The blood-brain barrier (BBB) is comprised of brain microvascular endothelial cells (BMEC) that are endowed with unique barrier properties restricting diffusion of solutes and drugs between the blood and brain. It is therefore often desired to study BMEC in vitro to facilitate the development of brain drug delivery strategies. However, the utility of in vitro BBB models consisting of cultured BMEC has been limited by BMEC de-differentiation and loss of BBB properties. We addressed the loss of in vivo characteristics by applying genomics to investigate the molecular level differences between in vivo and in vitro BMEC. This study led to the identification of 25 differentially expressed gene transcripts, many of which had not previously been reported to change upon removing BMEC from the brain. Quantitative analysis of these target genes confirmed that loss of phenotype in vitro was the result of significant down-regulation of transporter, neural signaling and proliferation-related genes. This genomic "fingerprint" of the in vivo situation was then used as an assessment tool for validating in vitro BBB model quality.; First, the gene panel was utilized to validate a novel method for attaining high purity rat BMEC cultures. The generation of pure BMEC cultures is quite difficult in practice. Cell types, such as pericytes and smooth muscle cells, that help maintain the BBB in vivo, instead reduce in vitro barrier properties by preventing the complete formation of BMEC monolayers. We therefore developed an approach for reproducibly generating in vitro BMEC cultures with 99.8% purity by employing the translation inhibitor puromycin. Analysis using the gene panel showed that this purification method minimally affected the BMEC genotype, verifying its utility in obtaining high purity cultures without deleterious effects. Next, these puromycin-purified BMEC were shown to respond optimally to hydrocortisone induction of barrier properties, yielding a substantially improved in vitro BBB model. Importantly, using the gene panel as a diagnostic, it was apparent that hydrocortisone treatment stimulated gene expression towards in vivo levels. Taken together, these approaches validated that our new in vitro BBB model was indeed providing improved in vivo-like qualities, and hence could have major utility in neuropharmaceutical applications.
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