All reagents were purchased from Sigma, USA, unless otherwise indicated. Wistar rats were obtained from Japan SLC Inc., Japan. All animals were treated in strict accordance with the NIH Guide for Care and Use of Laboratory Animals (NIH Publications No. 80–23) and as approved by the Nagasaki University Animal Care Committee.
Primary cultures of rat brain capillary endothelial cells (RBEC) were prepared from 3-week-old rats, as previously described (Deli et al. 1997). Meninges were carefully removed from forebrains and gray matter was minced into small pieces of approximately 1 mm3 in ice-cold Dulbecco’s modified Eagle’s medium (DMEM), then dissociated by 25 times of up- and down-strokes with a 5-ml pipette in DMEM containing collagenase type 2 (1 mg/ml, Worthington, USA), 300 μl DNase (15 μg/ml), gentamicin (50 μg/ml), and then digested in a shaker for 1.5 h at 37°C. The cell pellet was separated by centrifugation in 20% bovine serum albumin (BSA)-DMEM (1,000g, 20 min). The microvessels obtained in the pellet were further digested with collagenase-dispase (1 mg/ml, Roche, Switzerland) and DNase (6.7 μg/ml in DMEM for 1 h at 37°C. Microvessel endothelial cell clusters were separated on a 33% continuous Percoll (Pharmacia, Sweden) gradient, collected and washed twice in DMEM before plating on 35-mm plastic dishes coated with collagen type IV and fibronectin (both 0.1 mg/ml). RBEC cultures were maintained in DMEM/F12 supplemented with 10% plasma derived serum (PDS, Animal Technologies, USA), basic fibroblast growth factor (bFGF, Roche, Switzerland, 1.5 ng/ml), heparin (100 μg/ml), insulin (5 μg/ml), transferrin (5 μg/ml), sodium selenite (5 ng/ml) (insulin-transferrin-sodium selenite media supplement), gentamicin (50 μg/ml), and puromycin (4 μg/ml) (Perrière et al. 2005) (RBEC medium I) at 37°C with a humidified atmosphere of 5% CO2/95% air, for 2 days. On the third day, the cells received a new medium which contained all the components of RBEC medium I except puromycin (RBEC medium II). When the cultures reached 80% confluency (fourth day in vitro), the purified endothelial cells were passaged by a brief treatment with trypsin (0.05% wt/vol)-EDTA (0.02% wt/vol) solution, and used to construct various types of in vitro BBB models.
Rat cerebral astrocytes were obtained from neonatal Wistar rats. Meninges were removed and cortical pieces mechanically dissociated in astrocyte culture medium (DMEM supplemented with 10% fetal bovine serum). Dissociated cells were seeded into cell culture flasks. In order to obtain type 1 astrocytes, flasks with confluent cultures were shaken at 37°C overnight. The purity of astrocytes was checked by immunostaining for glial fibrillary acidic protein (GFAP), and the cells were used at passage 2.
Pure cultures of rat cerebral pericytes were obtained by a prolonged, 2-week culture of isolated brain microvessel fragments, that contain pericytes beside endothelial cells. The same preparations yield primary RBEC after puromycin-treatment. Pericyte survival and proliferation was favored by selective culture conditions, using uncoated dishes, and DMEM supplemented with 10% fetal bovine serum and antibiotics. Culture medium was changed every 3 days. Pericytes were characterized by their large size and branched morphology, positive immunostaining for α-smooth muscle actin, and absence of von Willebrand factor and GFAP staining. Pericytes and astrocytes were frozen in cryo-medium CELLBANKER® (BCL-1, ZENOAQ, Japan), and stored at −80°C.
Construction of In vitro BBB models
The day when the endothelial cells were plated and models were established was defined as day zero in vitro (Day 0). To construct various in vitro models of BBB, pericytes or astrocytes (1.5 × 104 cells/cm2) were seeded on the bottom side of the collagen-coated polyester membrane of the Transwell® inserts. The cells were let to adhere firmly for overnight, then endothelial cells (1.5 × 105 cells/cm2) were seeded on the upper side of the inserts placed in the well of the 12-well culture plates containing no cells, pericytes, or astrocytes (out-of-contact types, Fig. 1). BBB models were maintained in RBEC medium II. From Day 1, the culture medium was supplemented with 500 nM hydrocortisone (Hoheisel et al. 1998). Under these conditions, in vitro BBB models were established within 3 days after setting of the cells. Seven types of BBB models were constructed as shown on the scheme (Fig. 1). As negative controls, astrocytes and pericytes, which do not form barrier, were cultured on the inserts, respectively.
Evaluation of the Barrier Integrity
TEER, which reflects in culture conditions the flux of mainly sodium ions through cell layers, was measured by Epithelial-volt-ohm meter and Endohm-12 chamber electrodes (World Precision Instruments, USA). TEER of coated, but cell-free filters was subtracted from measured TEER values of the models shown as Ω × cm2.
The flux of sodium fluorescein (Na-F) across endothelial monolayers was determined as previously described (Kis et al. 2001). Cell culture inserts were transferred to 12-well plates containing 1.5 ml assay buffer (136 mM NaCl, 0.9 mM CaCl2, 0.5 mM MgCl2, 2.7 mM KCl, 1.5 mM KH2PO4, 10 mM NaH2PO4, 25 mM glucose, and 10 mM Hepes, pH 7.4) in the basolateral or lower compartments. In the inserts culture medium was replaced by 0.5 ml buffer containing 10 mg/ml Na-F (MW: 376 Da). The inserts were transferred at 5, 15, and 30 min to a new well containing assay buffer. The concentrations of the marker molecule in samples from the upper and lower compartments were determined by fluorescence multiwell plate reader (excitation: 485 nm, emission: 535 nm). Flux across cell-free inserts was also measured. Transport was expressed as μl of donor (luminal) compartment volume from which the tracer is completely cleared. Transendothelial permeability coefficient (Pe in 10−6 cm/s) was calculated as previously described (Deli et al. 2005).
All data presented are means ± SEM. The values were compared using the analysis of variance followed by Bonferroni–Dunn test. Changes were considered statistically significant at P < 0.05. All experiments were repeated at least three times, and the number of parallel inserts was four.