DEVELOPMENT OF AN IN VITRO BLOOD-BRAIN BARRIER MODEL FOR BRAIN DISPOSITION SCREENING OF PHARMACEUTICALS.

The study by scientists at MatTek Corp. describes initial results in development of an in vitro blood-brain barrier model derived from bovine brain capillary endothelial cells (BCEC). Solute distribution between blood and brain is strictly regulated by the blood-brain barrier (BBB). While the blood-brain barrier performs an important function in keeping unnecessary or harmful molecules from the brain, it poses a challenging problem in delivering therapeutics, including anticancer, antibiotic or antipsychotic drugs into the brain. Conversely, preventing potentially damaging molecules from overcoming the blood-brain barrier is also an increasing problem, especially when combinations of therapeutics are encountered. Thus, medical and pharmaceutical scientists have a growing need for rapid, reliable in vitro models of the blood-brain barrier for preclinical screening of pharmaceutical blood-brain barrier transport properties. Capillary vessels were first isolated from bovine brains. Individual BCECs were then released by further enzymatic digestion of the capillaries. BCEC thus obtained were cryopreserved. The isolation procedure produced a highly pure population of BCECs, as demonstrated by immunocytochemical staining for the endothelial cell marker von Willebrand factor. After recovery from cryopreservation, BCECs were cultured on microporous membrane culture inserts to produce the blood-brain barrier model. Transmission electron microscopy and H&E stained light microscopy of the cultures show uniform endothelial cell monolayers with evidence of tight junction formation. Immunocytochemical staining also demonstrated uniform expression of the tight junction protein ZO-1 localized along the BCEC borders. Permeation of Lucifer yellow across the blood-brain barrier culture was low, further demonstrating development of barrier function. Finally, Western blotting experiments were conducted to reveal the presence of the important blood-brain barrier efflux transporter p-glycoprotein. These results show significant progress in development of a reliable in vitro blood-brain barrier model that will be useful for preclinical screening of candidate pharmaceutical compounds. This work was supported by NCI Grant # R43 CA101703-02.

BBB, Blood-brain barrier (BBB), Bovine brain capillary endothelial cells (BCEC), Delivering therapeutics, Endothelial cells, Glutaraldehyde, Immunofluorescence, Lead citrate, Osmium tetroxide, Paraformaldehyde, Pharmaceutical BBB transport properties, Pre-clinical screening, Sodium cacodylate buffer, Spurr’s low-viscosity epoxy resin, TEER, TEM, Transepithelial electrical resistance (TEER), Transmission electron microscopy (TEM), Uranyl acetate, Western blot analysis

Glutaraldehyde, Lead citrate, Osmium tetroxide, Paraformaldehyde, Sodium cacodylate buffer, Uranyl acetate