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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References
Persidsky, Y., Ramirez, S.H., Haorah, J. & Kanmogne, G.D. Blood–brain barrier: structural components and function under physiologic and pathologic conditions. J. Neuroimmune Pharmacol. 1, 223–236 (2006).
Weiss, N., Miller, F., Cazaubon, S. & Couraud, P.O. The blood–brain barrier in brain homeostasis and neurological diseases. Biochim. Biophys. Acta 1788, 842–857 (2009).
Zlokovic, B.V. The blood–brain barrier in health and chronic neurodegenerative disorders. Neuron 57, 178–201 (2008).
Ehrlich, P. Das Sauerstoffbedürfnis des Organismus, Eine Farbenanalytische Studie. Hirschwald, Berlin, (1885).
Joó, F. & Karnushina, I. A procedure for the isolation of capillaries from rat brain. Cytobios 8, 41–48 (1973).
Deli, M.A., Abrahám, C.S., Kataoka, Y. & Niwa, M. Permeability studies on in vitro blood–brain barrier models: physiology, pathology, and pharmacology. Cell Mol. Neurobiol. 25, 59–127 (2005).
Bowman, C. et al. Cystic hygroma reconsidered: hamartoma or neoplasm? Primary culture of an endothelial cell line from a massive cervicomediastinal cystic hygroma with bony lymphangiomatosis. Lymphology 17, 15–22 (1984).
Way, D. et al. Lymphatic endothelial cell line (CH3) from a recurrent retroperitoneal lymphangioma. In Vitro 23, 647–652 (1987).
Bowman, P.D. et al. Primary culture of capillary endothelium from rat brain. In Vitro 17, 353–362 (1981).
Bowman, P.D., Ennis, S.R., Rarey, K.E., Betz, A.L. & Goldstein, G.W. Brain microvessel endothelial cells in tissue culture: a model for study of blood–brain barrier permeability. Ann. Neurol. 14, 396–402 (1983).
Stins, M.F., Gilles, F. & Kim, K.S. Selective expression of adhesion molecules on human brain microvascular endothelial cells. J. Neuroimmunol. 76, 81–90 (1997).
Dorovini-Zis, K., Prameya, R. & Bowman, P.D. Culture and characterization of microvascular endothelial cells derived from human brain. Lab. Invest. 64, 425–436 (1991).
Dombrowski, S.M. et al. Overexpression of multiple drug resistance genes in endothelial cells from patients with refractory epilepsy. Epilepsia 42, 1501–1506 (2001).
Weksler, B.B. et al. Blood–brain barrier-specific properties of a human adult brain endothelial cell line. FASEB J. 19, 1872–1874 (2005).
Hughes, C.C.W. & Lantos, P.L. Brain capillary endothelial cells in vitro lack surface IgG Fc receptors. Neurosci. Lett. 68, 100–106 (1986).
Hiu, T. et al. Tissue plasminogen activator enhances the hypoxia/reoxygenation-induced impairment of the blood–brain barrier in a primary culture of rat brain endothelial cells. Cell Mol. Neurobiol. 28, 1139–1146 (2008).
Coisne, C. et al. Mouse syngenic in vitro blood–brain barrier model: a new tool to examine inflammatory events in cerebral endothelium. Lab. Invest. 85, 734–746 (2005).
Malina, K.C., Cooper, I. & Teichberg, V.I. Closing the gap between the in-vivo and in-vitro blood–brain barrier tightness. Brain Res. 1284, 12–21 (2009).
Colgan, O.C. et al. Influence of basolateral condition on the regulation of brain microvascular endothelial tight junction properties and barrier function. Brain Res. 1193, 84–92 (2008).
Parkinson, F.E. & Hacking, C. Pericyte abundance affects sucrose permeability in cultures of rat brain microvascular endothelial cells. Brain Res. 1049, 8–14 (2005).
Perrière, N. et al. Puromycin-based purification of rat brain capillary endothelial cell cultures. Effect on the expression of blood–brain barrier-specific properties. J. Neurochem. 93, 279–289 (2005).
