4.2. hCMEC/D3 Cell Culture

The human brain endothelial cell line hCMEC/D3, an immortalized suitable in vitro model for BBB, was kindly provided by Dr. Pierre-Olivier Couraud, Institut Cochin, Paris. hCMEC/D3 was cultured following the protocol previously reported [17,18]. Cells were plated on rat tail collagen I (Life Technologies, Carlsbad, CA, USA) pre-coated Petri dishes (Life Technologies, USA), or Lab-Tek chamber slides (Dutscher, Bernolsheim, France). The cells were seeded at a density of 25,000 cells per cm² and grown until the cells reached 90 to 95% confluence.

Cells were cultured at 37 °C, 5% CO₂ atmosphere in EBM-2 basal medium (Lonza) containing 5% fetal calf serum (FCS), 10-mM HEPES (Dutscher, Bernolsheim, France), 1 ng/mL of basic fibroblast growth factor (bFGF) (Sigma-Aldrich, St. Louis, MO, USA), hydrocortisone (1.4 µM), ascorbic acid (5 µg/mL), chemically defined lipid concentrate (CONC, Life Technologies, USA), and 1% antibiotics (penicillin–streptomycin). The medium was supplemented with 10 mM lithium chloride and replaced every 3 to 4 days. The cells became completely confluent within 4 days. All experiments were performed on cultures of confluent cells between passage 29 and 35. Periodically, the culture was tested for mycoplasma contamination using the MycoProbe^® Mycoplasma Detection Kit (Catalog # CUL001B, R&D Systems, Abingdon, UK).