Cryopreservation of peripheral blood progenitor cells: characteristics of suitable techniques

The influence of 6 different cooling rates (1.4, 5, 10, 15, 20, 160 K/min) and 5 different compositions of the suspensions to be frozen (% DMSO/% HES: 10/0, 7.5/2.5, 5/6, 2.5/7.5, 0/10) were investigated in 30 aliquots from each of 12 peripheral blood progenitor cells (PBSC) products which had been obtained by leukapheresis from 11 patients with hematological malignancies and solid tumors and 1 healthy donor treated with 5-24 micrograms/kg body weight/day granulocyte colony stimulating factor (G-CSF) over 5 days. The MNC concentration in the products obtained amounted to 4.51 +/- 1.55 x 10(7)/ml (mean +/- SEM), range: 2.10-7.65 x 10(7)/ml). For freezing, cooling rates were generated by means of a liquid nitrogen(LN2)-operated, computer-controlled freezer, a mechanical -80 degrees C freezer, in the vapor phase over LN2, and by submerging into LN2. The statistical analysis of the results clearly indicates that optimum results compared with the prefreeze values for numerical recovery (80.6 +/- 20.1%, Coulter counter), viability (membrane integrity by Trypan blue exclusion 91.6 +/- 4.1%), and colony-forming potential (56.2 +/- 45.8% erythroid burst-forming units [BFU-E], 63.4 +/- 72.8% myeloid colonies [CFU-GM]) were achieved at a cooling rate of 1.4 K/min with 10% DMSO being present. The values obtained at a cooling rate of 5 K/min (-80 degrees C mechanical refrigerator) in the presence of 5% DMSO and 6% HES did not differ significantly (i.e., membrane integrity 91.8 +/- 3.9%, BFU-E 53.0 +/- 37.4%, CFU-GM 47.8 +/- 58.2%). At cooling rates above 5 K/min and DMSO concentrations lower than 5% (in spite of higher HES concentrations) there was a significant drop of CFU recovery (CFU-GM plus BFU-E) to almost 0%. In parallel, numerical recovery and viability dropped as well, but less pronounced, indicating that both methods are unsuitable for the prediction of CFU recovery when different freezing protocols are applied. We need at least 5% DMSO (in the presence of 6% HES) in spite of the undesirable histamine-releasing effect of this compound. The cooling rate is not critical in the range from 1 to 5 K/min and can easily be achieved by -80 degrees C freezers.