不同冻存液对长江鲟鳍条细胞冷冻效果的影响

为了探究不同浓度配比冻存液对长江鲟(Acipenser dabryanus)鳍条细胞(Dabry′s sturgeon fin-derived cells,DSFCs)冷冻效果的影响,选择传代培养处于对数生长期的DSFCs,将第8代DSFCs置于液氮(-196℃)中冻存。根据冻存液中培养基(MEM)、胎牛血清(Fetal bovine serum,FBS)及二甲基亚砜(Dimethyl sulfoxide,DMSO)的配比不同,分为8个实验组:①含不同浓度DMSO配比实验组:5%DMSO+45%MEM+50%FBS,10%DMSO+40%MEM+50%FBS,20%DMSO+30%MEM+50%FBS;②含不同浓度FBS配比实验组:10%DMSO+90%FBS+0%MEM,10%DMSO+70%FBS+20%MEM,10%DMSO+50%FBS+40%MEM,10%DMSO+30%FBS+60%MEM,10%DMSO+10%FBS+80%MEM,对各组分采用形态学观察、CCK-8法、流式细胞仪检测细胞凋亡和细胞周期,综合分析不同冻存液组分对DSFCs冻存后活力的影响。结果显示,不同浓度DMSO配比实验组中,DMSO的浓度为20%时,细胞复苏后难以贴壁,存活率下降非常明显,仅为38.3%;而5%和10%DMSO细胞生长状态良好,存活率分别为80.2%和78.7%。不同浓度FBS配比实验组中,90%、30%和10%实验组细胞活力显著高于70%和50%实验组。FBS浓度为30%和10%的冻存组细胞凋亡率显著高于90%的冻存组(8.24%,15.35%vs 3.54%),3组之间相互比较有明显差异(P<0.05);细胞周期结果显示,与FBS浓度为90%的冻存组比较,FBS浓度为30%和10%冻存组G 0/G 1期细胞比例均明显增加(P<0.05),S期和G 2+M期细胞比例均显著降低(P<0.05),FBS浓度30%与10%冻存组比较差异不明显(P>0.05)。研究表明,长江鲟细胞的长期冷冻保存宜采用稍低浓度的DMSO(5%~10%)和高浓度的FBS。

Efficacy of Different Cryoprotectants on the Cryopreservation of Dabry′s Sturgeon Fin Cells

The cryopreservation of cells from endangered species plays an important role in the preservation and rescue of germplasm resources.In this study,the effect of freezing solution composition on the cryopreservation of Dabry′s sturgeon fin-derived cells(DSFCs)was investigated.The objectives were to completely preserve the genetic information of Dabry′s sturgeon at the cellular level and provide technical support for continued applications of somatic cell lines.Eighth passage DSFCs in the logarithmic phase were cryopreserved in liquid nitrogen(-196℃)with eight different formulations of the freezing solution.The freezing solution ingredients were culture media(MEM)with dimethyl sulfoxide(DMSO)at three concentrations and bovine serum(FBS)at five concentrations.The concentration ratios were prepared as follows:(1)5%DMSO+45%MEM+50%FBS;(2)10%DMSO+40%MEM+50%FBS;(3)20%DMSO+30%MEM+50%FBS;(4)10%DMSO+90%FBS+0%MEM;(5)10%DMSO+70%FBS+20%MEM;(6)10%DMSO+50%FBS+40%MEM;(7)10%DMSO+30%FBS+60%MEM;(8)10%DMSO+10%FBS+80%MEM.Apoptosis and cell cycle phase were detected using morphological observations,CCK-8 assays and flow cytometry.The DSFCs treated with 20%DMSO did not adhere well and the survival rate was low(38.3%),but the survival rates were significantly higher when the DMSO concentration was reduced to 10%DMSO(78.7%)or 5%DMSO(80.2%).At the different FBS concentrations,cell viability was significantly higher at FBS concentrations of 90%FBS,30%FBS and 10%FBS than at 70%FBS and 50%FBS.Furthermore,the rates of cell apoptosis at 30%FBS(8.24%)and 10%FBS(15.35%)were significantly higher than that at 90%FBS(3.54%),and the differences were significant among all three groups(P<0.05).The cell cycle phase results showed that the proportion of cells in the G 0/G 1 phase at 30%FBS and 10%FBS were significantly higher(P<0.05)than at 90%FBS,while the proportion of cells in the S phase and G 2+M phase were significantly lower(P<0.05).There was no significant difference in the cell cycle of DSFCs between the 30%FBS group and the 10%FBS group(P>0.05).Our results show that a low concentration of DMSO(5%-10%)and high concentration of FBS should be used for the long-term cryopreservation of Dabry′s sturgeon cells.