Co-culture Model of Brain Microvascular Endothelial Cells and Astrocytes

Human brain microvascular endothelial cells isolated from human brain tissue (HBMECs; Catalog #1000, ScienCell, Carlsbad, CA, USA) and human astrocytes isolated from human cerebral cortex (HAs; Catalog #1800, ScienCell) were co-cultured in endothelial cell medium (Catalog #1001; concentrations, mL^–1; 10 μg apo-transferrin, 10 μg BSA, 2 ng fibroblast growth factor (FGF)-2, 1 μg hydrocortisone, 2 ng insulin-like growth factor 1 (IGF-I), 7.5 μg insulin and 20 nM progesterone, 2% FBS, 5.55 mM glucose and 10 000 units·mL⁻¹ of penicillin and streptomycin) and astrocyte medium (Catalog #1801; concentrations, mL^–1; 10 μg apo-transferrin, 10 μg BSA, 10 ng EGF, 10 ng FGF-2, 1 μg hydrocortisone, 5 μg insulin, 2 ng IGF-I, 0.5 ng IGF LR3 10-8 M retinoic acid and 2 ng VEGF, 5% FBS, glucose 5.55 mM and 10 000 units·mL⁻¹ of penicillin and streptomycin; both ScienCell). HAs were seeded on the outside of collagen-coated 0.4 μm pore PTFE membrane transwell inserts (12-well type; Corning Costar, Tewksbury, MA, USA) directed upside down and allowed to adhere to the membrane overnight. HBMECs were seeded on the inside of the insert and cells were grown to confluence to create a contact co-culture model (Allen and Bayraktutan, 2009 (link); Mestre et al., 2011 (link)).