Clear edge staining was also observed in P.1 PBECs, confirming the maintenance of BBB features after passaging. The loss of in vivo phenotype reported for many in vitro BBB models appears to be mainly due to the removal of endothelial cells from their natural Epacadostat environment. However, the changes can be counteracted to some degree using several inductive factors and co-cultures as discussed. Recently developed primary cultured in vitro BBB models offer advantages as assay systems since they express more features of the in vivo BBB (including membrane lipid and protein composition, expression of uptake and efflux transporters and drug metabolising enzymes) than Caco-2

(from human colon carcinoma) or MDCK (from canine PD0332991 mouse kidney epithelium) cell lines, which are commonly used in the pharmaceutical industry. Until around year 2000 the in vitro BBB model showing the best correlation with in vivo BBB permeability was the system using bovine brain endothelial cells co-cultured on filters above rat astrocytes

( Cecchelli et al., 1999), but over the last decade several groups have reported successful use of porcine brain endothelial cells as useful tools for drug screening ( Franke et al., 1999 Franke et al., 2000, Smith et al., 2007 and Zhang et al., 2006). Our results demonstrate that the PBEC model described here has the potential to be useful as a permeability screen to investigate BBB permeation of drugs of interest with a range of chemistries, including those that are substrates for transporters, whether or not the

particular transporters involved have been identified. With inclusion of sufficient passively permeating reference compounds, substrates for transporters can be identified as outliers, for further mechanistic study. If required and desirable, porcine brain endothelial cell production could be scaled MYO10 up for high/medium-throughput screening. However, it is possible to limit the numbers of compounds that need to be tested on living BBB models using better in silico (computer-based) screens. Thus a serial and parallel screening process can be used to bring the numbers to manageable level (e.g. 200 cf. >100,000) for testing on an in vitro BBB model ( Abbott, 2004). In conclusion, results confirm that this optimised in vitro porcine BBB model is relatively simple to prepare, reliable and repeatable compared to most other static BBB models, and gives high TEER without the need for astrocyte co-culture. The quality, simplicity and robustness of the porcine BBB model make it an attractive model for industry to use in CNS drug discovery programmes and also for a variety of basic scientific projects. Because the method generates PBECs with high TEER, it is likely to show good apical: basal differentiation for other important BBB features, including receptors, transporters, enzymes and ion channels.