Purified CD31-positive ciBECs and ECs were suspended with SFM medium supplemented with 20 ng/mL bFGF, 10 ng/mL EGF, and 10 μg/mL human plasma fibronectin. A total of 12 × 104 ciBECs were plated on 500 μg/mL human plasma fibronectin-coated cell-culture inserts in the 24-well Transwell at 200 μL/well (Costar; polyester membrane, 0.4 mm pore size). Pre-seeded astrocytes from D125 to D180 on the bottom side of the culture dish were switched from DMEM/Ham's F12 supplemented with N2 supplement into SFM medium supplemented with 20 ng/mL bFGF, 10 ng/mL EGF, and 10 μg/mL human plasma fibronectin, and the inserts with ciBECs were added. After 3 days the medium was changed to minimum essential medium (Life Technologies) with 10% FBS (Life Technologies) and 100 ng/mL VEGF (Wako) for 2 days and then changed again to SFM medium supplemented with 20 ng/mL bFGF, 10 ng/mL EGF, and 10 μg/mL human plasma fibronectin for 2 days, and were used for further experiments such as drug kinetics.
