Ware Carol B, Nelson Angelique M, Blau C Anthony
Department of Comparative Medicine, University of Washington, Seattle, WA 98195-7190, USA.
Biotechniques. 2005 Jun;38(6):879-80, 882-3. doi: 10.2144/05386ST01.
A significant obstacle to using human embryonic stem cells (hESCs) arises from extremely poor survival associated with freezing, typically in the range of 1%. This report describes a slow controlled-rate freezing technique commonly used for mammalian embryo cryopreservation. Using a combination of surviving colony number and colony diameter; survival was determined relative to untreated hESCs. Using a dimethyl sulfoxide (DMSO) cryoprotectant and either a homemade controlled-rate freezing device or a commercial freezing device, survival rates of 20%-80% were obtained. To achieve the highest levels of survival, the critical factors were an ice crystal seed (at -7 degrees to -10 degrees C), a freeze rate between 0.3 degrees and 1.8 degrees C/min, and a rapid thaw rate using room temperature water. Slow controlled-rate cooling allows a rapid, simple, and reproducible means of cryopreserving hESCs.
使用人类胚胎干细胞（hESCs）的一个重大障碍源于与冷冻相关的极低存活率，通常在1%的范围内。本报告描述了一种常用于哺乳动物胚胎冷冻保存的慢速控制速率冷冻技术。结合存活集落数和集落直径，相对于未处理的hESCs确定存活率。使用二甲基亚砜（DMSO）冷冻保护剂以及自制的控制速率冷冻装置或商业冷冻装置，获得了20% - 80%的存活率。为了实现最高水平的存活，关键因素是冰晶种子（在 -7摄氏度至 -10摄氏度）、0.3摄氏度至1.8摄氏度/分钟的冷冻速率以及使用室温水解冻的快速解冻速率。慢速控制速率冷却为冷冻保存hESCs提供了一种快速、简单且可重复的方法。
