Journal: bioRxiv

Article Title: Human iPSC-derived brain endothelial microvessels in a multi-well format enable permeability screens of anti-inflammatory drugs

doi: 10.1101/2021.05.03.442133

Figure Lengend Snippet: (A) Schemata of a single three-lane OrganoPlate® chamber containing a microvessel (red) inside the top channel. The microvessel grows in contact to the ECM gel channel (light blue). (B) Bright-field images of microvessels, 48 hrs after cell seeding. Representative images at 20 µm (vessel bottom) and 170 µm (vessel top) height from channel bottom are shown. Scalebar equals to 300 µm. (C) Immunocytochemistry of microvessels 48 hrs after cell seeding. Representative images (from the microvessel bottom) of tight-junction proteins (ZO-1, Occludin, Claudin-5), endothelial marker (VE-Cadherin, PECAM-1), nutrient transporter (Glut-1) and efflux transporter (P-gp, BCRP) expression are shown. Nuclei staining with Hoechst and cytoplasm staining with CellMask™ as indicated. Scalebar equals to 300 µm. (D-F) 3D imaging of microvessels stained with nuclei (Hoechst), tight-junction (ZO-1) and cytoplasm CellMask™ marker. (G) Subcellular localization of BCRP and Glut-1. Microvessel wall sections at the vessel/ECM contact are shown.

Article Snippet: Hoechst (Life Technologies), DRAQ5 (biostatus) or CellMask™ deep red stain (Life Technologies) was used according to manufacturer’s protocols.

Techniques: Immunocytochemistry, Marker, Expressing, Staining, Imaging

Journal: bioRxiv

Article Title: Human iPSC-derived brain endothelial microvessels in a multi-well format enable permeability screens of anti-inflammatory drugs

doi: 10.1101/2021.05.03.442133

Figure Lengend Snippet: (A) DEX-A647 mean intensity ratios (gel/top) over time with a different y-scale as depicted in . DEX-A647 leak-tight microvessels with a constant ratio over time (< 0.01) are shown (red line). Error bars indicate the SD (n=6). (B) Microvessel treated by 20 µM STS for 120 min were fixated by 4% PFA/PBS and stained by immunocytochemistry for nuclei (Hoechst), tight-junction marker (ZO-1) and cytoplasm marker (CellMask™). Loss of microvessel adhesion (yellow arrowheads) and vessel holes (white arrowheads) induced by STS are indicated. (C) NaF mean intensity ratios (gel/top) over time with a different y-scale as depicted in . NaF intensity ratios of preselected DEX-A647 leak-tight microvessels are shown (green line). Error bars indicate the SD (n=6). Slope ( m ) and R 2 from linear regression of data points are shown. (D) K-means cluster analysis by using tracer intensity data with 6 independent variables as described in the methods section. 2D scatterplot of principal component 1 (PC1) versus PC2 is indicated. Cluster members of cluster 1 are shown in red and members of cluster 2 in green. Leak-tight microvessels are shown as green dots which were defined after excluding those cluster members found at the outer boundaries of cluster 2 (fraction with a green cross). Leaky microvessels are indicated by a red or green cross. A total of n=36 microvessels were analyzed including n=10 control chambers without microvessel (no vessel). Chambers without a microvessel clustered without any exception into cluster 1 (leaky). From a total of 26 microvessels, 14 candidates were finally defined as leak-tight. (E-F) Layout and circuit of the customized microvessel TEER device. (G) Calibration and testing of the TEER device with known standard resistors. (H) Correlation of TEER versus NaF ratios measured in leak-tight microvessels after 150 min incubation. Green line indicates the linear regression of NaF/TEER data points. R 2 of the regression analysis and the calculated correlation coefficient r is indicated in the graph. (I) Average TEER of leak-tight microvessels calculated according to different NaF thresholds (R gel/top NaF 150min < 0.005 and R gel/top NaF 300min < 0.01). Error bars indicate the SD of n=6. (J) Scatter plot of average NaF (and DEX-A647) intensity ratios (on x-axis) versus average TEER (on y-axis). Data of leak-tight microvessels before (dots) and after 60 min STS treatment (triangles) are shown in the graph. Data from chambers without microvessel (no vessel) are shown as grey diamonds in comparison. Error bars show the SD of (n=2). Thresholds defined by K-mean clustering analysis were applied to distinguish between leak-tight and leaky microvessels by using NaF and DEX-A647 ratio data. Threshold for NaF (R gel/top NaF 300min < 0.02) indicated as green line and thresholds for DEX-A647 (R gel/top DEX 300min < 0.01) indicated as red line. By considering the fluorescent tracer data, previously leak-tight microvessels shifted into the leaky cluster after incubation with STS. The result was in line with a measured decrease in TEER after STS.

Article Snippet: Hoechst (Life Technologies), DRAQ5 (biostatus) or CellMask™ deep red stain (Life Technologies) was used according to manufacturer’s protocols.

Techniques: Staining, Immunocytochemistry, Marker, Incubation