CD133 stem/progenitor cells were isolated by magnetic separation with ferrite-conjugated antibody from bone marrow aspirates, one day before coronary artery bypass graft (CABG).
After density centrifugation, cells were transferred into a Cobe 2991 cell processor. The cells were washed twice with PBS containing 5% human serum albumin (HSA). After washing, the cell suspension was concentrated to a volume of 85 mL. Then the AC133 reagent (Miltenyi Biotec) was added to the cell processing bag for 30 minutes of incubation. The cell suspension was washed again twice with PBS with HSA and adjusted to a final volume of 150 mL. Samples were drawn again for quantification of stem cell number and viability to monitor the performance of the cell labeling procedure. CD133 cells were isolated with a CliniMACS Magnetic Cell Separation device. The cell product was processed according to the standard program for CD133 cell selection with maximum cell numbers of 6x10^10 leukocytes and 6x10^8 CD133 target cells. The CliniMACS column yielded a purified CD133 cell product suspended in approximately 70 mL PBS with HSA. After calculation of the number of viable stem cells, the CD133-enriched cell suspension was centrifuged for 10 minutes at 200g, resuspended in PBS with HSA, and then adjusted to a cell concentration according to the Fibonacci dose escalation scheme used in the safety study protocol. The cells were aliquoted into 2-mL vials.