Poller, B. et al. The human brain endothelial cell line hCMEC/D3 as a human blood–brain barrier model for drug transport studies. J. Neurochem. 107, 1358–1368 (2008).
Mkrtchyan, H. et al. Molecular cytogenetic characterization of the human cerebral microvessel endothelial cell line hCMEC/D3. Cytogenet. Genome Res. 126, 313–317 (2009).
Goldstein, G.W., Wolinsky, J.S. & Csejtey, J. Diamond, I. Isolation of metabolically active capillaries from rat brain. J. Neurochem. 25, 715–717 (1975).
Way, D.L. et al. In vitro models of angiotumotigenesis. Lymphology 27 (Suppl): 136–137 (1993).
Persidsky, Y. et al. A model for monocyte migration through the blood–brain barrier during HIV-1 encephalitis. J. Immunol. 158, 3499–3510 (1997).
Fiala, M. et al. TNF-α opens a paracellular route for HIV-1 invasion across the blood–brain barrier. Mol. Med. 3, 553–564 (1997).
Gan, X. et al. Cocaine enhances brain endothelial adhesion molecules and leukocyte migration. Clin. Immunol. 91, 68–76 (1999).
Persidsky, Y. et al. Microglial and astrocyte chemokines regulate monocyte migration through the blood–brain barrier in human immunodeficiency virus-1 encephalitis. Am. J. Pathol. 155, 1599–1611 (1999).
Liu, N.Q. et al. Human immunodeficiency virus type 1 enters brain microvascular endothelia by macropinocytosis dependent on lipid rafts and the mitogen-activated protein kinase signaling pathway. J. Virol. 76, 6689–6700 (2002).
Miebach, S. et al. Isolation and culture of microvascular endothelial cells from gliomas of different WHO grades. J. Neurooncol. 76, 39–48 (2006).
Shepherd, B.R., Chen, Y.S.H., Smith, C.M., Gruionu, G., Williams, S.K. & Hoying, J.B. Rapid perfusion and network remodeling in a microvascular construct after implantation. Arterioscler. Thromb. Vasc. Biol. 24, 898–904 (2004).
Fiala, M. et al. Cocaine increases human immunodeficiency virus type 1 neuroinvasion through remodeling brain microvascular endothelial cells. J. Neurovirol. 11, 281–291 (2005).
Lossinsky, A.S. et al. The histopathology of Candida albicans invasion in neonatal rat tissues and in the human blood–brain barrier in culture revealed by light, scanning, transmission and immunoelectron microscopy. Histol. Histopathol. 21, 1029–1041 (2006).
Liu, J. et al. T cell independent mechanism for copolymer-1-induced neuroprotection. Eur. J. Immunol. 37, 3143–3154 (2007).
Haorah, J., Schall, K., Ramirez, S.H. & Persidsky, Y. Activation of protein tyrosine kinases and matrix metalloproteinases causes blood–brain barrier injury: novel mechanism for neurodegeneration associated with alcohol abuse. Glia 56, 78–88 (2008).
Ramirez, S.H. et al. Activation of peroxisome proliferator-activated receptor gamma (PPARgamma) suppresses Rho GTPases in human brain microvascular endothelial cells and inhibits adhesion and transendothelial migration of HIV-1 infected monocytes. J. Immunol. 180, 1854–1865 (2008).
Zaghi, J. et al. Alzheimer disease macrophages shuttle amyloid-beta from neurons to vessels, contributing to amyloid angiopathy. Acta Neuropathol. 117, 111–124 (2009).
Ramirez, S.H. et al. Methamphetamine disrupts blood–brain barrier function by induction of oxidative stress in brain endothelial cells. J. Cereb. Blood Flow Metab. 29, 1933–1945 (2009).
Cardoso, FL. Establishment and characterization of a human model of the blood–brain barrier. Master Degree Thesis, New University of Lisbon 2009.
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.
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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.
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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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DOI: https://doi.org/10.1038/nprot.2010.76
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