Human brain microvascular endothelial cells (dhBMECs) were differentiated from the BC1 human induced pluripotent cell (hiPSC) line (provided by Dr. Linzhao Cheng, Johns Hopkins University). Details of the differentiation and characterization of the hBMECs have been reported elsewhere [
4]. Briefly, all cells were cultured in T25 and T75 flasks (Falcon, Tewksbury, MA, USA) with daily media changes. BC1-hiPSCs were cultured in colonies on 40 µg mL
−1 Matrigel-treated tissue culture dishes (Corning, Tewksbury, MA, USA) and maintained in TeSR-E8 media, changed daily (Stem Cell Technologies, Vancouver, Canada). BC1-hiPSCs were passaged using StemPro
® Accutase
® solution (Life Technologies, Waltham, MA, USA). 10 µM ROCK inhibitor Y27632 (ATCC, Manassas, VA, USA) was included in the TeSR-E8 culture media for the first 24 h after passaging. After culture for 3–4 days in TeSR-E8, the media was switched to unconditioned media without basic fibroblast growth factor (bFGF) (UM/F- media) to induce the differentiation. The cells were maintained in this media for 6 days with daily media replacement. The UM/F- media is composed of DMEM/F12 (Life Technologies) supplemented with 20% KnockOut Serum Replacement (Life Technologies), 1% non-essential amino acids (Life Technologies), 0.5%
l-glutamine (Sigma-Aldrich, St. Louis, MO, USA), and 0.84 µM beta-mercaptoethanol (Life Technologies). The media was then switched to endothelial cell media (EC) for 2 days to promote growth of the endothelial cells. The EC media is composed of endothelial cell serum-free media (Life Technologies), supplemented with 1% human platelet poor derived serum (Sigma-Aldrich), 20 ng mL
−1 bFGF (R&D Systems), and 10 µM all-trans retinoic acid (Sigma-Aldrich). After 2 days in EC media, the cells were sub-cultured into the microfluidic devices.