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Background  The complexity of the neurovascular unit (NVU) poses a challenge in the investigations of drug transport across the blood–brain barrier (BBB) and the function of the brain capillary endothelium. Several in vitro models of the brain capillary endothelium have been developed. In vitro culture of primary endothelial cells has, however, been reported to alter the expression levels of various brain endothelial proteins. Only a limited number of studies have addressed this in detail. The aim of the present study was to investigate mRNA levels of selected BBB transporters and markers in in vitro models of the BBB based on bovine primary endothelial cells and compare these to the levels estimated in freshly isolated bovine brain capillaries.    Methods  Brain capillaries were isolated from bovine cerebral cortex grey matter. Capillaries were seeded in culture flasks and endothelial cells were obtained using a brief trypsinization. They were seeded onto permeable supports and cultured in mono-, non-contact- or contact co-culture with/without primary rat astrocytes. mRNA-expression levels of the selected BBB markers and transporters were evaluated using qPCR and monolayer integrity of resulting monolayers was evaluated by measuring the transendothelial electrical resistance (TEER).    Results  The capillary mRNA transcript profile indicated low expression of  ABCC1  and  CLDN1 . The mRNA expression levels of  TPA ,  OCLN ,  ABCB1 ,  SLC2A1 ,  SLC16A1  and  SLC7A5  were significantly decreased in all culture configurations compared to freshly isolated bovine brain capillaries.  ALP ,  VWF ,  ABCC1  and  ABCC4  were upregulated during culture, while the mRNA expression levels of  F11R ,  TJP1 ,  CLDN5 ,  CLDN1  and  ABCG2  were found to be unaltered. The mRNA expression levels of  VWF ,  ALP ,  ABCB1  and  ABCC1  were affected by the presence of rat astrocytes.    Conclusion  The endothelial mRNA transcript profile in bovine capillaries obtained in this study correlated nicely with profiles reported in mice and humans. Cultured endothelial cells drastically downregulated the mRNA expression of the investigated SLC transporters but maintained expression of efflux transporter and junctional protein mRNA, implying that the bovine in vitro BBB models may serve well to investigate basic barrier biology and in vivo permeation of passively permeating compounds and efflux transporter substrates but may be less well suited for investigations of SLC-mediated transport.

Culture-induced changes in mRNA expression levels of efflux and SLC-transporters in brain endothelial cells