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VALIDATION OF AN ALTERNATIVE METHOD FOR FREEZING HUMAN HEMATOPOIETIC CELLS IN CASES WHERE A PROGRAMMABLE FREEZER IS NOT AVAILABLE. G. Cameron, C. Robertson, H. Nakamoto, M. Taller, S. Abraham, C. Eaves. Terry Fox Laboratory, BC Cancer Agency, Vancouver, BC, Canada.
Cryopreservation of primitive hematopoietic cells is a critical part of most autologous transplant procedures. Much effort over the last 3 decades has led to the identification of conditions that allow a high recovery after thawing of viable primitive cells. The standard procedure in use at our centre involves first adding an equal volume of sterile 20% dimethyl sulfoxide (DMSO) in PlasmaLyte A® to the cell suspension and then immediately transferring the cells to a Kryo 10 controlled rate freezer preprogrammed to decrease the temperature 1°C/minute to -60°C and then 5°C/minute to -90°C. The cells are then placed directly in a liquid nitrogen freezer. Upon rapid thawing, patients’ cells frozen according to this procedure show a viability of 92±7% (n=151) as assessed in colony-forming cell (CFC) assays (for CFU-GM, BFU-E and CFU-GEMM). To evaluate an alternative freezing strategy in case of failure of the Kryo 10 freezer, the following study was undertaken. Parallel test vials of cells from patients’ bone marrow (n=3) or mobilized peripheral blood stem cells (n=2) in the final DMSO solution were placed in a Styrofoam container in a -86°C freezer for a minimum of 2 hours. These vials were then transferred to the same liquid nitrogen freezer as the control vials frozen using the Kryo 10 freezer. CFC and 6-week long term culture-initiating cell (LTC-IC) assays were then performed on the test and control cells thawed simultaneously. CFC and LTC-IC values in the test-frozen vials were 94±11% and 138±62% of control values. These findings suggest that the alternative method described here is suitable for freezing progenitor cells for use in clinical protocols.