Primitive hematopoietic progenitors within mobilized blood are spared by uncontrolled rate freezing

Abstract

Uncontrolled-rate freezing techniques represent an attractive alternative to controlled-rate cryopreservation procedures which are time-consuming and require high-level technical expertise. In this study, we report our experience using uncontrolled-rate cryopreservation and mechanical freezer storage at −140°C. Twenty-eight PBPC samples (10 cryovials, 18 freezing bags) from 23 patients were cryopreserved in a cryoprotectant solution composed of phosphate-buffered saline (80%, v/v) supplemented with human serum albumin (10%, v/v) and dimethylsulfoxide (10%, v/v). The cryopreservation procedure required on average 1.5 h. The mean (± s.e.m.) storage time of cryovials and bags was 344 ± 40 and 299 + 57 days, respectively. Although cell thawing was associated with a statistically significant reduction of the absolute number of nucleated cells (vials: 0.3 × 10⁹ vs 0.2 × 10⁹, P ⩽ 0.02; bags: 14 × 10⁹ vs 11 × 10⁹, P ⩽ 0.0003), the growth of committed progenitors was substantially unaffected by the freezing–thawing procedure, with mean recoveries of CFU-Mix, BFU-E, and CFU-GM ranging from 60 ± 29% to 134 ± 15%. Mean recoveries of LTC-IC from cryovials and bags were 262 ± 101% and 155 ± 27% (P ⩽ 0.2), respectively. In 14 out of 23 patients who underwent high-dose chemotherapy and PBPC reinfusion, the pre- and post-freezing absolute numbers of hematopoietic progenitors cryopreserved in bags were compared. A significant reduction was detected for CFU-Mix (11 vs 7.4 × 10⁵), but no significant loss of BFU-E (180 vs 150 × 10⁵), CFU-GM (400 vs 290 × 10⁵) and LTC-IC (15 vs 16 × 10⁵) could be demonstrated. When these patients were reinfused with uncontrolled-rate cryopreserved PBPC, the mean number of days to reach 1 × 10⁹/l white blood cells and 50 × 10⁹/l platelets were 9 and 13, respectively. In conclusion, the procedure described here is characterized by short execution time, allows a substantial recovery of primitive and committed progenitors and is associated with prompt hematopoietic recovery following myeloablative therapy even after long-term storage.