Neuroinflammation Model Using hCMEC/D3 Cells

The human brain EC line hCMEC/D3 was obtained at Tebubio. Cells were used up to passage 35. Cells were cultured in collagen coated (rat tail, type I, Sigma-Aldrich) culture flasks in growth medium (EGM-2MV medium [Lonza] supplemented with 2.5% FBS [Thermo Fisher Scientific]). Cells were collected using trypsin and cultured in 24-well plates or in Thincerts (both Greiner Bio-One) for migration assays as described further. After reaching near confluency (90%), cells were replenished with experimental medium (basal EBM2 [Lonza] supplemented with Gentamicin [10 μg/mL], Amphotericin B [1 μg/mL], fibroblast growth factor [FGF, 1 ng/mL], hydrocortisone [HC, 1.4 μM; all Sigma-Aldrich], 2.5% FBS [Thermo Fisher Scientific]). One day later, cells were treated with TNF-α (100 ng/mL) and IFN-γ (10 ng/mL, both from Peprotech) (72 (link)) for 24 hours in reduced medium (basal EBM2 [Lonza] supplemented with Gentamicin, Amphotericin, FGF, 0.25% FBS) (73 (link)).