2.1 Dynamic Co-Culture Model of the BBB
2.1.1 Coating of Cell Culture Plates, Flasks, and Filters
1. All disposable cell culture plastics are from Corning Costar BV (Schiphol, The Netherlands).
2. Collagen type IV (Sigma-Aldrich BV, Zwijndrecht, The Netherlands, Cat. C-5533, nonsterile) coating solution is prepared in a 100 µg/ml stock solution in 0.1% v/v acetic acid (J.T. Baker, Deventer, The Netherlands, Cat. 6001) solution. The acetic acid solution is prepared in Milli-Q water (from Milli-Q system, Millipore, Etten- Leur, The Netherlands) and sterilized over 0.2-µm filter (Corning Costar BV). The collagen stock solution should not be sterile filtered and can be stored at 4°C no longer than 2 months. Collagen working solution of 10 µg/ml is prepared by dilution in 0.1% v/v acetic acid, kept at 4°C, and can be used for no longer than 2 weeks.
3. Fibronectin (Boehringer Mannheim BV, Almere, The Netherlands, Cat. 1080 938) coating solution is prepared in a 1 mg/ml stock solution in sterile Milli-Q water and stored in 1 ml aliquots at −20°C in 100-ml bottles. Fibronectin working solution of 10 µg/ml is prepared by dilution in phosphate buffered saline (PBS, Cambrex, Verviers, Belgium, Cat. 17-516F), kept at 4°C, and can be used for no longer than 6 weeks.
4. Poly-d-lysine (Sigma-Aldrich BV, Cat. P-7280) coating solution is prepared in a 1 mg/ml stock solution in sterile Milli-Q water and stored in 1 ml aliquots at
−20°C in 100-ml bottles. Poly-d-lysine working solution of 10 µg/ml is prepared by dilution in PBS, kept at 4°C, and can be used for no longer than 6 weeks.
2.1.2 Isolation of Astrocytes
1. Newborn (no older than 5 days) Wistar rat pups are obtained from Harlan B.V.
(Zeist, The Netherlands).
2. Dulbecco’s modified Eagle’s medium (DMEM) is supplemented (DMEM + S) with 10% (v/v) heat inactivated (30 min at 56°C) fetal calf serum (FCS, Cambrex, Cat. BE14-603F (see Note 1)). The DMEM, formulated with high d- glucose (4.5 g/l), NaHCO3 (3.7 g/l), and HEPES (25 mM), contained extra MEM nonessential amino acids (Cambrex, Cat. 13-114E), 2 mM l-glutamine (Cambrex, 17–605E), streptomycin sulfate (0.1 g/l), and penicillin G sodium (100,000 U/l) mixture (Cambrex, 17–602E).
3. Fully (50 mM) HEPES buffered DMEM (i.e., without NaHCO3) is prepared with DMEM from Cambrex (Cat. 15-604D) as described earlier.
4. 0.25% trypsin–0.02% EDTA is from Sigma-Aldrich BV (Cat. T-4049).
5. Nylon mesh filters of 120 and 45 µm are hand-cut from fabric obtained at Merrem & la Porte BV (Zaltbommel, The Netherlands) and placed in filter holders from Millipore (Cat. SX00 047 00).
2.1.3 Isolation of Bovine Brain Capillaries
1. Bovine (calf) brains are obtained at the slaughterhouse from freshly killed ani- mals (see Note 2).
2. Nylon mesh filters of 200 and 150 µm are hand-cut from fabric obtained at Merrem & la Porte BV and placed in filter holders from Millipore (Cat. SX00 047 00).
3. Collagenase type 3 (Gibco, Breda, The Netherlands, Cat. 17102-013) is prepared in a 2,000 U/ml stock solution in PBS, sterilized over 0.2-µm filter and stored at −20°C.
4. Trypsin TRL (Worthington, Cat. LS003702) is prepared in a 900 U/ml stock solution in PBS, sterilized over 0.2-µm filter, and stored at −20°C.
5. DNAse I (Worthington, Cat. LS002138) is prepared in a 3,400 U/ml stock solution in PBS, sterilized over 0.2-µm filter, and stored at −20°C.
