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  • br BBB physiological structure hBMVECs were cultured under a flu


    BBB physiological structure. hBMVECs were cultured under a flu-
    idic shear of 0.1 ll/min, which was introduced by continuous per-fusion of cell culture medium on the vascular channel. To evaluate the BBB structure, we quantified TJ formation through permeation studies and protein analyses of the cultured hBMVECs. Permeation studies were conducted by tracking FITC (fluorescein isothio-cyanate) conjugated-dextran, while protein analyses were per-formed using immunofluorescence staining.
    Permeability of the endothelial barrier against small com-pounds was assessed by detecting the fluorescence intensity of FITC-dextran (40 kDa, Sigma-Aldrich, St. Louis, MO, USA). After the formation of the barrier, FITC-dextran (0.5 mg/ml) was added the chip to diffuse across the BBB with a flow rate of 0.1 ll/min on the vascular channel. Time-course images of FITC-dextran per-meation on the chip were collected, and the absolute fluorescence intensities of FITC from the vascular channel and three random regions of interest (ROIs) in the Fluorouracil side were quantified using the Image J software (National Institutes of Health, USA). The ROIs were of equal sizes and evenly distributed along the circular curves as shown in Fig. 2E. To evaluate and compare the permeability of BBB, FITC-dextran permeation was quantitated by calculating the ratio of the fluorescence intensities on the brain side to that of the vascular channel, while the fluorescence intensities of the vas-cular channel were defined as 100% for standardization.
    2.2.5. Transendothelial electric resistance (TEER) analysis
    Electrical resistance was measured as described previously
    [19,22]. The chip was balanced to room temperature and con-nected with a Cell Resistance Analyzer (Millicell ERS-2, Merck, USA) according to the manufacturer’s instructions. Each resistance 
    value (R) was read three times. The resistance of ECM-coated only was detected as the initial background resistance (R0) in the same way. The resistance value was normalized for area (A), where the cell culture area of the BBB was 1.884 10 3 cm2, and was calcu-lated in X cm2 as following equation (1):
    2.2.6. Immunofluorescence staining
    After washing in phosphate buffered saline (PBS) 3 times, hBMVECs were fixed in 4% paraformaldehyde (Beijing Solarbio Science & Technology Co., Ltd., China) for 10 min and permeabi-lized in 0.2% Triton X-100 solution (Sigma, USA) for 15 min. The samples were then blocked with goat serum (Sigma, USA) and incubated with antibodies against VE-Cadherin (1:100 dilution; Abcam, UK), ZO-1 (1:100 dilution; Abcam, UK), CD31 (1:100 dilu-tion; Abcam, UK), E-cad (1:100 dilution; Proteintech, USA), N-cad (1:100 dilution; Proteintech, USA), Vimentin (1:100 dilution; Proteintech, USA), a-SMA (1:100 dilution; Proteintech, USA), and GFAP (1:100 dilution; Proteintech, USA) and AKR1B10 (1:100 dilu-tion; Abcam, UK) separately at 4 LC overnight. To detect the tar-geted proteins, samples were incubated with FITC-conjugated secondary antibodies (1:100 dilution; Proteintech, USA) for 1 h at room temperature. Actin filaments (F-actin) were identified by incubation with rhodamine phalloidin (1:1,000; Sigma, USA) for 1 h at room temperature. Cell nuclei were stained with 1 lg/ml DAPI (40,6-diamidino-2-phenylindole; Sigma, USA) for 10 min at room temperature. Images were obtained with an Olympus IX81 fluorescence microscope (Olympus Corporation, Japan) and an Leica TCS SP5II confocal microscopy (Leica, German), and analyzed using Image J software.
    2.2.7. Monitoring the extravasation of tumor cells through the BBB To visually monitor the trans-endothelial process, we used PC9
    cells stably expressing GFP and the red live labeled endothelial cells. GFP-positive PC9 cells were generated by transfection of GFP-geneticin plasmid using the polybrene (Sigma, USA) reagent, and subsequent resistance selection with G418 (TransGen Biotech, China). hBMVECs were labeled with the Cell TrackerTM CM-Dil dye (Invitrogen, USA) according to the manufacturer’s instructions. The growth, transition, and invasion of lung cancer cells on the upper stream ‘‘lung” were determined as previously reported [35]. Once the GFP-PC9 cells invaded the circulation and were transported to the vascular downstream channel along with the fluid cell medium, the syringe pump was turned off to allow the metastatic cells to attach to and trans-migrate through the BBB. Extravasation events were observed using an inverted fluorescent microscope at time points of 0, 18, 24, 36, and 48 h. The observa-tion starting time point 0 h was designated as the time when the first cell reached the downstream vascular channel along with the fluid.