Differentiation of Primary Human Endothelial Cells

Primary human Endothelial cells (phECs) were differentiated from CD34⁺ cells isolated from human cord blood as previously described [24 (link)] and were provided in frozen aliquots of 1 million cells at passage 5 by Prof. Gosselet, Université d´Artois, France. After thawing, cells were seeded onto gelatine (0.2% in PBS) coated 10 cm-dishes in ECM-5 medium (ECM from Sciencell with 5 mL of Endothelial cell growth supplement (ECGS), 2.5 mL of Gentamycin 10 mg/mL (BiochromAG, Ref. A-2712) and 25 mL of pre-selected, heat-inactivated FBS; and cultivated at 37 °C, 5% CO₂. When cells reached confluency, they were washed three times with PBS, detached with Trypsin/EDTA solution, counted and re-seeded at a concentration of ∼20 000 cells/cm². Expression of EC marker CD31 was confirmed by flow cytometry and immunofluorescence.