Figure 3

An in vivo-like barrier function in ECs cultured on an BM-COL1 layer was stably generated following the deposition of BM onto COL1 nanofibers.

(a) Absolute permeability values of EC monolayers in the COL1 system (left) and the synthetic endothelial ECM system (right) were measured by introducing growth medium supplemented with 10 μM FITC-dextran (40 kDa, green) into the EC channel and monitoring FITC fluorescence. Inset graphs show intensity profiles of each dotted line (blue dotted line, COL1; red dotted line, synthetic endothelial ECM) and white arrows indicate the direction of diffusion. The images were taken 2 hours after the introduction of FITC-dextran-supplemented growth medium. Scale bar, 100 μm. (b) Simulated diffusion profiles at 2 hours after the introduction of FITC-dextran-supplemented growth medium in the COL1 (blue line) and the synthetic endothelial ECM (red line) systems containing an EC monolayer on each gel surface. (c) The fluorescence microscopic image shows the diffusion of FITC-dextran from an EC channel to a side channel passing through the BM-COL1 layer in the absence of an EC monolayer. The image was taken 1 hour after the addition of FITC-dextran-supplemented growth medium. The inset graph indicates the intensity profile of the white dotted line. Scale bar, 100 μm. (d) Absolute vascular permeability values for the synthetic endothelial ECM system (red bars) were dramatically reduced compared with those for the COL1 (*P = 0.029) and approximated in vivo levels. Error bars, ± SEM (n = 4). (e) Absolute vascular permeability values for the MAT system were much higher than those for the synthetic endothelial ECM system (***P < 0.001 for the MAT on culture day4; **P = 0.002 for the MAT and **P = 0.005 for the COL1 on culture day5). Error bars, ± SEM (n = 8 ~ 24). (f) Absolute vascular permeability values of the synthetic endothelial ECM (BM-COL1), the COL1 (COL1) and the LN-coated COL1 (L-COL1) systems. For the generation of optimal layer for the L-COL1, we coated LNs onto the COL1 fibers for 20, 40, 60 and 80 min. in a humidified 37 °C incubator. Error bars, ± SEM (**P < 0.01 and ***P < 0.001, n = 5 ~ 18). (g) Levels of mRNA for the TJ proteins CLDN1, CLDN5 and OCLN in the COL1 (blue bars) and the synthetic endothelial ECM (red bars) systems, normalized to those of GAPDH, were acquired on culture days 1, 2 and 3 (*P = 0.016 and ***P < 0.001). Error bars, ± SEM (n = 3 ~ 4). (h–j) Expression levels of CLDN1, CLDN5 and OCLN proteins in the COL1 and the synthetic endothelial ECM systems on culture day5 were determined by fluorescence microscopy (green, CLDN1/CLDN5/OCLN; blue, nuclei). Scale bars, 50 μm.