Osmometric and permeability characteristics of human placental/umbilical cord blood CD34+ cells and their application to cryopreservation
Osmometric and permeability characteristics of human placental/umbilical cord blood CD34+ cells and their application to cryopreservation
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DOI：
10.1089/152581600319379
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发表时间：
2000-04-01
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影响因子：
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通讯作者：
Critser, JK
中科院分区：
文献类型：
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作者：
Woods, EJ;Liu, J;Critser, JK
胎盘/脐带血来源的HPC的移植（例如，CD 34（+）细胞）已成为一种有用的治疗恶性和非恶性疾病的广谱。高效冷冻保存这种细胞类型的能力为脐带血移植增加了相当大的灵活性。本研究的目的是了解这些细胞的基本低温生物学因素，包括渗透/渗透特性，并使用理论方法来优化冷冻程序。为此，使用改进的库尔特计数器（Coulter Electronics，Inc.，希亚利亚，佛罗里达州）在22摄氏度。此外，使用集落形成试验评估PCB CD 34（+）细胞的渗透耐受性。这些实验确定的参数用于数学模型中，以预测最佳的冷冻保护剂添加和去除程序。结果表明，对于DMSO或丙二醇，V-b为0.32 x V-iso，平均L-p为0.17 +/- 0.03（μ m/min/atm +/- SD），P-CPA分别为0.94 +/- 0.004或1.0 +/- 0.004 cm/min（x10（-3））。所用的两种冷冻保护剂之间没有显著差异。渗透耐受限度确定为200和600 mOsm/kg（分别为1.29和0.62 x V-iso）。这些结果表明，对Rubinstein等，Proc Natl Acad Sci USA 92：10119-10122，1995）的广泛使用的脐带血CD 34（+）细胞冷冻保存方法进行修改的潜在益处。与Rubinstein的方法（在15分钟间隔内将DMSO加入冷却的细胞悬液中）相反，我们的数据表明，通过在环境温度下在5分钟内引入和去除冷冻保护剂可以获得更好的结果，从而通过避免来自渗透压休克的不必要风险来增加活力，并简化方案。此外，在实际冷冻和解冻过程中，丙二醇替代DMSO可能是有益的。
The transplantation of placental/cord blood-derived HPC (e.g., CD34(+) cells) has become a useful treatment for a broad spectrum of malignant and nonmalignant diseases. The ability to cryopreserve this cell type with high efficiency adds considerable flexibility to cord blood transplantation. The purpose of this study was to develop an understanding of the fundamental cryobiologic factors of these cells, including the osmotic/permeability characteristics, and to use a theoretical approach to optimize freezing procedures. To that end, biophysical parameters, including the osmotically inactive cell volume (V-b), hydraulic conductivity (L-p), and cryoprotectant permeability coefficient (P-CPA) for DMSO and propylene glycol were measured using a modified Coulter Counter (Coulter Electronics, Inc., Hialeah, FL) at 22 degrees C. In addition, the osmotic tolerance of PCB CD34(+) cells was assessed using a colony-forming assay. These experimentally determined parameters were used in a mathematical model to predict optimal cryoprotectant addition and removal procedures. The results demonstrate a V-b, of 0.32 x V-iso, an average L-p of 0.17 +/- 0.03 (mu m/min/atm +/- SD), and a P-CPA of 0.94 +/- 0.004 or 1.0 +/- 0.004 cm/min (x10(-3)) for DMSO or propylene glycol, respectively. No significant difference was determined between the two cryoprotectants used. The osmotic tolerance limits were determined to be 200 and 600 mOsm/kg (1.29 and 0.62 x V-iso, respectively). These results indicate potential benefits of modifications to the widely used method of Rubinstein et al. Proc Natl Acad Sci USA 92:10119-10122, 1995) for cord blood CD34(+) cell cryopreservation. As opposed to Rubinstein's method in which DMSO is added to cooled cell suspensions over a 15-min interval, our data indicate that better results may be obtained by introducing and removing the cryoprotectant at ambient temperature over 5 min both to increase viability by avoiding unnecessary risks from osmotic shock and to simplify the protocol. In addition, substitution of propylene glycol for DMSO may be of benefit during the actual freezing and thawing process.