海藻酸微囊替代DMSO和FBS的STO细胞冷冻保存研究

Research on Alginate Microcapsules Replacing DMSO and FBS on the Cryopreservation of STO Cells

目的:改进现有的细胞冷冻保存方法,建立一个不含二甲基亚砜(DMSO)和血清(FBS)的高效冷冻保存方法,为细胞治疗等临床实践提供优质细胞。方法:海藻酸微囊包埋鼠胚成纤维细胞(STO细胞)后用不含DMSO和FBS的冷冻保存液进行冷冻保存。设四个对照组:添加10%DMSO和20%FBS的组、仅添加10%DMSO的组、仅添加20%FBS、DMSO和FBS均不添加组。在冷冻前后对各实验组细胞用台盼兰染色,进行细胞计数,计算细胞存活率,同时利用溴乙锭的二聚物(EthD)、钙黄绿素-AM(Calcein-AM)进行染色观察细胞的形态,且进一步验证细胞存活率;解冻复苏后用MTT法评估细胞的增殖速度和生长活力。结果:冷冻保存30天后对各组的细胞数量、细胞存活率、细胞形态和解冻复苏后细胞的生长活力进行比较发现,海藻酸微囊包埋冷冻组的细胞数、细胞存活率、细胞形态和生长活力均与添加DMSO和FBS的组之间无显著性差异,而与其它三个对照组呈显著性差异。结论:使用海藻酸微囊替代DMSO和FBS保存STO细胞,能有效的维持细胞形态、数量、存活率,同时不影响细胞的生长活力,从而建立了一个不含DMSO和FBS的高效冷冻保存方法。

Objective： To improve cryopreservation method and to establish an efficient cryopreservation system without DMSO and FBS, in order to provide high-quality cells for cell-therapy and other clinical applications. Methods： STO cells were embedded into alginate microCapsules and cryopreserved in freezing solution. Four groups of cell suspension were set up as control： one group with 10 % DMSO and 20 % FBS, one group only with l 0 % DMSO, one group only with 20% FBS, and one group without DMSO and FBS. Cell number and the survival rate of STO cells were counted by trypan blue staining, cell morphology and the survival were assayed by double staining using EthD and Calcein-AM. Cell proliferation rate and growth activity was evaluated by using Methabenzthiazuron （MTT） assay. Results： For 30 days cryopreservation, then thawed the STO cells and analyzed cell number, cell viability and growth activity of five groups. The results showed that there was no significant difference between the group of alginate microcapsules embedded cells and the group with DMSO and FBS after cryopreserving and thawing. Conclusion： Alginate microcapsules replacing DMSO and FBS in the cryopreservation of STO cells can effectively maintaining cell morphology, cell number, viability and growth activity were not affected. An efficient cryopreservation system without DMSO and FBS was established.