Primary Mouse Brain Endothelial Cell Isolation

Primary mouse brain endothelial cell isolation was performed using 6-month-old C57BL/6 wild type and transgenic mice based on methods from our group [12 (link)–14 (link)]. Animals were sacrificed with cervical dislocation. Mouse forebrains were collected in ice-cold sterile phosphate buffered saline (PBS). Meninges were removed using sterile filter paper and the tissue was cut into 1 mm³ using a scalpel and digested with enzymes (1 mg/ml collagenase type II and 15 µg/ml deoxyribonuclease type I, Roche, Switzerland) in Dulbecco’s modified Eagle medium (DMEM/F12, Gibco, Life Technologies, USA) at 37°C for 50 min. Microvessels were separated from myelin by centrifugation on a 20% bovine serum albumin (BSA)-DMEM gradient (1,000×g, 20 min, 3 times). The collected vessels were further digested by enzymes (1 mg/ml collagenase–dispase and deoxyribonuclease type I in DMEM, both from Roche) for 35 min. Digested cerebral vessels were washed three times in cell culture medium then seeded onto Petri dishes (60 mm; Orange Scientific, Belgium) coated with collagen type IV and fibronectin. Mouse brain endothelial cells were cultured in DMEM/F12 supplemented with plasma-derived bovine serum (15%; First Link, UK), heparin (100 µg/ml), basic fibroblast growth factor (1 ng/ml; Roche), insulin (5 µg/ml), transferrin (5 µg/ml), sodium selenite (5 ng/ml), and gentamycin (50 µg/ml). Cells were grown under selective culture conditions for the first 4 days. Culture medium contained puromycin (3 µg/ml) to eliminate P-glycoprotein negative contaminating cell types [15 (link)]. When endothelial cells reached 90% confluency on the 4th–5th day after seeding to the Petri dish, cells were subcultivated into 96-well plates (Corning, USA; ACEA, USA), cover slips (VWR, USA) and hanging cell culture inserts for different experiments, respectively. All surfaces were coated with collagen type IV and fibronectin. Endothelial cells were passaged at a cell number of 5 × 10³ cells/well for 96-well plates and 3.5 × 10⁴ cells/cover slips. Since primary brain endothelial cell isolation yield from mice is low compared to other species, 24-well format culture inserts were used for permeability studies (ThinCert, 24-well format, polyethylene terephthalate membrane, 0.33 cm² surface, 3 µm pore size, Greiner Bio-one, Germany). These culture inserts were found to be appropriate for BBB permeability studies [16 (link)]. For barrier integrity tests endothelial cells were seeded to the luminal side of culture inserts at a cell number of 2.5 × 10⁴ cells/insert. To enhance BBB characteristics, primary mouse endothelial cells were co-cultured with primary mouse cerebral glial cells and pericytes [17 (link)].
Primary cultures of mouse pericytes were prepared by the same protocol as primary mouse brain endothelial cells, except for a shorter time of the second enzymatic digestion (15 min) and the omission of the puromycin treatment. At the end of the isolation process cerebral microvessels containing pericytes beside endothelial cells were seeded into collagen type IV coated 60 mm culture dishes (Orange Scientific, Belgium). After 4 days of culture in DMEM, 10% fetal bovine serum (FBS), and gentamycin (50 µg/ml) attached cells reached 70% of confluency. Pericytes were passaged into bigger, uncoated dishes (Orange Scientific, Belgium). Mouse pericyte cultures were used at second passage and seeded at a cell number of 5 × 10³ cells/well for 96-well plates, 3 × 10⁴ cells/cover slips and 5 × 10³ cells/culture inserts. Primary mouse pericytes stain positive for α-smooth muscle actin but not for von Willebrand factor or glial fibrillary acidic protein (GFAP) [17 (link), 18 (link)].
Primary mouse glial cells were obtained from 1 or 2-day-old wild type or ApoB-100 transgenic mice [12 (link), 13 (link)]. Meninges were removed by fine forceps from brains. Little pieces of cortices were minced and mechanically dissociated by pressing the tissue through a nylon mesh (40 µm, Millipore, USA). Cell clusters were seeded onto uncoated 75 cm² flasks (TPP, Germany) and cultured in DMEM containing FBS (10%; Lonza, Switzerland) and gentamycin (50 µg/ml) until 90% confluency. Glial cells were passaged at a cell number of 5 × 10³ cells/well for 96-well plates, 3 × 10⁴ cells/cover slips and 10⁵ cells/wells for 24-well plates (Greiner Bio-One, Germany). For the BBB co-culture model mixed glial cells were cultured for 2 weeks before use. Confluent glia cultures contained 90% of astroglia (positive for GFAP), and 10% microglia (positive for CD11b).
To establish the triple co-culture BBB model culture inserts were put into 24-well plates containing confluent glia cultures. Mouse pericytes were subcultured to the under side of the culture membranes and mouse brain endothelial cells were passaged to the upper side of the coated inserts. Both compartments received endothelial culture medium [17 (link)]. After 2 days of co-culture, cells were kept in culture medium containing 550 nM hydrocortisone and 10 mM lithium chloride [19 (link), 20 (link)].