Ficoll paque gradient

Whole blood was collected from trial participants and controls, diluted in an equal volume of PBS (1:1 ratio), and loaded onto a Ficoll-Paque gradient (VWR Scientific) at a 2:1 blood:Ficoll ratio. Gradients were centrifuged for 30 minutes at 540g. Peripheral blood mononuclear cells (PBMCs) were aspirated and washed in PBS prior to cryopreservation.

The FDA encouraged development of a culture protocol without FBS. Thus marrow from the initial five control donors was divided prior to MNC isolation to allow parallel MSC expansion in either hMSC media or Mosaic XF medium (Becton Dickinson), a serum/xeno-free medium. For Mosaic XF expansion, the marrow suspension was diluted 1:1 in PBS and the mixture was loaded onto a Ficoll-Paque gradient (VWR Scientific) at a 2:1 ratio and centrifuged for 30 minutes at 540g. MNCs were washed with DPBS. For cell culture using the Mosaic medium, flasks were coated with a collagen/fibronectin blend up to passage 1, and fibronectin alone thereafter. Cells were initially plated at 30 × 10³ cells/cm² in a 175 cm² flask containing Mosaic XF medium and processed as described above, but using this medium. Three control donors were aspirated after the manufacturer terminated production of this medium, so their aspirates were isolated and expanded as described for trial participants.