Isolation and Characterization of Human Bone Marrow-Derived Mesenchymal Stromal Cells

Human bone marrow (BM) was obtained from the iliac crest of healthy donors (N = 7) that were either subjects in a clinical trial or undergoing orthopaedic surgery for other than BM related problems. The unused surplus of mononuclear cell fraction obtained from BM after erythrocyte depletion using Gelofusine (B. Braun, Melsungen AG, Germany) sedimentation were supplied by Bioinova, Ltd. (Prague, Czech Republic) and processed as described previously^{59 (link),60 (link)}. Mononuclear cells were cultured in a complete culture medium (CCM) containing α-MEM (LONZA, Basel, Switzerland), 5% pooled PL (Bioinova, Ltd.), and 10 µg/ml gentamicin (Sandoz, Holzkirchen, Germany) at 37 °C, in a humidified atmosphere, containing 5% CO₂ with regular media changes twice a week. After reaching near-confluence, cells were harvested using the 0.05% Trypsin/EDTA solution (Life Technologies, Carlsbad, CA, USA) and reseeded onto a fresh plastic surface (Nunc, Roskilde, Denmark) at a density of 5 × 10³ cells/cm². Cells derived at passage 3 were used for the evaluation of immunophenotype, differentiation properties, gene expression and secretome profile in normoxic and hypoxic (5% O₂) conditions.