Application of in vitro Blood-Brain Barrier model for assessment of drug delivery to central nervous system.

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(1) Establishment of immortal bovine brain endothelial cellTo establish immortal brain endothelial cell, pSV3-neo which contains SV40 T antigen was introduced to primary cultured bovine brain endothelial cells. After selection by G418, we obtained six clones that exhibited the spindle-shaped appearance of normal brain endothelial cell. One of them, t-BBEC-117, retained the brain endothelial cell phenotype.(2) Establishment of immortal rat astrocytepSV3-neo was introduced to rat primary cultured astrocytes. We obtained four immortal colony, which exhibited glial fibrillary acidic protein. A single clone, ACT-57, exhibited stable growth in a chemically defined medium and a stronger expression of NGF mRNA than that of normal astrocyte.(3) Identification of astrocytic factor which induce blood-brain barrier phenotype.Astrocyte in co-culture was found to tighten the intercellular contacts of t-BBEC-117. Among known astrocytic factors, only fibroblast growth factor-basic (bFGF) could mimic the actions of astrocytes. These data suggest that bFGF is one of the most plausible astorcytic factors to induce the blood-brain barrier properties of immortal brain endothelial cells.