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

Michael J Bernas; Filipa L. Cardoso; Sarah K. Daley; Martin E. Weinand; Alexandre R. Campos; António J.Gonçalves Ferreira; James B. Hoying; Marlys H. Witte; Dora Brites; Yuri Persidsky; Servio H. Ramirez; Maria A. Brito

doi:10.1038/nprot.2010.76

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

Michael J Bernas, Filipa L. Cardoso, Sarah K. Daley, Martin E. Weinand, Alexandre R. Campos, António J.Gonçalves Ferreira, James B. Hoying, Marlys H. Witte, Dora Brites, Yuri Persidsky, Servio H. Ramirez, Maria A. Brito

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161 Scopus citations

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.

Original language	English (US)
Pages (from-to)	1265-1272
Number of pages	8
Journal	Nature Protocols
Volume	5
Issue number	7
DOIs	https://doi.org/10.1038/nprot.2010.76
State	Published - Jul 2010

ASJC Scopus subject areas

Biochemistry, Genetics and Molecular Biology(all)

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Bernas, M. J., Cardoso, F. L., Daley, S. K., Weinand, M. E., Campos, A. R., Ferreira, A. J. G., Hoying, J. B., Witte, M. H., Brites, D., Persidsky, Y., Ramirez, S. H., & Brito, M. A. (2010). Establishment of primary cultures of human brain microvascular endothelial cells to provide an in vitro cellular model of the blood-brain barrier. Nature Protocols, 5(7), 1265-1272. https://doi.org/10.1038/nprot.2010.76

Bernas, MJ, Cardoso, FL, Daley, SK, Weinand, ME, Campos, AR, Ferreira, AJG, Hoying, JB, Witte, MH, Brites, D, Persidsky, Y, Ramirez, SH & Brito, MA 2010, 'Establishment of primary cultures of human brain microvascular endothelial cells to provide an in vitro cellular model of the blood-brain barrier', Nature Protocols, vol. 5, no. 7, pp. 1265-1272. https://doi.org/10.1038/nprot.2010.76

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title = "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.",

author = "Bernas, {Michael J} and Cardoso, {Filipa L.} and Daley, {Sarah K.} and Weinand, {Martin E.} and Campos, {Alexandre R.} and Ferreira, {Ant{\'o}nio J.Gon{\c c}alves} and Hoying, {James B.} and Witte, {Marlys H.} and Dora Brites and Yuri Persidsky and Ramirez, {Servio H.} and Brito, {Maria A.}",

note = "Funding Information: acknoWleDGMents 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.",

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AU - Bernas, Michael J

AU - Cardoso, Filipa L.

AU - Daley, Sarah K.

AU - Weinand, Martin E.

AU - Campos, Alexandre R.

AU - Ferreira, António J.Gonçalves

AU - Hoying, James B.

AU - Witte, Marlys H.

AU - Brites, Dora

AU - Persidsky, Yuri

AU - Ramirez, Servio H.

AU - Brito, Maria A.

N1 - Funding Information: acknoWleDGMents 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.

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N2 - 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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Establishment of primary cultures of human brain microvascular endothelial cells to provide an in vitro cellular model of the blood-brain barrier

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