Development and Preliminary Testing of Porcine Blood-Derived Endothelial-like Cells for Vascular Tissue Engineering Applications: Protocol Optimisation and Seeding of Decellularised Human Saphenous Veins

Abstract

Functional endothelial cells (EC) are a critical interface between blood vessels and the thrombogenic flowing blood. Disruption of this layer can lead to early thrombosis, inflammation, vessel restenosis, and, following coronary (CABG) or peripheral (PABG) artery bypass graft surgery, vein graft failure. Blood-derived ECs have shown potential for vascular tissue engineering applications. Here, we show the development and preliminary testing of a method for deriving porcine endothelial-like cells from blood obtained under clinical conditions for use in translational research. The derived cells show cobblestone morphology and expression of EC markers, similar to those seen in isolated porcine aortic ECs (PAEC), and when exposed to increasing shear stress, they remain viable and show mRNA expression of EC markers similar to PAEC. In addition, we confirm the feasibility of seeding endothelial-like cells onto a decellularised human vein scaffold with approximately 90% lumen coverage at lower passages, and show that increasing cell passage results in reduced endothelial coverage.

Keywords: bioengineering; cell seeding; endothelial colony forming cells; endothelium; vascular graft.

Figure A1

Colony forming ability of endothelial-like cells (ELC) at (a) passage 3, and (b) passage 4. Phase contrast images. Scale bars are 1000 μm.

Figure 1

Representative images of porcine endothelial-like cells (ELC; (a–e)) and porcine aortic endothelial cells (PAEC; (g–k)) stained for common endothelial cell markers (green): CD31 (a,g), VE-Cadherin (b,h), DBA-Lectin (c,i), von Willebrand factor (vWF, (d,j)), and vimentin (e,k); (f) phase contrast image of ELC showing cobblestone morphology. Markers of interest are shown in green. Nuclei are stained with DAPI (blue). White bars represent 200 μm. The black bar in (f) represents 400 μm.

Figure 2

Representative images of porcine blood outgrowth cells (BOC) stained for common endothelial cell markers (green; (a–e)) and mesenchymal cell markers (red; (f,g)): (a) CD31, (b) VE-Cadherin, (c) DBA-Lectin, (d) von Willebrand factor (vWF), (e) vimentin, (f) CD90/Thy-1, (g) α-smooth muscle actin, (h) phase contrast image of blood outgrowth cells showing spindle-like morphology. Nuclei are stained with DAPI (blue). All bars represent 400 μm.

Figure 3

mRNA expression (arbitrary units) of endothelial cell markers CD31 (a), VE-Cadherin (b), endothelial nitric oxide synthase (eNOS) (c) and von willebrand factor (vWF) (d) of porcine endothelial-like cells (ELC) and aortic endothelial cells (PAEC) under different flow conditions on the orbital shaker. Error bars are standard errors of the mean.

Figure 4

Seeding endothelial-like cells (ELC) onto decellularised human saphenous vein. (a) Percentage of lumen covered by ELC following 96 h on a roller at 1 rpm and a further 72 h in static culture in (a) all seeded vessels and (b) lumen coverage for each cell passage used. Error bars indicate standard error of the mean. Representative serial section images of ELC on lumen, stained with (c,f) DBA-Lectin (green) and DAPI (blue), (d) CD31 (dark brown), and (e) H&E for nuclei. (f) Unseeded decellularised human saphenous vein. Black and white scale bars represent 100 μm.