Collagen 4

Brain microvascular endothelial-like cells (iBMECs) were prepared as previously described [8 (link)]. In Brief, human iPS cells were cultured in iPSC medium (Dulbecco’s Modified Eagle’s Medium/Ham’s F12 (Wako Pure Chemical Industries (Wako), Osaka, Japan) containing 20% knockout serum replacement (Invitrogen, Carlsbad, CA, USA), 2 mM l-glutamine (Wako), 1% minimal essential medium with non-essential amino acids (Invitrogen), and 0.1 mM β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA)) at 37℃ in 5% CO₂ for 6 days. Then the culture medium was changed to EC medium (Human Endothelial-SFM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 1% platelet-poor plasma derived bovine serum (PDS) (Alfa Aesar, Haverhill, MA, USA), 20 ng/mL FGF2, and 10 µM all-trans retinoic acid (RA) (Tocris Bioscience, Bristol, UK)). After 2 days, the cell were detached by Accutase (Nacalai Tesque, Kyoto, Japan) and re-prated on 0.3-cm² Transwelll-Clear permeable inserts (0.4 µm pore size, Sabeu GmbH & CO. KG, Northeim, Germany) coated with a mixture of fibronectin (100 µg/mL; Wako) and collagen IV (400 µg/mL; Nitta gelatin, Osaka, Japan). These cells were then used to construct the in vitro BBB model.