Wang S Y, Ho C K, Chen P M, Yung C H, Chong L L, Chen L Y
Cryobiology. 1987 Jun;24(3):229-37. doi: 10.1016/0011-2240(87)90025-3.
Two different cryogenic methods were used to study the preservation of murine bone marrow cells. Compared to the classical methods, in which separated mononuclear marrow cells in 10% dimethyl sulfoxide (DMSO) were cryopreserved in liquid nitrogen (-196 degrees C), a modified technique was carried out by cryopreservation of unfractionated marrow cells in a mixed protectant of 5% DMSO and 6% hydroxyethyl starch (HES) at -80 degrees C. Samples that were separately thawed after storage for 1, 4, 8, and 12 weeks were assayed for cell viability and recovery of CFU-GM and CFU-S. No macroscopic clumping of cells was noted either in fractionated or in unfractionated marrow cell cryopreservations. A mild damage, about 25% reduction of stem cells, was found at 1 week and did not deepen further. It seems that the greatest loss of stem cells occurred in the process of cryopreservation itself. Compared to prefreeze values, both a high number of cells that excluded trypan blue (87 +/- 3.4%) and a high recovery of CFU-GM (75 +/- 9.8%) and CFU-S (74 +/- 11.2) were observed in unfractionated marrow samples cryopreserved with the DMSO/HES mixture at -80 degrees C for 3 months and these results were very similar to those obtained from fractionated mononuclear marrow cells cryopreserved at -196 degrees C. The DMSO/HES protectant provides a simplified bone marrow cryopreservation technique that should be favorable to clinical application because of its high stem cell recovery and avoidance of cell-separation manipulation.
采用两种不同的低温保存方法来研究小鼠骨髓细胞的保存。与传统方法相比，传统方法是将分离的单核骨髓细胞置于10%二甲基亚砜（DMSO）中于液氮（-196℃）冷冻保存，而改良技术是将未分级的骨髓细胞置于5%DMSO和6%羟乙基淀粉（HES）的混合保护剂中于-80℃冷冻保存。分别在储存1、4、8和12周后解冻的样本进行细胞活力检测以及CFU-GM和CFU-S的回收率检测。在分级或未分级的骨髓细胞冷冻保存中均未观察到细胞的宏观聚集。在第1周发现有轻度损伤，干细胞减少约25%，且未进一步加重。似乎干细胞的最大损失发生在冷冻保存过程本身。与冷冻前的值相比，用DMSO/HES混合物在-80℃冷冻保存3个月的未分级骨髓样本中，观察到大量排斥台盼蓝的细胞（87±3.4%）以及CFU-GM（75±9.8%）和CFU-S（74±11.2）的高回收率，这些结果与在-196℃冷冻保存的分级单核骨髓细胞所获得的结果非常相似。DMSO/HES保护剂提供了一种简化的骨髓冷冻保存技术，因其高干细胞回收率和避免细胞分离操作，应该有利于临床应用。
