Histological and Immunofluorescence Characterization of Engineered Vessels

For histological characterization, engineered vessels were fixed in formalin (Fisher), dehydrated through a graded ethanol series using a Sakura Tissue Processor (Sakura), embedded in paraffin and sectioned at 7 μm thickness. Sections were deparaffinised, rehydrated through a graded ethanol series and stained using haematoxylin & eosin (SigmaAldrich) following the manufacture’s instructions. For immunofluorescence analyses, tissues were cryopreserved in Cryomatrix (ThermoFisher) and sectioned at 20 μm thickness using a cryotome (Leica). Immunostaining was performed as described [17 (link)] using antibodies specific for the EC marker CD31 (clone JC/70A Biolegend, 1:50) and α-smooth muscle actin (clone 1A4 SigmaAldrich, 1:200). Sections were counter-stained with DAPI and imaged with an inverted microscope (Carl Zeiss). Endothelium barrier integrity was analyzed by injecting Evans blue (Sigma Aldrich) at a final concentration of 0.5% in the circulation loop of the bioreactor for 10 min followed by continuous PBS washing for 20 min. Vessels were cut open longitudinally and en face preparations were analysed macroscopically with photo documentation.