Culture of murine brain microvascular endothelial cells that maintain expression and cytoskeletal association of tight junction-associated proteins.
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 beta-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.
Song
Li
L
Blood-Brain Barrier Laboratory, Department of Pharmacology, University of Connecticut Health Center, Farmington, Connecticut 06030, USA.
Pachter
Joel S
JS
D000818:Animals; D015415:Biomarkers; D001812:Blood-Brain Barrier / Q000502:physiology; D001921:Brain / Q000033:anatomy & histology*; D018929:Cell Culture Techniques / Q000379:methods*; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism*; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology*; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D019108:Tight Junctions / Q000378:metabolism*
D015415:Biomarkers; D008565:Membrane Proteins