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Establishment of primary cultures of human brain microvascular endothelial cells to provide an in vitro cellular model of the blood-brain barrier

Abstract

We describe a method for generating primary cultures of human brain microvascular endothelial cells (HBMVECs). HBMVECs are derived from microvessels isolated from temporal tissue removed during operative treatment of epilepsy. The tissue is mechanically fragmented and size filtered using polyester meshes. The resulting microvessel fragments are placed onto type I collagen-coated flasks to allow HBMVECs to migrate and proliferate. The overall process takes less than 3 h and does not require specialized equipment or enzymatic processes. HBMVECs are typically cultured for approximately 1 month until confluent. Cultures are highly pure (97% endothelial cells; 3% pericytes), are reproducible, and show characteristic brain endothelial markers (von Willebrand factor, glucose transporter-1) and robust expression of tight and adherens junction proteins as well as caveolin-1 and efflux protein P-glycoprotein. Monolayers of HBMVECs show characteristically high transendothelial electric resistance and have proven useful in multiple functional studies for in vitro modeling of the human blood-brain barrier.

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Figure 1: Representative steps of the procedure to isolate and culture human brain microvascular endothelial cells.
Figure 2: Phase-contrast microscopy of human brain microvascular endothelial cells.
Figure 3: Characterization of primary cultures of human brain microvascular endothelial cells by immunofluorescence microscopy.
Figure 4: Immunocytochemical characteristics of confluent human brain microvascular endothelial cell monolayers.
Figure 5: Western blot and quantitative RT-PCR analyses of protein and mRNA expression in confluent monolayers of human brain microvascular endothelial cells (HBMVECs) between passages 1 and 3, from three donors.
Figure 6: Transendothelial electric resistance (TEER) measured with the Electrical Cell-Substrate Impedance Sensing (ECIS) system (Applied Biophysics, model 1600R).

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Acknowledgements

This work was supported by Grants NIH R01AA017398, R01MH065151 and AA015913 (YP-Temple) with subcontract (MHW-Arizona), and by Grant FCT-PTDC/SAU-FCF/68819/2006 (MAB-Lisbon). We also thank Dr. Shongshan Fan and Holly Dykstra for the technical contributions.

Author information

Authors and Affiliations

Authors

Contributions

M.J.B. developed the protocol; F.L.C. implemented the protocol in the Lisbon laboratory and performed cell culture characterization; S.K.D. performed and validated the protocol in Arizona; M.E.W. contributed to the developmental inception of the project, maintains IRB approval and provided brain tissue in Arizona; A.R.C. and A.J.G.F. were responsible for IRB approval as well as for selecting and providing human brain tissue for culture in Lisbon; J.B.H. provided key technical improvements to the protocol; M.H.W. inspired and conceived the project and established and maintains the United States collaborations; D.B. assisted in the Lisbon protocol and provided helpful discussion of the work and of the article; Y.P. helped develop the method and provided funding and support for the United States collaboration; S.H.R. contributed experience in the use and characterization of these cells; M.A.B. worked on the implementation of the protocol in the Lisbon laboratory and on the cell culture characterization, provided financial support for this work, established the international collaboration and put together the article for publication.

Corresponding authors

Correspondence to Servio H Ramirez or Maria A Brito.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Table 1

Antibodies and dilutions (PDF 17 kb)

Supplementary Method

Characterization of human brain microvascular endothelial cells by immunocytochemical analysis (PDF 45 kb)

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Bernas, M., Cardoso, F., Daley, S. et al. Establishment of primary cultures of human brain microvascular endothelial cells to provide an in vitro cellular model of the blood-brain barrier. Nat Protoc 5, 1265–1272 (2010). https://doi.org/10.1038/nprot.2010.76

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