Primary porcine brain microvascular endothelial cells: biochemical and functional characterisation as a model for drug transport and targeting.

The blood-brain barrier (BBB) remains a significant obstacle to the delivery of therapeutic agents into the central nervous system (CNS). Primary cell cultures of brain capillary endothelial cells represent the closest possible phenotype to the in vivo BBB cell providing a convenient model for the study of transport systems and events that mediate solute delivery to the CNS. In this investigation we have characterized an in vitro primary BBB model from porcine brain microvascular endothelial capillary (PBMVEC) cells after recovery from cryopreservation of upto 12 months and studied their modulation by astrocytes. Co-cultures of PBMVECs with astrocytes (C6 astroglioma) resulted in trans-endothelial electrical resistance of up to approximately 900Omega cm2 and marked discrimination between the para- and trans- cellular markers sucrose and propranolol. Micrographs of confluent monolayers of PBMVECs showed the presence of tight junction complexes and vesicles with the morphological characteristics of either caveolae or clathrin coated pits. Extensive RT-PCR evaluation highlighted the expression of tight junction transcripts, ABC transporters, leptin receptor and select nutrient transporters. Functional studies examined the kinetics of transport of glucose, large neutral amino acids and p-glycoprotein (P-gp). Our findings indicate primary PBMVECs retain many barrier characteristics and transport pathways of the in vivo BBB. Further, primary cells can be stored as frozen stocks which can be thawed and cultured without phenotypic drift many months after isolation. Frozen PBMVECs therefore serve as a robust and convenient in vitro cell culture tool for research programs involving CNS drug delivery and targeting and in studies addressing blood-brain barrier transport mechanisms.