Culturing hCMEC/D3 Cells for Barrier Assays

Cultures of hCMEC/D3 cells (≤P35) were grown in MCDB 131 medium (Pan-Biotech, Aidenbach, Germany) supplemented with 5% FBS, GlutaMAX (100×, Life Technologies, Carlsbad, CA, USA), lipid supplement (100×, Life Technologies, Carlsbad, CA, USA), 10 µg/mL ascorbic acid, 550 nM hydrocortisone, 100 µg/mL heparin, 1 ng/mL basic fibroblast growth factor (bFGF, Roche, San Francisco, CA, USA), 2.5 µg/mL insulin, 2.5 µg/mL transferrin, 2.5 ng/mL sodium selenite (ITS), and 50 µg/mL gentamicin [84 (link)]. All of the plastic surfaces were coated with 0.05% collagen in sterile distilled water before cell seeding and the medium was changed every 2 days. Before each experiment, the medium of hCMEC/D3 cells was supplemented with 10 mM LiCl for 24 h to improve the barrier properties [85 (link)].