Bone marrow-derived mesenchymal stem cells (University of Cambridge)

Bone-marrow mononuclear cells were separated by density gradient centrifugation in Ficoll-Paque™ PREMIUM. Washed cells were re-suspended in PBS/EDTA and cultured in Dulbecco's modified Eagle's low glucose medium supplemented with foetal bovine serum (10%), and plated at a density of 1 × 10⁸ cells per cell-factory. Cultures were maintained in cell factories, at 37°C in a humidified atmosphere containing CO₂ (5%). When the cultures were near confluence (>80%), cells were detached, by treatment with trypsin-EDTA (0.25%), and re-plated at 3.5 × 10⁶ cells per cell factory. When 2 × 10⁶ cells/kg patient body weight, or more, were obtained, MSCs were harvested and cryopreserved in human albumin solution (4.5%) with dimethyl sulphoxide (10%).