Rat collagen 1

To establish the brain endothelium barrier model, mouse ECs (b.End5, BECs) (5 × 10⁴ cells/well) were seeded on the top of a PET transwell insert (0.33 cm², 8 µm pore size, Corning) previously coated with rat collagen-I (50 μg/ml) and fibronectin (20 μg/ml, Invitrogen). For hBLECs, inserts with cells from co-cultures were placed into new wells without pericytes. Cells were grown to confluence in their respective culture media. We considered that endothelial cell barrier integrity in murine monocultures was established when the transendothelial electrical resistance (EVOM² Epithelial Voltohmmeter, World Precision Instruments) was above 200 Ω × cm². TEER measurements under NX/NG conditions were evaluated in random transwells from each assay prior to any experimental procedure for b.End5 (550 ± 25.17 Ω × cm²), BECs isolated from Cort^+/+ (585.7 ± 22.15 Ω × cm²), Cort^+/− (505.5 ± 27.78 Ω × cm²), and Cort^−/− (525 ± 25.28 Ω × cm²) mice. Human barrier integrity was also evaluated by measuring the permeability of Lucifer yellow (Pc < 15·10^–6 cm/s) as described below and detailed in Additional file 1.

To establish the brain endothelium barrier model, mouse ECs (b.End5, BECs) (5 × 10⁴ cells/well) were seeded on the top of a PET transwell insert (0.33 cm², 8 µm pore size, Corning) previously coated with rat collagen-I (50 μg/ml) and fibronectin (20 μg/ml, Invitrogen). For hBLECs, inserts with cells from co-cultures were placed into new wells without pericytes. Cells were grown to confluence in their respective culture media. We considered that endothelial cell barrier integrity in murine monocultures was established when the transendothelial electrical resistance (EVOM² Epithelial Voltohmmeter, World Precision Instruments) was above 200 Ω × cm². TEER measurements under NX/NG conditions were evaluated in random transwells from each assay prior to any experimental procedure for b.End5 (550 ± 25.17 Ω × cm²), BECs isolated from Cort^+/+ (585.7 ± 22.15 Ω × cm²), Cort^+/− (505.5 ± 27.78 Ω × cm²), and Cort^−/− (525 ± 25.28 Ω × cm²) mice. Human barrier integrity was also evaluated by measuring the permeability of Lucifer yellow (Pc < 15·10^–6 cm/s) as described below and detailed in Additional file 1.

Silk films were coated with rat collagen I (Gibco, Thermo Fisher), fibronectin (BD Biosciences, Bedford, MA, USA), mouse laminin (Invitrogen, Thermo Fisher) or Poly-d-Lysine (Gibco, Thermo Fisher) respectively in a 50 μg/ml concentration diluted in dH₂O. We used 200 μl ECM to coat the silk film surfaces and incubated for 2 h at room temperature (RT). Then washed with 1 ml PBS for 3 times. These coated silk films were prepared freshly before seeding cells^{34 (link)–36 (link)}.