Isolation and Characterization of Primary HUVECs

Umbilical cords were collected and stored at 4 °C and processed within 24 of collection. Primary female HUVECs (FHUVECs) and male HUVEC (MHUVECs) were isolated by collagenase treatment (Sigma-Aldrich, Milano, Italy), as previously described by Addis et al. [25 (link)] and cultured in plates pre-coated with 1% gelatine (Sigma-Aldrich, Milano, Italy) in an M199 medium (Life Technologies, Monza, Italy) supplemented with 10% fetal bovine serum (FBS) (Life Technologies, Monza, Italy), 10% newborn calf serum (NBCS) (Life Technologies, Monza, Italy), 1% antibiotic/antimycotic (Sigma-Aldrich, Milano, Italy) and 2 mM of L-glutamine (Sigma-Aldrich, Milano, Italy), until confluence, in a 5% CO2 humidified atmosphere. As previously described (Addis), cultured cells were characterized as endothelial cells by the exhibition of cobblestone morphology when they were contact-inhibited and by an evaluation of the expression of von Willebrand factor, a glycoprotein that is constitutively stored in intra-endothelial Weibel–Palade granules.
FHUVECs and MHUVECs were used in passage 3 to ensure their endothelial characteristics, and all experiments were conducted in duplicate or triplicate.