Figure 4.
Platelets preserve integrity of angiogenic vessels. (A) Perfusable angiogenic vessels can be induced into collagen I ECM. Vessels were cultured against Geltrex mix (i) or collagen I (ii) for 2 days in a 3-lane OrganoPlate before addition of an angiogenesis-inducing cocktail of vascular endothelial growth factor (VEGF), S1P, basic fibroblast growth factor (bFGF), and PMA to the bottom channel for 8 days. Vessels were fixed, blocked, and stained with Hoechst-33342 (blue), phalloidin-FITC (green), and α-CD31 (red). Z-stacks were taken on a Leica SP5 confocal microscope. (iii) After 8 days of angiogenesis induction, collagen I vessels were perfused with 250 kDa FITC-dextran (green) or RBCs for 45 minutes and imaged on an EVOS M5000/bright-field microscope. (B) Platelets reduce angiogenic vessel permeability. Platelets were isolated from human blood and resuspended in EGM-FCS. They were then perfused through the vessels for 1.5 hours 250 kDa FITC-dextran was added to the vessels for the last 45 minutes of platelet perfusion, and then fluorescence imaged on an EVOS M5000. Dextran leakage was calculated by dividing the fluorescence in the ECM by the fluorescence in the vessel. Data were then normalized to the media only control. (C) Platelets prevent RBC leakage from angiogenic vessels. (i) Vessels were prestained with CellMask Deep Red Plasma Membrane stain (red). After the process described in panel B, RBCs were added to the vessels for 30 minutes. Vessels were then fixed, blocked, and stained with Hoechst-33342 (blue), and α-CD235a (green). Z-stacks were taken on a Leica SP5 confocal microscope. (ii) RBC leakage was quantified in FIJI. (D) Platelets bind singly to angiogenic vessels. Vessels (red) were fixed, blocked, and stained with Hoechst-33342 (blue), and α-CD41a (green). Z-stacks were taken on a Leica SP5 confocal microscope. 3D reconstructions were generated in FIJI. N = 4 independent experiments per blood donor, n = 3 chips per condition. Mean ± SEM; ∗P < .05 and ∗∗P < .01. Scale bars are representative of 50 μm.

Platelets preserve integrity of angiogenic vessels. (A) Perfusable angiogenic vessels can be induced into collagen I ECM. Vessels were cultured against Geltrex mix (i) or collagen I (ii) for 2 days in a 3-lane OrganoPlate before addition of an angiogenesis-inducing cocktail of vascular endothelial growth factor (VEGF), S1P, basic fibroblast growth factor (bFGF), and PMA to the bottom channel for 8 days. Vessels were fixed, blocked, and stained with Hoechst-33342 (blue), phalloidin-FITC (green), and α-CD31 (red). Z-stacks were taken on a Leica SP5 confocal microscope. (iii) After 8 days of angiogenesis induction, collagen I vessels were perfused with 250 kDa FITC-dextran (green) or RBCs for 45 minutes and imaged on an EVOS M5000/bright-field microscope. (B) Platelets reduce angiogenic vessel permeability. Platelets were isolated from human blood and resuspended in EGM-FCS. They were then perfused through the vessels for 1.5 hours 250 kDa FITC-dextran was added to the vessels for the last 45 minutes of platelet perfusion, and then fluorescence imaged on an EVOS M5000. Dextran leakage was calculated by dividing the fluorescence in the ECM by the fluorescence in the vessel. Data were then normalized to the media only control. (C) Platelets prevent RBC leakage from angiogenic vessels. (i) Vessels were prestained with CellMask Deep Red Plasma Membrane stain (red). After the process described in panel B, RBCs were added to the vessels for 30 minutes. Vessels were then fixed, blocked, and stained with Hoechst-33342 (blue), and α-CD235a (green). Z-stacks were taken on a Leica SP5 confocal microscope. (ii) RBC leakage was quantified in FIJI. (D) Platelets bind singly to angiogenic vessels. Vessels (red) were fixed, blocked, and stained with Hoechst-33342 (blue), and α-CD41a (green). Z-stacks were taken on a Leica SP5 confocal microscope. 3D reconstructions were generated in FIJI. N = 4 independent experiments per blood donor, n = 3 chips per condition. Mean ± SEM; ∗P < .05 and ∗∗P < .01. Scale bars are representative of 50 μm.

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