Bone marrow-derived mononuclear cells (Barts & The London NHS Trust)

Bone marrow was aspirated (50 ml) from three separate sites in the right iliac crest and equally aliquoted into 10 x 10 ml heparin-treated syringes. The entire volume (50 ml) was passed through a blood component transfusion set with a 200 um filter. 25 ml was layered on 25 ml of Ficoll-Paque (density gradient: 1.077 [+ or -] 0.001 g/ml) and the remaining 25 ml were layered on 25 ml of Lymphoprep (density gradient: 1.077 [+ or -] 0.001 g/ml). Both preparations were centrifuged in a Sorvall RT Legend centrifuge at 2500 rpm for 30 min with the brake off. The bone marrow-derived mononuclear cell layer from each preparation was extracted and spun again at 2500 rpm for 10 min with the brake off. The resulting cell pellets were resuspended and underwent two cycles of washing in 0.9% saline. The final cell pellets were resuspended in 2 ml autologous serum.