Endogro mv complete culture media

Human induced pluripotent stem cell (iPSC) line IMR90-4 (WiCell) was maintained on Matrigel (Corning) coated 6-well plates (Sarstedt) in StemFlex medium (Gibco), passaging twice a week and changing medium daily as previously described [33 (link), 44 (link)–46 , 48 (link)]. Meningioma cells of the meningothelial histological subtype were derived from tumor biopsies and characterized as previously described [36 (link)]. Meningioma cells were grown in DMEM + GlutaMAX (Gibco) with 10% FCS (Gibco) and 1% Pen/Strep (Gibco) and passaged twice a week with a seeding density of 4 × 10³ cells/cm². The cells were expanded and used for experiments up to passage 10 or until showing signs of senescence. hCMEC/D3 cells (Sigma, SCC066) were cultured in EndoGRO-MV Complete Culture Media (Sigma) splitting as needed. hCMEC/D3 and meningioma cells were grown on 30 µg/ml collagen 1 (rat tail, Gibco) coated tissue culture flasks.

The in vitro BBB models were prepared according to the method previously reported.^[10] Briefly, the insert of 24‐Trans‐well plate (Corning, Corning, NY) was coated with Collagen I (Corning) for 1 h so that endothelial cells could attach to the surface. Afterward, the insert cultured with 10 000 cells of hCMEC/D3 cells (Sigma‐Aldrich) in 100 µL of a EndoGRO‐MV complete culture media (Sigma‐Aldrich) was placed in the Trans‐well plate containing 500 µL of the same culture media in the bottom well. Once the cells reached to 90% of confluency (after ≈2 days), they were serum‐starved for 3 days by incubation in a serum‐free EndoGRO‐MV complete culture media to enhance the tight junction formation between adjacent endothelial cells and prevent multilayered paracellular barriers on the surface of Trans‐well insert. The BBB models were accepted if the effective permeability coefficient of a fluorescent‐labeled 40 kDa dextran (Thermo Fisher Scientific) was less than ≈1.0 × 10⁻⁵ cm s⁻¹, which validated the reasonable integrity of BBB tight junctions. Details were described in the “Assessment of BBB Permeability” subsection.