Collagenase 4

For endothelial cell isolation the resected specimens containing normal or malignant tissue were dissociated using a standardised, semi-automated protocol based on a combination of mechanical tissue disruption and collagenase IV digestion, using a gentleMACS dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). Briefly, tissues were cut in small pieces (2–4 mm) in a petri dish prior to collection in a dedicated gentleMACS tube, to which 10 ml of RPMI 1640 (Lonza, BioWhittaker, Walkersville, MD, USA) with 1% L-Glutamin, 1% Penicillin/Streptomycin, 10% heat-inactivated FBS (Sigma Aldrich, St Louis, MO, USA), as well as collagenase IV (Sigma Aldrich), 1 mg ml⁻¹, and DNAse I (Sigma Aldrich), 10 μg ml⁻¹, had been added. The tissues were mechanically minced on the gentleMACS after incubation for 30 min at 37 °C in a shaking water-bath. This procedure was repeated once. After the second dissociation step at the gentleMACS, the cell suspension was passed through a 70 μm Falcon cell strainer (BD Biosciences, San Jose, CA, USA). The cell suspension was then collected in a 50 ml conical tube, and centrifuged at 1000 g for 5 min. The resulting pellet was washed with 50 ml phosphate buffered saline (PBS) and concentrated at 2 × 10⁶ cells ml⁻¹ in PBS for flow cytometry to detect and characterise the tissue-derived ECs.