Biocoll density gradient centrifugation

Human bone marrow was harvested from the iliac crest of 2 young healthy donors between 20 and 30 years old. 40 mL bone marrow aspirate were collected in a syringe containing 10,000 IU heparin to prevent coagulation. The mononuclear cell fraction was isolated by Biocoll density-gradient centrifugation (d = 1.077 g/cm³; Biochrom, Berlin, Germany). Cells were plated on fibronectin (10 ng/mL, Sigma, Taufkirchen, Germany)-coated cell culture flasks and selected by plastic adherence upon culture in MSC-NH expansion medium (Miltenyi Biotec, Bergisch Gladbach, Germany).

Following overnight fasting, venous blood from healthy volunteers (up to 50 mL) was drawn in EDTA tubes and processed within 2 hours from sampling. Peripheral blood mononuclear cells (PBMCs) were fractionated using Biocoll density-gradient centrifugation (Biochrom AG; density = 1.077 g/mL). PBMCs were seeded on 2 μg/cm² fibronectin coated culture dishes (BD Falcon) or Lab-Tek II chamber slides system (Sigma-Aldrich, Ltd., Poole, Dorset, UK) after erythrocytes lysis. Cells were cultured in endothelial basal medium (EBM-2, Lonza Sales AG, Basel, Switzerland) supplemented with EGM-2-MV-SingleQuots containing human endothelial growth factor, hydrocortisone, insulin-like growth factor, fibroblast growth factor, vascular endothelial growth factor (VEGF), antibiotics, and 5% fetal bovine serum (FBS, Lonza Sales AG). After 3-day culture, nonadherent cells were discarded by washing with PBS and the culture medium was replenished daily. Finally, on day 5, adherent cells, displaying an elongated spindle-shaped morphology, were identified as “early” EPCs.

Following overnight fasting, venous blood from healthy volunteers was drawn in EDTA tubes and processed within 2 hours from collection. Peripheral blood mononuclear cells (PBMCs) were fractionated using Biocoll density-gradient centrifugation (Biochrom AG; density = 1.077 g/ml). PBMCs (1×10⁶ cells/cm²) were seeded on 2 µg/cm² fibronectin coated culture dishes (BD Falcon) or Lab-Tek II chamber slides system (Sigma-Aldrich Ltd, Poole, Dorset, UK) after red cell lysis. Cells were cultured in endothelial basal medium (EBM-2, Lonza Sales AG, Basel, Switzerland) supplemented with EGM-2-MV-SingleQuots containing human endothelial growth factor, hydrocortisone, insulin-like growth factor, fibroblast growth factor, vascular endothelial growth factor (VEGF), antibiotics and 5% fetal bovine serum (FBS, Lonza Sales AG). After 3 days culture, non-adherent cells were discarded by washing with PBS and the culture medium replenished daily. On day 5, adherent cells, displaying an elongated spindle-shaped morphology, were identified as early EPCs.