After the last passage of marrow or PBSC over the magnets, sterile, pyrogen-free DMSO (Cryoserv, Edwards Lifesciences, Irvine, CA) is diluted to 20% in L-15 medium with 1.5% hetastarch and 2.5% human serum albumin.

Cells are resuspended in L-15 medium with 2.5% human serum albumin at a concentration of 5-10 x 107/ml. The 20% DMSO solution (ice cold) is then added slowly to the cell suspension while the cells are on ice.

The cell suspension is then aliquoted into 100 ml Cryocyte freezing bags (Nexel). We always freeze in a minimum of 2 bags (usually 3) in case of breakage.

Several test vials of 2 mls are prepared for subsequent thaw-recovery analysis, and 2 to 4 vials are saved for future analyses that may contribute to the understanding of autologous PBSCT (e.g., more sensitive assay for tumor cells, assay, or future assays that may correlate with engraftment).

The vials and bags are frozen using a Cryo-Med freezing system (Mt. Clemens, Michigan) with a cooling program provided in hardware with the freezer. The cooling rate is -1°C/min, and the program maintains the cooling rate as close to -1°/min during phase change. The cooling rate is maintained at -2°/min after phase change until -40°C to avoid the damage shown to result from more rapid cooling rates after release of fusion heat. Frozen bags are placed into racks and stored in the vapor phase of a liquid nitrogen freezer ^(5,6).