Establishment and characterization of a human model of the blood-brain barrier

Dissertação apresentada para a obtenção do Grau de Mestre em Genética Molecular e Biomedicina, pela Universidade N ova de Lisboa, Faculdade de Ciências e Tecnologia

The discovery of the blood-brain barrier has allowed several studies focused on its properties throughout the last decades. The available study methods are both expensive and time-consuming. We describe a new simplified method for isolation of primary cultures of human brain microvascular endothelial cells (HBMVEC). Human samples of temporal lobe, obtained as discarded tissue during operative treatment for epilepsy, were mechanically fragmented and filtered through adequate pore-size polyester meshes. Resulting microvessel fragments were placed on type-I collagen-coated flasks to allow HBMVEC to migrate from vessels and proliferate. Usually, it takes approximately 1 month until confluence. The resulting cultures are highly pure and comprise important barrier properties such as: specific endothelial cell markers (von Willebrand factor and glucose transporter-1), expression of tight and adherens junctions (zonula occludens-1 and D-catenin, respectively), vesicular transport machinery(caveolae, through the expression of caveolin-1), ABC transporters (P-glycoprotein), as well as high transendothelial electric resistance, characteristics that are maintained for two passages. Furthermore, we have successfully cryopreserved primary HBMVEC maintaining the expression of the previously mentioned proteins important to barrier properties.