Brain is well protected against blood-borne xenobiotics (drugs, nutrients, and toxins) by two major barriers, i.e., blood-brain barrier (BBB) and blood-cerebrospinal fluid (CSF) barrier (BCB). The BCB, whose surface area is about one-half of the BBB, is located in brain ventricles and functions to produce CSF and transport xenobiotics between blood and CSF. We have recently established a novel immortalized choroidal epithelial cell line, named Z310 cell line. This ceil line possesses the essential morphology of the parent primary cells and, upon growing on a semipermeable membrane, forms a monolayer that restricts the free movement of paracellular leakage marker, [14C]sucrose. While the tightness of the cell monolayer, as measured by trans-epithelial electrical resistance (TEER) or paracellular leakage of [14C]sucrose, remains to be improved, we are convinced that this cell line shows a great promise as a unique in vitro blood-brain barrier transport system, as there has been no such brain cell-derived transport model in the current neurotoxicology and neuropharmacology research field. To create this novel system, we hypothesize that the tightness of the monolayer of Z310 epithelial cells can be improved by alteration of the chemical components of the culture media, by induction and promotion of tight junction assembly, and/or by genetic modulation of the expression of tight junction proteins. Thus, our specific aims are to reduce the paracellular permeability of the Z310 barrier model by modifying the culture medium components, such as using serum-free or astrocyte-conditioned culture media, to improve the tightness of the barrier structure by application of tight-junction inducing agents in cell culture medium, and to knock-in the specific gene fragments that encode the proteins or regulatory proteins associated with tight junctions in existing Z310 cells. We further design a series of experiments to validate this model system. The studies proposed in this application, if successful despite the notable risk, will lead to the creation of a novel in vitro blood-brain/CSF model for transport study of materials into brain and should have significant impact on pharmacological and toxicological investigation of CNS transport of drugs and toxicants, CNS drug development, and etiological research of brain diseases.
