In vitro BBB model set up

The BBB model was composed of bEnd.3 cells (immortalized mouse brain endothelial cells) and C6 cells. All cells were used before passage number 30 (passages 12–28 for bEnd.3 cells and 10–30 for C6 cells), which corresponded to the time where these cells may begin to lose their BBB properties, as cited by the supplier and in a previous study [23].

Once the cells reached subconfluency, they were placed onto cell culture inserts for 24-well plates. Fig 1 shows a schematic description of the in vitro BBB model.

For the procedure of contact coculture, the transwell filter was inverted and C6 cells were seeded onto the abluminal side of the filter at a density of 4x10⁴ cells/cm². Then cells were placed at 37°C for 6 h (time necessary for the cells to adhere to the membrane of the insert). Afterwards, the insert was flipped back and C6 cells were cultured for two days in DMEM. At the end of two days, bEnd.3 cells were seeded onto the luminal side of the transwell filter at a density of 4x10⁵ cells/cm² and cocultured with C6 cells for eight days.