Alternatives to dimethylsulfoxide for serum-free cryopreservation of human mesenchymal stem cells
Alternatives to dimethylsulfoxide for serum-free cryopreservation of human mesenchymal stem cells
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DOI：
10.1177/039139881003300605
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发表时间：
2010-06-01
影响因子：
1.7
通讯作者：
Czermak, Peter
中科院分区：
文献类型：
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作者：
Grein, Tanja A.;Freimark, Denise;Czermak, Peter
人骨髓间充质干细胞（human mesenchymal stem cell，hMSCs）具有可塑性强、多向分化潜能大、体外培养操作简便等特点，在临床和治疗中具有广泛的应用前景。对于常规应用，这些细胞必须储存一段时间而不丧失细胞活力和功能。保存细胞的一种简单方法是将它们储存在-80摄氏度和-196摄氏度之间的温度（液氮）。为了防止细胞受到冷冻保存过程造成的损害，并实现高细胞回收率和活力，使用冷冻保护剂。通常二甲亚砜，通常与血清组合，用作冷冻保护剂。然而，对于临床方法，由于几个原因，在患者中使用二甲亚砜和血清是有问题的。因此，在没有二甲亚砜和血清的情况下，用于细胞治疗应用的人间充质干细胞的冷冻保存需要调查。在这项工作中，无毒的替代二甲亚砜，如甘油或相容的溶质，脯氨酸和依克多因，在无血清的冷冻介质中分析其冷冻保护性能。研究了不同浓度的冷冻保护剂（分别为1-10%（w/v）依克多因或脯氨酸，或5-20%（v/v）甘油）和特定孵育时间（0-60分钟）对解冻后细胞活力和细胞生长的影响。我们的结果表明，在一般情况下，冷冻保存与依克多因导致高解冻后细胞存活率高达72%，而冷冻保存后与甘油和脯氨酸，hMSC细胞完全死亡（甘油）或只有差的细胞存活（脯氨酸，22%）。此外，形态学的hMSC细胞改变为一个大的和扁平的表型后，冷冻保存与脯氨酸。这些结果表明甘油和脯氨酸不适合于hMSC的冷冻保存。相比之下，依克多因有可能取代二甲基亚砜作为无血清冷冻介质中的冷冻保护剂。
Human mesenchymal stem cells (hMSCs) have some favorable characteristics like high plasticity, multilineage differentiation potential, and comparably easy handling in vitro, making them of interest for many clinical and therapeutic approaches including cell therapy. For routine applications, these cells have to be stored over a certain period of time without loss of cell vitality and function. An easy way to preserve cells is to store them at temperatures between -80 degrees C and -196 degrees C (liquid nitrogen). To prevent cells from the damage caused by the cryopreservation process and to achieve high cell recovery and vitality, cryoprotectants are used. Typically dimethylsulfoxide, often in combination with serum, is used as a cryoprotectant. However, for clinical approaches, the use of dimethylsulfoxide and serum in patients is problematic for several reasons. Therefore, the cryopreservation of human mesenchymal stem cells for cell therapeutic applications without dimethylsulfoxide and serum demands investigation. In this work, non-toxic alternatives to dimethylsulfoxide such as glycerol or the compatible solutes, proline and ectoin, were analyzed in a serum-free cryomedium with respect to their cryoprotective properties. Different concentrations of the cryoprotectants (1-10% (w/v) ectoin or proline, respectively, or 5-20% (v/v) glycerol) and certain incubation times (0-60 minutes) were investigated with regard to post-thaw cell vitality and cell growth. Our results showed that, in general, cryopreservation with ectoin led to high post-thaw cell survival of up to 72% whereas after cryopreservation with glycerol and proline, the hMSC cells were completely dead (glycerol) or had only poor cell survival (proline, 22%). Moreover, the morphology of the hMSC cells changed to a large and flat phenotype after cryopreservation with proline. These results indicate that glycerol and proline are not suitable for cryopreservation of hMSC. In contrast, ectoin has the potential to replace dimethylsulfoxide as a cryoprotectant in a serum-free cryomedium.