不同冻存液对人脐带间充质干细胞生物学特性的影响

Influence of different freezing mediums on biological characteristics of human umbilical cord mesenchymal stem cell

目的观察不同冻存液对人脐带间充质干细胞(human umbilical cord mesenchymal stem cell,hUC-MSC)冻存复苏后生物学特性的影响,优化冻存方法。方法脐带取自2011年1月至2011年12月在中山大学附属第三医院产科剖宫产的足月健康产妇。从脐带分离得到的hUC-MSC培养传代,取第3代细胞。根据冻存液不同分为3组,A组冻存液为二甲基亚砜(dimethyl sulfoxide,DMSO)∶胎牛血清(fetal bovine serum,FBS)∶低糖杜氏培养基(low glucose Dulbecco's modified Eagle's medium,LG-DMEM)为1∶2∶7,B组为DMSO∶FBS∶LG-DMEM为1∶5∶4,C组为DMSO∶FBS为1∶9,不含LG-DMEM。经冻存12个月,复苏细胞,以第3代未冻存细胞悬液作为对照组,采用台盼蓝染色法比较hUC-MSC的细胞存活率,采用噻唑蓝(methyl thiazolyl tetrazolium,MTT)法比较细胞的生长情况绘制生长曲线,倒置显微镜下观察细胞的生长状况,采用流式细胞仪检测各组细胞的表面标记。结果A、B、C三组的细胞存活率分别为(20±4)%、(71±8)%和(85±7)%。与C组比较,A组和B组的细胞存活率降低,差异有统计学意义(均为P<0.05)。细胞生长曲线显示,与对照组相比,C组hUC-MSC在各时间点细胞生长迅速,但差异无统计学意义(P>0.05);B组hUC-MSC在24 h、48 h、72 h细胞生长差异无统计学意义(P>0.05),在96 h时间点生长速率低于对照组,差异有统计学意义(P<0.05);而A组hUC-MSC在各个时间点细胞生长减慢,差异有统计学意义(均为P<0.05)。形态学观察显示,A组细胞胞体宽大多角,并且较多分支,B组和C组细胞饱满,折光性好,呈长梭形、纺锤形或多角形。A组细胞复苏后细胞数目过少无法进行表面标记,B组和C组hUC-MSC表面标记物CD31、CD34、CD45、人类白细胞抗原(HLA)-DR表达阴性,CD105、CD90、CD44表达阳性。结论含高浓度血清的冻存液适合hUC-MSC的长期保存,含90%FBS的冻存液为hUC-MSC的最佳冻存保护液。

Objective To investigate the difference of biological characteristics of human umbilical cord mesenchymal stem cell （hUC-MSC） after cryopreservation and resuscitation in different freezing mediums, and to optimize the suitable freezing medium. Methods Umbilical cords were taken from healthy full-term puerperas who underwent cesarean section in Department of Gynecology and Obstetrics of the Third Affiliated Hospital of Sun Yat-sen University from January 2011 to December 2011. hUC-MSCs were isolated from human umbilical cord and the cells of third generation were taken after passage culture. According to different freezing mediums, hUC-MSCs were divided into 3 group [ Group A： dimethyl sulfoxide （DMSO） ： fetal bovine serum （FBS） ：low glucose Dulbecco＇s modified Eagle＇s medium （LG-DMEM） = 1：2： 7; Group B： DMSO： FBS：LG-DMEM = 1 ： 5：4 ; Group C ： DMSO： FBS = 1 ： 9 without LG-DMEM ]. After cryopreservation for 12 months, hUC-MSCs were recovered and hUC-MSCs of third generation without cryopreservation were chosen as control group. Survival rate of hUC-MSCs was detected by trypan blue staining. Growth situation of hUC-MSCs was detected by methyl thiazolyl tetrazolium （MTF） and growth curve was drawn. Growth condition of hUC-MSCs was observed under inverted microscope. Surface markers of hUC-MSCs were detected by flow cytometry. Results The survival rates of hUC-MSCs were （20 ± 4）% in Group A, （71 ± 8 ）% in Group B and （85 ± 7 ） % in Group C. Compared with Group C, the survival rates of hUC-MSCs were lower than those of Group A and B, and there was significant difference （ both in P 〈 0. 05 ）. The results of growth curve showed that compared with control group, hUC-MSCs in Group C grew quickly at every time point without significant difference （P 〉 0. 05 ）. There was no significant difference in growth rate of hUC-MSCs between Group B and control group at 24 h, 48 h, 72 h （P 〉 0. 05）. But the growth rate of hUC-MSCs in Group B was lower than that in control group at 96 h with significant difference （P 〈 0. 05 ）. And hUC-MSC in Group A grew slowly at every time point with significant difference （P 〉 0. 05 ）. The results of morphological observation showed that the cell body in Group A was wide and multangular with many branches. And the cell body in Group B and C was plump with good refraction in the shape of long fusiform, spindle or polygon. After resuscitation, there was few cells to detect surface marker in Group A. The surface markers of hUC-MSC in Group B and C were negative in CD31, CD34, CIMS, HLA-DR and positive in CD105, CD90, CD44. Conclusions Freezing medium with high concentration of serum is suitable for long-term cryopreservation of hUC-MSC, and freezing medium with 90% FBS is most suitable.