Development of BBB Model.

Preparation of the in vitro BBB model was done using primary astrocytes and bEnd.3 cells as previously reported.^56,57 Primary astrocytes (1.5 × 10⁴/cm²) were cultured on the abluminal side of the culture insert, whereas bEnd.3 cells (1.5 × 10⁶/cm²) were cultured on the top layer using 20% serum containing media to form the in vitro BBB. Epithelial Volt/Ohm Meter 2 (EVOM) was used to determine transendothelial electrical resistance (TEER) to establish barrier integrity. Flux of the BBB impermeable dye sodium–fluorescein (Na–F) was used to estimate paracellular transport across the coculture model.^58,59 This coculture and bEnd.3 cells monolayer models were moved to different wells containing 500 μL of PBS. Na–F in PBS (10 μg/mL) was added to each culture insert, and its transport was evaluated at various time points up to 1 h. The fluorescence intensity of Na–F in the culture insert and 24-well plate was determined using a microplate reader at 485/535 nm. Quantification of Na–F dye flux across the blank culture insert was also performed and transendothelial permeability coefficients (Pe) were measured for coculture as well as monolayer models.^56,59