Fig. 2
Assessment of barrier function in TY10 brain endothelial microvessels. a Schematic representation of the barrier integrity assay. A perfused TY10 microvessel is grown against an ECM gel. A fluorescent dye is added to the medium inlets and outlets and perfused through the lumen of the microvessel. In case of a leaktight vessel, all dye is retained in the vessel. In case of a leaky vessel, the dye leaks into the adjacent gel channel. b Fluorescent dye distribution for a TY10 microvessel (top image) and a cell-free control (middle image) during a barrier integrity assay (FITC-dextran, 20 kDa, t = 30 min). To quantify leakage of the fluorescent dye in a chip, the fluorescence intensity measured in the medium channel and the gel channel (FluoMed and FluoGel, respectively) are measured over time (bottom image). c For each condition the ratio between the fluorescence signals measured in the two channels is plotted over time. In case of a leaktight TY10 microvessel, the ratio FluoGel/FluoMed is constant, resulting in a flat horizontal line. In case of a leaky microvessel or a cell-free control, the fluorescence signal measured in the gel channel increases over time, resulting in an increase in the ratio FluoGel/FluoMed. d TY10 microvessels were grown under perfusion or static conditions for 7 days, after which barrier integrity assays (for FITC-dextran, 20 kDa) were performed. n = 6 for TY10 microvessels, n = 2 for cell-free controls. Error bars show standard deviation of the mean. e Assessment of barrier function (for FITC-dextran, 20 kDa) of TY10 microvessels cultured under perfusion for 7 days in various commercially available cell culture media. n = 7 for all conditions. Error bars show standard deviation of the mean