Human brain vascular endothelial cells (HBECs) were isolated from a 49-year-old white male with no known neuronal diseases and provided at passage 2 (500,000 cells) as a gift by Clonetics (BioWhittaker, Walkersville, MD). Bovine retinal vascular endothelial cells (BRECs) from VEC Technologies, Inc. (Rensselaer, NY) were isolated from a pool of bovine retinas. Cells were cultured in endothelial basic medium (EBM) or endothelial growth medium (EGM, which is EBM supplemented with growth factors and 5% serum; BioWhittaker). Primary cultures of rat cerebral astrocytes (RCAs) were obtained from the cerebra of 2- to 3-day-old Holtzman Sprague-Dawley rats (Harlan Sprague-Dawley, Inc., Indianapolis, IN) by the method of McCarthy and de Vellis.
18 19 Cerebra were removed from newborn pups and immersed in saline solution (138 mM NaCl, 5.4 mM KCl, 1.1 mM Na
2HPO
4, 22 mM glucose, and 0.9 mM CaCl
2) at 4°C. Tissue was mechanically disrupted and resuspended in culture medium (DMEM-Ham’s F-12 medium; DMEM/F-12, 1:1, vol/vol) containing 5% fetal calf serum, 5% horse serum, and 1% penicillin-streptomycin. Suspended cells were filtered through cell strainers, and cells in the filtrate were plated at high density in 75-cm
2 flasks and grown in complete culture medium in an atmosphere of 5% CO
2-95% O
2 at 37°C. On day 10, cell cultures were shaken for 18 hours in an atmosphere of 5% CO
2-95% O
2 at 37°C (250 rpm, stroke diameter = 1.5 inches) to remove contaminating oligodendrocytes and neurons. After shaking, astrocytes were recovered by trypsin-EDTA and replaced at one third of their confluent density. On reaching confluence, astrocytes were plated for astrocyte-EC coculture or plated in 75-cm
2 flasks for collecting conditioned media.