Optimization of the freezing process for hematopoietic progenitor cells: effect of precooling, initial dimethyl sulfoxide concentration, freezing program, and storage in vapor-phase or liquid nitrogen on in vitro white blood cell quality.

Abstract

Adding dimethyl sulfoxide (DMSO) to hematopoietic progenitor cells (HPCs) causes an exothermic reaction, potentially affecting their viability. The freezing method might also influence this. The aim was to investigate the effect of 1) precooling of DMSO and plasma (D/P) and white blood cell (WBC)-enriched product, 2) DMSO concentration of D/P, 3) freezing program, and 4) storage method on WBC quality. WBC-enriched product without CD34+ cells was used instead of HPCs. This was divided into six or eight portions. D/P (20 or 50%; precooled or room temperature [RT]) was added to the WBC-enriched product (precooled or RT), resulting in 10% DMSO, while monitoring temperature. The product was frozen using controlled-rate freezing ("fast-rate" or "slow-rate") and placed in vapor-phase or liquid nitrogen. After thawing, WBC recovery and viability were determined. Temperature increased most for precooled D/P to precooled WBC-enriched product, without influence of 20 or 50% D/P, but remained for all variations below 30°C. WBC recovery for both freezing programs was more than 95%. Recovery of WBC viability was higher for slow-rate freezing compared to fast-rate freezing (74% vs. 61%; p < 0.05) and also for 50% compared to 20% D/P (two test variations). Effect of precooling D/P or WBC-enriched product and of storage in vapor-phase or liquid nitrogen was marginal. Based on these results, precooling is not necessary. Fifty percent D/P is preferred over 20% D/P. Slow-rate freezing is preferred over fast-rate freezing. For safety reasons storage in vapor-phase nitrogen is preferred over storage in liquid nitrogen. Additional testing using real HPCs might be necessary.