Figure 1.
Platelets reduce leakage of small molecules from 3D HUVEC vessels. (A) Vessel formation in the 2-lane OrganoPlate. (i) The 2-lane OrganoPlate consists of 96 chips in a 384-well microplate format. Each chip has 2 lanes separated by a phase guide which supports patterning of ECM and cells via inlets and outlets. (ii) Addition of ECM into the bottom channel forms a barrier against which cells can be seeded in the top channel. (iii) HUVEC were seeded into the top channel and cultured for 1 week. Vessels were fixed and then stained with Hoechst-33342 (blue), α-CD31 (red), and phalloidin-FITC to identify actin (green). Images were taken on a Leica SP5 confocal microscope. 3D reconstructions of Z-stacks were produced in FIJI. The PhaseGuide is autofluorescent at 405 nm, and therefore is visible as a blue cuboid structure adjacent to the vessel. (B) Platelets reduce permeability of unstimulated vessels. Vessels were cultured as described for panel A, against Geltrex mix (i) or collagen I (ii). Platelets (plts) and neutrophils (neuts) were isolated from human blood and resuspended in EGM-FCS. They were then perfused through the vessels for 1.5 hours. FITC-dextran (250 kDa) was added for the last 45 minutes and then fluorescence was imaged on an EVOS M5000. Dextran leakage was calculated by dividing the fluorescence in the ECM channel by the fluorescence in the vessel channel. Data were then normalized to the media only control. (C) Platelet adherence is not observed in unstimulated vessels. After the process described in panel B, vessels were fixed, blocked, and stained with Hoechst-33342 (blue), α-CD41a (green), and α-CD31 (red). Z-stacks were taken on a Leica SP5 confocal microscope. (D) Collagen I vessels are leakier to RBCs than Geltrex mix vessels. After neutrophil and platelet perfusion, RBCs were added to the vessels for 30 minutes. RBC leakage was imaged using an iPhone through a bright-field microscope and quantified in FIJI. N = 5 independent experiments per blood donor (N = 4 in panel Ci), n = 2 chips per condition. Mean ± standard error of the mean (SEM); ∗P < .05 and ∗∗P < .01. Scale bars are representative of 50 μm.

Platelets reduce leakage of small molecules from 3D HUVEC vessels. (A) Vessel formation in the 2-lane OrganoPlate. (i) The 2-lane OrganoPlate consists of 96 chips in a 384-well microplate format. Each chip has 2 lanes separated by a phase guide which supports patterning of ECM and cells via inlets and outlets. (ii) Addition of ECM into the bottom channel forms a barrier against which cells can be seeded in the top channel. (iii) HUVEC were seeded into the top channel and cultured for 1 week. Vessels were fixed and then stained with Hoechst-33342 (blue), α-CD31 (red), and phalloidin-FITC to identify actin (green). Images were taken on a Leica SP5 confocal microscope. 3D reconstructions of Z-stacks were produced in FIJI. The PhaseGuide is autofluorescent at 405 nm, and therefore is visible as a blue cuboid structure adjacent to the vessel. (B) Platelets reduce permeability of unstimulated vessels. Vessels were cultured as described for panel A, against Geltrex mix (i) or collagen I (ii). Platelets (plts) and neutrophils (neuts) were isolated from human blood and resuspended in EGM-FCS. They were then perfused through the vessels for 1.5 hours. FITC-dextran (250 kDa) was added for the last 45 minutes and then fluorescence was imaged on an EVOS M5000. Dextran leakage was calculated by dividing the fluorescence in the ECM channel by the fluorescence in the vessel channel. Data were then normalized to the media only control. (C) Platelet adherence is not observed in unstimulated vessels. After the process described in panel B, vessels were fixed, blocked, and stained with Hoechst-33342 (blue), α-CD41a (green), and α-CD31 (red). Z-stacks were taken on a Leica SP5 confocal microscope. (D) Collagen I vessels are leakier to RBCs than Geltrex mix vessels. After neutrophil and platelet perfusion, RBCs were added to the vessels for 30 minutes. RBC leakage was imaged using an iPhone through a bright-field microscope and quantified in FIJI. N = 5 independent experiments per blood donor (N = 4 in panel Ci), n = 2 chips per condition. Mean ± standard error of the mean (SEM); ∗P < .05 and ∗∗P < .01. Scale bars are representative of 50 μm.

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