Grein Tanja A, Freimark Denise, Weber Christian, Hudel Klaus, Wallrapp Christine, Czermak Peter
University of Applied Sciences Giessen-Friedberg, Institute of Biopharmaceutical Technology, Giessen, Germany.
Int J Artif Organs. 2010 Jun;33(6):370-80.
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.
人骨髓间充质干细胞（hMSCs）具有一些良好的特性，如高可塑性、多向分化潜能以及在体外相对易于操作，这使得它们在包括细胞治疗在内的许多临床和治疗方法中备受关注。对于常规应用，这些细胞必须在一定时间内储存而不丧失细胞活力和功能。保存细胞的一种简便方法是将它们储存在-80℃至-196℃（液氮）之间的温度下。为防止细胞受到冷冻保存过程造成的损伤并实现高细胞回收率和活力，需使用冷冻保护剂。通常，二甲基亚砜常与血清联合用作冷冻保护剂。然而，对于临床应用，在患者中使用二甲基亚砜和血清存在诸多问题。因此，研究不使用二甲基亚砜和血清进行细胞治疗应用的人骨髓间充质干细胞的冷冻保存很有必要。在这项工作中，分析了二甲基亚砜的无毒替代物，如甘油或相容性溶质脯氨酸和四氢嘧啶，在无血清冷冻培养基中的冷冻保护特性。研究了不同浓度的冷冻保护剂（分别为1-10%（w/v）的四氢嘧啶或脯氨酸，或5-20%（v/v）的甘油）以及特定的孵育时间（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，总体而言，用四氢嘧啶冷冻保存可使解冻后细胞存活率高达72%，而用甘油和脯氨酸冷冻保存后，hMSC细胞完全死亡（甘油）或细胞存活率极低（脯氨酸，22%）。此外，用脯氨酸冷冻保存后，hMSC细胞的形态变为大而扁平的表型。这些结果表明，甘油和脯氨酸不适用于hMSC的冷冻保存。相比之下，四氢嘧啶有潜力在无血清冷冻培养基中替代二甲基亚砜作为冷冻保护剂。
