脂肪间充质干细胞外泌体保存策略研究

On the Preservation Strategy of Exosomes Derived from Adipose Mesenchymal Stem Cells

对超高速离心法获得的脂肪间充质干细胞外泌体进行浓度、粒径、形态和蛋白标志物验证,分别研究-80℃条件下不同保存时间节点(1周、1月)外泌体和细胞样本浓度、粒径和蛋白释放浓度的变化,4种保存策略(-80℃冻存;添加物质的量浓度为25 mM的海藻糖,-80℃冻存;添加体积分数为10%的二甲基亚砜(Dimethylsulfoxide,简称DMSO),-80℃冻存;添加体积分数为30%的甘油,-80℃冻存)在不同时间节点(1周、1月)对外泌体浓度、粒径和蛋白释放浓度的影响以及快速冻融循环(3次)对外泌体浓度及粒径的影响。结果显示,脂肪间充质干细胞在上述存储条件下浓度、粒径和完整性都发生改变,实验应尽可能提取新鲜外泌体,当实验条件不完备时,可采用细胞冻存后回收外泌体的保存方式,但切忌反复冻融。

The concentration,particle size,morphology and protein markers of exosomes derived from adipose mesenchymal stem cells obtained through ultracentrifugation were verified to respectively study the change of both exosomes and cell samples in their concentration,particle size and protein concentration under the condition of-80℃in 2 different periods(1 week and 1 month),the effects of four storage strategies(-80℃storage;25mM trehalose and-80℃storage;DMSO(Dimethylsulfoxide)of 10%volume fraction and-80℃storage;glycerol of 30%volume fraction and-80℃storage)on the concentration and the particle size of exosomes and on the protein concentration in 2 different time periods(1 week and 1 month),and the effects of rapid freeze-thaw cycle(3 times)on the concentration and the particle size of exosomes.The results show that changes have taken place in the concentration,particle size and morphology of the exosomes under all the above-mentioned storage conditions.It is of great necessity to extract fresh exosomes for experiments as far as possible.If the experimental conditions are incomplete,recovering exosomes after cell cryopreservation can be adopted,but repeated freezing and thawing should be avoided.