Gao D Y, Chang Q, Liu C, Farris K, Harvey K, McGann L E, English D, Jansen J, Critser J K
Cryobiology Research Institute, Methodist Hospital of Indiana, Inc., Indianapolis 46202, USA.
Cryobiology. 1998 Feb;36(1):40-8. doi: 10.1006/cryo.1997.2060.
While methods for the cryopreservation of hematopoietic stem cells are well established, new sources of progenitor cells, such as umbilical cord blood, fetal tissue, and ex vivo expanded progenitor cells, may require refined protocols to achieve optimal recovery after freezing. To predict optimal protocols for cryopreservation of human hematopoietic progenitors, knowledge of fundamental cryobiological characteristics including cell osmotic characteristics, water and cryoprotectant permeability coefficients of cell membrane, and activation energies of these coefficients is required. In this study, we used CD34+CD33- cells isolated from human bone marrow as hematopoietic progenitor cell models/representatives to study the osmotic characteristics of the progenitor cells. Volume distribution and osmotic behavior of the CD34+CD33- cells were determined using two different methods: (a) a shape-independent electronic sizing technique and (b) a shape-dependent optical image analysis. The cell diameter was measured to be 8.2 +/- 1.1 microns (mean +/- SD, n = 1,091,475, the number of donors = 8) using the electronic sizing technique or 8.7 +/- 1.2 microns (mean +/- SD, n = 1508, the number of donors = 6) by image analysis at initial (isotonic) osmolality, 325 mosm/kg. The cell volume change was measured after the cells were exposed and equilibrated to different anisosmotic conditions. The cell volume was found to be a linear function of the reciprocal of the extracellular osmolality (Boyle van't Hoff plot) ranging from 163 to 1505 mosm/kg. The volume fraction of intracellular water which is osmotically active was determined to be 79.5% of the cell volume. It was concluded that human CD34+CD33- cells osmotically behave as ideal osmometers. This information coupled with cell water and cryoprotectant permeability coefficients as well as their activation energies (to be determined in the ongoing research projects) will be used to design optimum conditions for cryopreservation of human hematopoietic progenitor cells.
虽然造血干细胞的冷冻保存方法已经成熟，但新的祖细胞来源，如脐带血、胎儿组织和体外扩增的祖细胞，可能需要完善的方案以在冷冻后实现最佳复苏。为了预测人类造血祖细胞冷冻保存的最佳方案，需要了解包括细胞渗透特性、细胞膜的水和冷冻保护剂渗透系数以及这些系数的活化能等基本低温生物学特性。在本研究中，我们使用从人骨髓中分离出的CD34+CD33-细胞作为造血祖细胞模型/代表来研究祖细胞的渗透特性。使用两种不同方法测定CD34+CD33-细胞的体积分布和渗透行为：(a)一种与形状无关的电子大小测量技术和(b)一种与形状有关的光学图像分析。在初始（等渗）渗透压为325 mosm/kg时，使用电子大小测量技术测得细胞直径为8.2±1.1微米（平均值±标准差，n = 1,091,475，供体数量 = 8），通过图像分析测得为8.7±1.2微米（平均值±标准差，n = 1508，供体数量 = 6）。在细胞暴露于不同的非等渗条件并达到平衡后，测量细胞体积变化。发现细胞体积是细胞外渗透压倒数的线性函数（玻意耳-范特霍夫图），范围为163至1505 mosm/kg。具有渗透活性的细胞内水的体积分数被确定为细胞体积的79.5%。得出的结论是，人类CD34+CD33-细胞在渗透方面表现为理想的渗透计。这些信息与细胞水和冷冻保护剂渗透系数及其活化能（将在正在进行的研究项目中确定）相结合，将用于设计人类造血祖细胞冷冻保存的最佳条件。
