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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 the current cryopreservation of cells and to establish an efficient cryopreservation method without dimethyl sulfoxide (DMSO) and serum (FBS) to provide high quality cells for clinical practice such as cell therapy. Methods: Alginate microencapsulated rat embryo fibroblasts (STO cells) were cryopreserved with cryopreservation solution without DMSO and FBS. Four control groups were set up: groups with 10% DMSO and 20% FBS, groups with 10% DMSO only, groups with 20% FBS alone, DMSO and FBS. The cells in each experimental group were stained with trypan blue before and after freezing, and the cell count was counted to calculate the cell viability. At the same time, the cells were stained with ethylenediamine dimer (EthD) and Calcein-AM (Calcein-AM) Morphology, and further verify the cell survival rate; Thawing and resuscitation MTT method to assess the proliferation rate and cell viability. Results: After 30 days of cryopreservation, the cell number, cell viability, cell morphology and cell viability after thawing and thawing were compared. The results showed that the cell number, cell viability, cell morphology, There was no significant difference in growth vigor between the groups supplemented with DMSO and FBS, but significant differences with the other three control groups. Conclusion: The use of alginate microcapsules instead of DMSO and FBS for the preservation of STO cells can effectively maintain cell morphology, number and survival rate without affecting the cell viability. Thus, a high-efficiency cryopreservation method without DMSO and FBS was established.