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Culture of murine brain microvascular endothelial cells that maintain expression and cytoskeletal association of tight junction-associated proteins

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Summary

A readily obtainable in vitro paradigm of the blood-brain barrier (BBB) would offer considerable benefits. Toward this end, in this study, we describe a novel method for purifying murine brain microvascular endothelial cells (BMEC) for culture. The method uses limited collagenase-dispase digestion of enriched brain microvessels, followed by immunoisolation of digested, microvascular fragments by magnetic beads coated with antibody to platelet-endothelial cell adhesion molecule-1. When plated onto collagen IV-coated surfaces, these fragments elaborated confluent monolayers of BMEC that expressed, as judged by immunocytochemistry, the adherens junction-associated proteins, VE-cadherin and β-catenin, as well as the tight junction (TJ)-associated proteins, claudin-5, occludin, and zonula occludin-1 (ZO-1), in concentrated fashion along intercellular borders. In contrast, cultures of an immortalized and transformed line of murine brain capillary-derived endothelial cells, bEND.3, displayed diffuse cytoplasmic localization of occludin and ZO-1. This difference in occludin and ZO-1 staining between the two endothelial cell types was also reflected in the extent of association of these proteins with the detergent-resistant cytoskeletal framework (CSK). Although both occludin and ZO-1 largely partitioned with the CSK fraction in BMEC, they were found predominantly in the soluble fraction of bEND.3 cells, and claudin-5 was found associated equally with both fractions in BMEC and bEND.3 cells. Moreover, detergent-extracted cultures of the BMEC retained pronounced immunostaining of occludin and ZO-1, but not claudin-5, along intercellular borders. Because both occludin and ZO-1 are thought to be functionally coupled to the detergent-resistant CSK and high expression of TJs is considered a seminal characteristic of the BBB, these results impart that this method of purifying murine BMEC provides a suitable platform to investigate BBB properties in vitro.

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  1. Blood-Brain Barrier Laboratory, Department of Pharmacology, University of Connecticut Health Center, 06030, Farmington, Connecticut

    Li Song & Joel S. Pachter

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  1. Li Song
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  2. Joel S. Pachter
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Correspondence to Joel S. Pachter.

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Song, L., Pachter, J.S. Culture of murine brain microvascular endothelial cells that maintain expression and cytoskeletal association of tight junction-associated proteins. In Vitro Cell.Dev.Biol.-Animal 39, 313–320 (2003). https://doi.org/10.1290/1543-706X(2003)039<0313:COMBME>2.0.CO;2

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  • DOI: https://doi.org/10.1290/1543-706X(2003)039<0313:COMBME>2.0.CO;2

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