6. Digest mix for one calf brain homogenate is freshly prepared by mixing 2 ml of Collagenase stock, 2 ml Trypsin stock, and 1 ml DNAse stock in 15 ml of DMEM + S.
7. Freeze mix is prepared by adding 10% DMSO (J.T. Baker, Cat. 7033) to 90% FCS.
2.1.4 Differential Seeding of Brain Capillaries and Culture of Bovine Brain Capillary Endothelial Cells
1. Heparin solution (Sigma, Cat. H-3149) is prepared in a 0.5% (w/v) stock solu- tion in PBS, sterilized over 0.2-µm filter, and kept at 4°C.
2. Growth medium is freshly prepared with either 50% DMEM + S, 50% astrocyte conditioned medium (ACM, see below) and 125 µg/ml heparin (GM+), or with 100% DMEM + S and 125 µg/ml heparin (GM−).
2.1.5 Preparation of the In Vitro BBB on Filter Inserts
1. 24-wells Transwell polycarbonate filters are used with a surface area of 0.33 cm2 and pore-size of 0.4 µm (Corning Costar, Cat. 93413), collagen coated as described below, with astrocytes on the bottom of the filter (as described below).
2. EDTA solution (J.T. Baker, Cat. 1073) is prepared in a 0.02% (w/v) solution in PBS, sterilized over 0.2-µm filter and kept at 4°C.
3. Trypsin-EDTA solution for endothelial cells (500 BAEE units porcine trypsin and 180 µg EDTA per ml) from Sigma (Cat. T-4299) is aliquoted in 5 ml and stored at −20°C.
4. 8-(4-chlorophenylthio (CPT) )-cAMP (Sigma, Cat. C-3912) is prepared as a 25 mM stock solution in Milli-Q water (not PBS), sterilized over 0.2-µm filter, aliquoted in 0.5 ml, and stored at −20°C.
5. RO-20-1724 (Calbiochem, Cat. 557502) is prepared as a 35 mM stock solution in DMSO, aliquoted in 25 µl and stored at −20°C.
6. Differentiation medium is freshly prepared by addition of 12.5 µl CPT-cAMP stock solution per ml DMEM + S and 0.5 µl RO-20-1724 stock solution per ml DMEM + S.
2.2 2B-Trans™ Technology
2.2.1 Characterization of Transport Receptor on the BBB In Vitro
1. Used proteins for these studies are: diphtheria toxin (DT, Sigma-Aldrich BV, Cat.
D-0564), nontoxic mutant protein of diphtheria toxin CRM197 (“cross-reactive material” 197, or [Glu52]-Diphtheria toxin from Sigma-Aldrich BV, Cat. D-2189) and soluble recombinant human heparin-binding EGF-like growth factor (HB-EGF, carrier free from R&D Systems, Cat. 259-HE/CF).
2.2.2 Receptor Targeted Cell Uptake Studies on the BBB In Vitro
1. Used proteins for these studies are: CRM197 (Sigma-Aldrich BV), HB-EGF (R&D Systems), CRM197, and bovine serum albumin fraction V (BSA, ICN Biomedicals, Cat. 160069).
2. For conjugation of proteins to HRP, a HRP conjugation kit from Alpha Diagnostic International (San Antonio, TX, Cat. 80220) is used according to the manufacturer’s instructions.
3. Conjugates are purified on a HiPrep 16/60 column packed with Sephacryl S-200 HR matrix from Amersham Biosciences (Cat. 17-1166-01).
4. HRP activity is determined with TMB liquid substrate (Sigma-Aldrich BV, Cat.
T-8665), with H2SO4 (J.T. Baker) to stop the reaction, and HRP type VI-A (Sigma-Aldrich BV, Cat. P-6782) for a standard curve (0–2 ng/ml) and absorption is read at 450 nm.
5. Cells are lysated by an aqueous solution of 0.1% Na-deoxycholate (Sigma-Aldrich BV, Cat. D-6750).
6. Cellular protein content is determined using Bio-Rad DC reagents (Bio-Rad Laboratories, Veenendaal, The Netherlands, Cat. 500-0111) and BSA for a standard curve (0–400 µg/ml). Absorption is read at 690 nm.
2.2.3 Receptor Targeted Transcytosis Studies across the BBB In Vitro 1. Materials are used as described earlier.