Cilloni D, Garau D, Regazzi E, Sammarelli G, Savoldo B, Caramatti C, Mangoni L, Rizzoli V, Carlo-Stella C
Department of Hematology, University of Parma, Italy.
Bone Marrow Transplant. 1999 Mar;23(5):497-503. doi: 10.1038/sj.bmt.1701601.
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 degrees 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: 0.3x10(9) vs. 0.2x10(9), P< or =0.02; bags: 14x10(9) vs. 11x10(9), P< or =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< or =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. 7.4x10(5)), but no significant loss of BFU-E (180 vs. 150x10(5)), CFU-GM (400 vs. 290x10(5)) and LTC-IC (15 vs. 16x10(5)) could be demonstrated. When these patients were reinfused with uncontrolled-rate cryopreserved PBPC, the mean number of days to reach 1x10(9)/l white blood cells and 50x10(9)/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.
非控速冷冻技术是一种颇具吸引力的替代方法，可替代耗时且需要高水平技术专长的控速冷冻保存程序。在本研究中，我们报告了使用非控速冷冻保存及在-140℃的机械冷冻柜储存的经验。来自23名患者的28份外周血祖细胞样本（10个冻存管，18个冷冻袋）在由磷酸盐缓冲盐水（80%，v/v）补充人血清白蛋白（10%，v/v）和二甲基亚砜（10%，v/v）组成的冷冻保护剂溶液中进行冷冻保存。冷冻保存程序平均需要1.5小时。冻存管和冷冻袋的平均（±标准误）储存时间分别为344±40天和299±57天。尽管细胞解冻与有核细胞绝对数量的统计学显著减少相关（冻存管：0.3×10⁹对0.2×10⁹，P≤0.02；冷冻袋：14×10⁹对11×10⁹，P≤0.0003），但定向祖细胞的生长基本上不受冻融程序的影响，CFU-Mix、BFU-E和CFU-GM的平均回收率在60±29%至134±15%之间。来自冻存管和冷冻袋的长期培养启动细胞（LTC-IC）的平均回收率分别为262±101%和155±27%（P≤0.2）。在23名接受大剂量化疗和外周血祖细胞回输的患者中，对14名患者冷冻保存于冷冻袋中的造血祖细胞冷冻前后的绝对数量进行了比较。检测到CFU-Mix有显著减少（11对7.4×10⁵），但未显示BFU-E（180对150×10⁵）、CFU-GM（400对290×10⁵）和LTC-IC（15对16×10⁵）有显著损失。当这些患者接受非控速冷冻保存的外周血祖细胞回输时，达到1×10⁹/L白细胞和50×10⁹/L血小板的平均天数分别为9天和13天。总之，此处描述的程序具有执行时间短的特点，能使原始祖细胞和定向祖细胞大量恢复，并且即使在长期储存后，与清髓性治疗后的快速造血恢复相关。
