Endothelial tube formation assay

The pro-angiogenic properties of exosomes released from HDFs grown on 3D-SFnws scaffolds were assessed using HDMVECs and the PromoKine Angiogenesis Assay Kit (Cat. No. PK-CA577-K905, PromoCell, Germany) according to the manufacturer’s instructions. Briefly, HDMVECs were grown at 37°C in air with CO₂ 5% v/v until reaching about 90% confluency in endothelial cell growth basal medium (Cat. No C-22210, PromoCell, Germany) with added supplement mix (Cat. No C-39215, PromoCell, Germany). Next, HDMVECs were harvested using trypsin 0.025% v/v and resuspended in endothelial cell growth basal medium fortified with 2.5% v/v exosome-depleted FBS. Then, an aliquot (50 μL) of ECM solution was added to each well of a 96-well sterile cell culture plate kept on ice that was thereafter incubated at 37°C to form a gel. After that, 20 × 10³ HDMVECs suspended in 100 μL culture medium were mixed with different concentrations (i.e. 0.625, 1.25, 2.5, 5.0 and 10.0 μg/ml) of exosomes from 3D-SFnws-adhering HDFs or with no exosomes (controls on polystyrene ± ECM gel) and directly added to each well. Finally, the plates were incubated at 37°C in air with CO₂ 5% v/v for 5 hours. HDMVECs were checked and photographed at ×400 magnification under an IM35 microscope with an Olympus DP10 digital camera (Olympus, Japan). The mean tube lengths in μm per microscopic field at ×100 magnification were quantified via morphometric methods [30]. Triplicate results were averaged and expressed as the means ± SDs.