摘要
目的:筛选冻存人羊膜间充质干细胞效果较好的冻存液。方法:①不同配方的细胞冻存液对细胞冻存效果的影响。人羊膜间充质干细胞培养至第三代后分为10组,分别加入以下10种不同组成的冻存液。A组:70%DMEM/F12+20%胎牛血清+10%DMSO;B组:60%DMEM/F12+30%胎牛血清+10%DMSO;C组:40%DMEM/F12+50%胎牛血清+10%DMSO;D组:90%胎牛血清+10%DMSO;E组:70%DMEM/F12+20%胎牛血清+10%甘油;F组:60%DMEM/F12+30%胎牛血清+10%甘油;G组:40%DMEM/F12+50%胎牛血清+10%甘油;H:90%胎牛血清+10%甘油;I组:90%DMEM/F12+10%DMSO;J组:90%DMEM/F12+10%甘油。调整细胞密度为5×106mL-1,放入程序降温盒中,入-80℃低温冰箱保存。1个月后复苏,MTT法筛选出吸光光度值较高的一种冻存液。②相同血清含量细胞冻存液对细胞冻存效果的影响。含体积分数为50%胎牛血清的细胞冻存液分3组保存细胞。K组:45%DMEM/F12+50%胎牛血清+5%DMSO;L组:40%DMEM/F12+50%胎牛血清+10%DMSO;M组:35%DMEM/F12+50%胎牛血清+15%DMSO;复苏冻存的细胞后,将细胞按冻存前的数量调整至5×105mL-1,台盼蓝染色后,计算细胞的成活率。在倒置相差显微镜下观察细胞的生长状况。结果及结论:配方为45%DMEM/F12+50%胎牛血清+5%DMSO的冻存液冻存复苏人羊膜间充质干细胞后细胞的成活率最高(P<0.05);冻存复苏后细胞的生长状况较好。
Aim ： By comparing the number of the recovery cells of different cryopreservation liquid through cryopreserration,to select a better-performing cryoprescrvation liquid of human amniotic mesenchymal stem cells. Methods：（1）The effect of different formulations of the cells cryopreservation liquid on cell cryopreservation. Cultivate human amniotic mem- brane-derived mesenchymal stem cells to the third generation, then divide into 10 groups, and add the following 10 kinds of different components of cryopreservation fluid respectively, group A： 70% DMEM/F12 ＋ 20% FBS ＋ 10% DMSO; group B： 60% DMEM/FI2 ＋30% FBS ＋ 10% DMSO;group C： 40% DMEM/F12 ＋50% fetal bovine serum ＋ 10% DMSO; group D： 90% FBS ＋10% DMSO;group E： 70% DMEM/F12 ＋20% FBS ＋10% glycerol;group F： 60% DMEM/ F12 ＋30 % FBS ＋10% glycerol;group G： 40% DMEM/FI2 ＋50% FBS ＋10% glycerol;group H： 90% fetal bovine serum ＋10% glycerol;group I： 90% DMEM / Fl2 ＋10% DMSO;group J： 90% DMEM/Fl2 ＋10% glycerol. Adjust the cell density to 5×10^6 mL ^-1 ,put in procedure cooling box, low-temperature of -80 ℃ refrigerator to cryopreserve,rccovery the cells One months later, MTT method selected the highest value-ray absorption of the frozen liquids. （2）The effect of same content serum of cryopreservation liquid on cell cryopreservation, cryopreservation fluid of the serum volume fraction of 50% were divided into 3 groups, group K： 45% DMEM/F12 ＋50% FBS ＋5% DMSO;group L： 40% DMEM/ FI2 ＋50% FBS ＋10% DMSO;group M： 35% DMEM/F12 ＋50% fetal bovine serum ＋ 15% DMSO; recovery the cells, Adjust the cell density to 5 ×10^5 mL^-1 acording to cell desity of precryopreservation, Taiwan blue staining, capare the survival rate of cells. Observe cell growth situation under the inverted phase contrast microscope. Results and Conclusions：Formulation of 45% DMEM/FI2 ＋ 50% FBS ＋ 5% DMSO cryopreservation fluid have the highest cell survival rate （ P 〈 0.05 ） ; cells grow better after cryopreservation.
出处
《郑州大学学报（医学版）》
CAS
北大核心
2009年第3期568-572,共5页
Journal of Zhengzhou University(Medical Sciences)
关键词
人羊膜
间充质干细胞
冻存
复苏
冻存液
human amniotic
mesenchymal stem cells
frozen
recovery of frozen
frozen liquid
