Establishment of primary cultures of human brain microvascular endothelial cells: a new and simplified method to obtain cells for an in vitro model of the blood-brain barrier

摘要

We describe a method for generating primary cultures of human brain microvascular endothelial cells (HBMVEC). HBMVEC 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 HBMVEC to migrate and proliferate. The overall process takes under 3 h and does not require specialized equipment or enzymatic processes. HBMVEC are typically cultured for approximately 1 month until confluence. Cultures are highly pure (~97% endothelial cells; ~3% pericytes), reproducible, and display characteristic brain endothelial markers (von Willebrand factor, glucose transporter-1), robust expression of tight and adherens junction proteins, caveolin-1, and efflux protein P-glycoprotein. Monolayers of HBMVEC display characteristic high transendothelial electric resistance and have proven useful in multiple functional studies for in-vitro modeling of the human blood-brain barrier.