Constructing a reliable in vitro blood-brain barrier (BBB) model using human primary cells has been considered a major challenge during the past decades. These systems could provide valuable information regarding the effect of therapeutic compounds on different BBB cell types (endothelial cells, astrocytes, pericytes) and their ability to cross the barrier in order to reach the brain. Several attempts have been made to develop in vitro BBB models, but these studies mainly used rat, bovine, and porcine cells rather than human primary cells. Genetically modified cell lines have also been used, but they do not appear to maintain physiological properties of the BBB. Here, we describe a detailed protocol for co-culturing and maintaining human brain primary endothelial cells, pericytes, and astrocytes under flow to create an in vitro human BBB model, which can be used for toxicity testing and for studying cross-interaction among different cell types involved in the BBB formation.
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