Efficient method for slow cryopreservation of human embryonic stem cells in xeno-free conditions
Efficient method for slow cryopreservation of human embryonic stem cells in xeno-free conditions
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DOI：
10.1016/s1472-6483(10)60302-1
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发表时间：
2008-07-01
影响因子：
4
通讯作者：
Simon, Carlos
中科院分区：
文献类型：
--
作者：
Valbuena, Diana;Sanchez-Luengo, Silvia;Simon, Carlos
对于任何具有临床应用前景的人类胚胎干细胞(HESC)实验室来说，有效、一致和无异种的冷冻保存技术是至关重要的。这项研究提出了一种新的无血清条件下的缓慢冷冻-快速解冻方法，允许在不使用可编程冰柜的情况下对大量克隆进行冷冻保存。为了验证其有效性，将该方法与现有的两种玻璃化冷冻方法进行了比较，并应用于三种不同的hESC株(H9、Val-3和Val-5)。该方法基于增加二甲基亚砜的浓度(1.0、1.2、1.5和2.0mol/L)，并采用缓慢或快速冷却系统。用这种方法，每个冷冻瓶大约可以冷冻60个克隆，根据所用细胞系的不同，存活率在15%到68%之间，大多数存活的克隆是A级克隆。解冻后，细胞保留了hESC固有的未分化特性和生长速度曲线，注射到严重联合免疫缺陷动物体内，核型、端粒酶活性和畸胎瘤形成稳定，与新鲜品系相当。该方法已经在两个不同的实验室进行了三年的测试。
An effective, consistent and xeno-free cryopreservation technique is crucial for any human embryonic stem cell (hESC) laboratory with future perspectives for clinical application. This study presents a new slow freezing-rapid thawing method in serum-free conditions that allows the cryopreservation of a large number of colonies without the use of a programmable freezer. To test its efficacy, this method has been compared with two established vitrification methods and applied to three different hESC lines (H9, VAL-3 and VAL-5). The method is based on an increasing concentration of dimethylsulphoxide (1.0, 1.2, 1.5 and 2.0 mol/l) with a slow or a rapid cooling system. Using this method, approximately 60 colonies per cryovial could be cryopreserved, the survival rate ranged between 15 and 68% depending on the cell line used, and the majority of the surviving colonies were grade A. Post-cryopreserved hESC have been cultured for 20 passages, re-cryopreserved and re-thawed with consistent results. After thawing, cells retained the inherent undifferentiated characteristics of hESC :and growth rate curve, with a stable karyotype, telomerase activity and teratoma formation when injected into severe combined immunodeficient animals, which was comparable with the fresh lines. This method has been tested for 3 years in two different laboratories.