND: 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. 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. METHODS: Several 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. RESULTS: Our 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. CONCLUSIONS: Our 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.