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

• Published in: Nature protocols Vol. 5; no. 7; pp. 1265 - 1272
• Main Authors: Ramirez, Servio H, Brito, Maria A, Bernas, Michael J, Cardoso, Filipa L, Daley, Sarah K, Weinand, Martin E, Campos, Alexandre R, Ferreira, António J Gonçalves, Hoying, James B, Witte, Marlys H, Brites, Dora, Persidsky, Yuri
• Format: Journal Article
• Language: English
• Published: England Nature Publishing Group 01.07.2010
• Subjects: ATP-Binding Cassette, Sub-Family B, Member 1; Blood vessels; Blood-brain barrier; Blood-Brain Barrier - cytology; Blood-Brain Barrier - physiology; Brain; Caveolin 1; Cell culture; Cell Culture Techniques; Cell research; Cells, Cultured; Collaboration; Collagen Type I; Electric Impedance; Endothelial Cells - metabolism; Endothelium; Enzymes; Epilepsy; Glucose; Glucose Transporter Type 1; Glycoproteins; Hippocampus - cytology; Humans; Laboratories; Methods; Models, Biological; Pericytes; Temporal Lobe - cytology; Tight Junctions; von Willebrand Factor; United States; Portugal; Lisbon Portugal; United States > US
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/nprot.2010.76

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 ( approximately 97% endothelial cells; approximately 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.