The alternative co-culture variant of this method described provides considerable flexibility for experimental design, depending on the application. The ultimate goal for most BBB researchers is to be able to study the human BBB. However, the difficulties associated with developing robust and realistic in vitro human BBB models have led to the use of animal models ( Patabendige, 2012). A porcine BBB model is a good alternative as the biology of the pig is closer than that of other laboratory animals to the biology of the human ( Everolimus research buy Walters et al., 2011). The PBEC model presented in this paper is one of the best BBB models giving high TEER. However, as with all BBB models, there

are some limitations. Strict adherence to the experimental procedure is required to produce high yields of pure PBEC cultures and to minimise variation between batches. Only limited in vivo data is available for porcine models compared to rodent models; however, with the increased use of transgenic and miniature pigs this will improve in future. Availability of good porcine primers and

antibodies is Venetoclax manufacturer currently an issue, but this also will improve with the recent publication of a high-quality draft pig genome sequence ( Groenen et al., 2012). Further examination of expression and function of transporters and receptors on the PBEC model is currently under way. In summary, this method combines simplicity and reproducibility with optimum cell yield and purity, making the resulting PBEC model robust, reliable 3-mercaptopyruvate sulfurtransferase and flexible, with good preservation of BBB features, suitable for a range of appli-cations. 8 h isolation of brain capillaries and freezing (from 6 pig brains) Culture medium L-15

Leibovitz (L-15); medium 199 (M199); DMEM; Penicillin (10,000 U/mL)/Streptomycin (10 mg/mL) (P/S); Glutamine (2 mM stock soln); Heparin; Puromycin; cell permeant cAMP analogue, CPT-cAMP; Hydrocortisone; Trypsin-EDTA for endothelial cells; Hanks’ balanced salt solution (HBSS) without (w/o) Ca2+,Mg2+; FCS; poly-D-lysine; human fibronectin; dimethyl sulfoxide (DMSO); all from Sigma. Type IV phosphodiesterase inhibitor, RO 20-1724 from Calbiochem/Merck. Enzymes from Lorne Laboratories Limited, UK. Collagenase, Trypsin, DNase I. Minimal essential medium (MEM+HEPES) from MP Biomedicals. Phosphate buffered saline (PBS) with Ca2+ and Mg2+ from Cambrex Bio Science. BPDS from First Link UK. Nylon meshes (60 µm and 150 µm pore size) from Plastok Associates, UK. Rat tail collagen type I from Becton Dickinson. Tissue culture plastics (flasks, plates, Petri dishes). Filter inserts: Costar ‘Transwell Clear’ 12-well tissue-culture-treated sterile polyester membrane, 0.4 µm pore, 12 mm membrane, pre-loaded on cluster plates. [14C]sucrose (0.15 µCi/mL final concentration, specific activity 643 mCi/mmol) and [14C]mannitol (0.