Dulbecco s modified eagle s medium ham s f12

Primary neuronal cultures were prepared from the cortices of embryonic day 17 C57BL/6 mouse embryos.^{52 (link)} Briefly, cortices were dissected and the meninges were freed. The cortical fragments were incubated with 0.25% trypsin and 20 µg/ml DNase I in phosphate-buffered saline at 37 °C for 15 min, dissociated into single cells by pipetting, and then resuspended in serum-free Dulbecco’s modified Eagle’s medium/Ham’s F-12 (Wako Pure Chemical Industries, Osaka, Japan) supplemented with N2 Supplement with Transferrin (Wako Pure Chemical Industries, Osaka, Japan) supplemented with N2 Supplement with Transferrin (Wako Pure Chemical Industries, Osaka, Japan).^{53 (link)} Primary neuronal cells were plated in poly-ethylene-imine-coated 12-well plates at a density of 1 × 10⁶ cells per well. The neurons were stimulated with or without 0.1, 1, 10 µg/ml of P. gingivalis-derived LPS for 24 h. Then levels of Aβ40 and Aβ42 in the media were measured by an ELISA (Wako Pure Chemical Industries).

Human iPSCs (610B1, 606A1 and 648A1) were purchased from RIKEN (Saitama, Japan) and were maintained on a feeder layer of mitomycin C-treated mouse embryonic fibroblasts 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), 0.1 mM β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA), and 5 ng/mL human fibroblast growth factor-2 (FGF-2) (GenScript, Nanjing, China)] at 37 °C in 5% CO₂.

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.