Isolation and Culture of Brain Endothelial Cells

The primary cultures of brain endothelial cells (BECs) were prepared from 6–7-month-old WT and APOB-100 transgenic mice as described in detail by Lénárt et al. [32 (link)]. 3 male and 3 female mice were used in both the WT (n = 6) and the APOB-100 group (n = 6) for each isolation. Forebrains were collected in ice-cold sterile PBS; meninges were removed, grey matter was minced by scalpel into 1 mm³ pieces and digested with 10 mg/ml collagenase II and 1 mg/ml DNase I in Dulbecco’s modified Eagle’s medium (DMEM)/F12 for 50 min at 37 °C. Microvessels were separated from myelin containing elements by centrifugation (1000×g, 20 min) in 20% bovine serum albumin (BSA)-DMEM and further digested with 10 mg/ml collagenase-dispase (Roche, Basel, Switzerland) and 1 mg/ml DNase I in DMEM/F12 for 35 min at 37 °C. Then they were washed twice in DMEM/F12 before plating on collagen type IV and fibronectin-coated (100 µg/ml each) dishes, 6 well plates (Corning Costar Co., Lowell, MA, USA) or cell culture inserts (Transwell clear, 1 cm²; pore size of 0.4 μm; Corning Costar Co.). Cultures were maintained in DMEM/F12 supplemented with 15% plasma-derived bovine serum (PDS; First Link, Wolverhampton, UK), 1 ng/ml basic fibroblast growth factor (Roche) and 100 μg/ml heparin. During the first 2 days, the culture medium contained puromycin (4 μg/ml) in order to selectively remove P-glycoprotein-negative contaminating cells [33 (link)]. Cultures reached confluency within a week and were used for experiments. To induce BBB characteristics, BECs were co-cultured with mouse astroglial cells. The resulting double co-culture model was used for permeability studies and transendothelial electrical resistance measurements [34 (link), 35 (link)].