side of the scaffolds. As a control, HUVECs were cultured in absence of SVPs ‘luminal’/Adh on both of scaffold. Quantification of endothelial cell nuclei showed that the combination of the presence of pericytes and the conjugates had a significantly higher impact on endothelial coverage, as compared to each of the factors alone (Figure 4). 2.4 Effect of pericytes on endothelial cell migration Pericytes are known to release a plethora of pro-angiogenic factors; hence we hypothesize that SVPs can improve the migration of endothelial cells. This is particularly relevant in the context of vascular grafting to encourage the migration of the endothelial cells from neighbouring vessels and reach the complete endothelialization of the lumen of the implanted scaffolds. Indeed, the transwell migration assay showed that the presence of pericytes in the lower chamber increased the migration of endothelial cells by 2.5 times in the 8 hours timeframe of the assay (Figure 5a and Figure 5b). Furthermore, we confirmed an increased wound closure by endothelial cells in a modified scratch assay performed in a co-culture system on PCL where the endothelial monolayer was interrupted by forming a gap with a polydimethylsiloxane (PDMS) mask (Figure 5c). Fluorescence imaging of the whole side of the scaffolds indicated increased ‘luminal’ presence of endothelial cells in the gap of the scaffolds pre-seeded with pericytes the (Figure 5d-g). Quantification of the mean fluorescence within the gap ‘abluminal/Hep’ showed an average increase of 67.6±20.8% in endothelial cell density (P<0.05; N = 3) in the SVP-seeded scaffolds. 2.5 Freezing of pericytes-coated scaffolds The effective deployment of the devised scaffold for the development of clinically relevant TEVG is highly dependent on its resilience to freezing/thawing for preservation/delivery
‘luminal’/Adh side of the scaffolds. As a control, HUVECs were cultured in absence of SVPs on both of scaffold. Quantification of endothelial cell nuclei showed that the combination of the presence of pericytes and the conjugates had a significantly higher impact on endothelial coverage, as compared to each of the factors alone (Figure 4). 2.4 Effect of pericytes on endothelial cell migration Pericytes are known to release a plethora of pro-angiogenic factors; hence we hypothesize that SVPs can improve the migration of endothelial cells. This is particularly relevant in the context of vascular grafting to encourage the migration of the endothelial cells from neighbouring vessels and reach the complete endothelialization of the lumen of the implanted scaffolds. Indeed, the transwell migration assay showed that the presence of pericytes in the lower chamber increased the migration of endothelial cells by 2.5 times in the 8 hours timeframe of the assay (Figure 5a and Figure 5b). Furthermore, we confirmed an increased wound closure by endothelial cells in a modified scratch assay performed in a co-culture system on PCL where the endothelial monolayer was interrupted by forming a gap with a polydimethylsiloxane (PDMS) mask (Figure 5c). Fluorescence imaging of the whole ‘luminal’ side of the scaffolds indicated increased presence of endothelial cells in the gap of the scaffolds pre-seeded with pericytes the ‘abluminal/Hep’ (Figure 5d-g). Quantification of the mean fluorescence within the gap showed an average increase of 67.6±20.8% in endothelial cell density (P<0.05; N = 3) in the SVP-seeded scaffolds. 2.5 Freezing of pericytes-coated scaffolds The effective deployment of the devised scaffold for the development of clinically relevant TEVG is highly dependent on its resilience to freezing/thawing for preservation/delivery