In vitro transwell BBB model

B End.3 and HT22 cells were purchased from the Cell Bank of Typical Culture Preservation Committee of the Chinese Academy of Sciences. Cells were maintained in a cell incubator (37° C and 5% CO₂).

The Transwell BBB model was established as previously reported. Briefly, the b End.3 cells (0.2 mL of 5×10^5 cells/mL) were seeded into the upper chamber of an insert (Transwell, polycarbonate membrane, 0.4 μm pore size, 6.5 mm diameter, 24-well plate, Costar, Corning, NY). Next, 0.8 mL of complete media was added to the lower chamber of each well. The system was incubated for 2 days to allow cells to form tight junctions. Subsequently, the HT22 cells was seeded in the lower chamber of each well and cultured for another day [30]. The model was considered mature when trans-endothelial electrical resistance (TEER) values reached 60 Ω*cm², and the permeability coefficient remained below 0.02 [31].