Analysis of Treated and Cryopreserved Marrow and PBSC
for Hematopoietic Cells

The total number of viable cells and CFU-G,M that can be recovered from a cryopreserved test vial will be compared to the numbers that were frozen ^(5,6). Although the presence of adequate viable cells (>0.5 x 108 per kg) and CFU-G,M (>104 per kg) in the cryopreserved marrow does not insure that sufficient pluripotent stem cells are present, these are the most practical tests currently available, and several studies have correlated CFU-G,M in marrow with marrow after transplant. The total number of CD34+ cells will also be determined prior to cryopreservation (see below).

The 2 ml test vial is thawed rapidly in a 37°C water bath, and the contents are quickly transferred to a 15 ml plastic centrifuge tube. The cell suspension is diluted 3-fold with Iscove's DMEM medium containing 2% fetal bovine serum. Nucleated cell is counted with Turk's solution, while an additional count with trypan blue is used to assess cell viability. The viability determined by trypan blue counting is used in conjunction with the total number of cells frozen to calculate the viable total cells per kg (bone marrow) or in conjunction with CD34+ quantification by flow cytometry and the total number of cells frozen, to determine the viable CD34+ cells available for infusion.

CFU-G,M are assayed using 0.5% agar-Iscove's DMEM medium, and recombinant human GM-CSF is used to stimulate colony formation. Cells are cultured at a concentration of 2 x 105 cells/35 mm dish. Colonies of more than 50 cells are enumerated with a dissecting microscope after 12 days.