Endogro mv complete culture media kit

hCMEC/D3 immortalized human cerebral microvascular endothelial cell line was purchased from Millipore Sigma and maintained using EndoGRO‐MV Complete Culture Media Kit supplemented with 1 ng/ml human animal‐free basic fibroblast growth factor (bFGF‐AF) and 1% Penicillin–Streptomycin. Tissue culture flasks were coated with 1:20 dilution of Corning® Collagen Type I, Rat Tail, at 37°C for 1 hr prior to use. Cells were incubated at 37°C, 95% humidity, and 5% CO₂ until confluent. All cells used were between passages 28 and 35.
Idealized coculture microfluidic devices made up the scaffold for the μHuB and were purchased from SynVivo, Inc. (Huntsville, AL). Figure 1a shows the device schematic. Devices were prepared as previously described.44 Briefly, devices were coated with 300 μg/ml human fibronectin for 1 hr, then perfused and primed with nitrogen gas to remove bubbles. Cells were then injected at ~5 × 10⁷ cells/ml, the device was inverted, and cells were allowed to adhere to the upper polydimethylsiloxane (PDMS) surface. This process was then repeated with the device in an upright position. Cells were fed daily by perfusion of media. A linear ramping protocol (100 nl/min to 5 μl/min over 12 hr) was utilized to condition the cells to physiological shear stress.

Human Cerebral Microvascular Endothelial Cell (hCMEC) were purchased from Cellutions Biosystems Inc.(Ontario, Canada) and were passed in endothelial cell growth medium EndoGRO-MV Complete Culture Media Kit (Millipore) supplemented with human basic fibroblast growth factor (Sigma) and penicillin-streptomycin (Life technologies) [47 (link)]. To generate stable IRS-1-overexpressing hCMEC, a vector pTagRFE-N harboring IRS-1cDNA was transfected into 5 × 10⁵ hCMEC cells using lipofectamine 2000 reagent (Invitrogen) at 80% confluence for 48 h. Different clones with IRS-1 overexpression were isolated after G418 selection (500ug/mL, Gibco) of the transfected cells over two weeks, and validated by immunoblotting.