冻存对犬脂肪干细胞特性的影响

Experimental study on the effect of cell cryopreservation on the biological characteristics of canine adipose-derived stem cells

目的研究冻存对犬脂肪干细胞(ADSC)生物学特征及体外诱导分化能力的影响。方法2017年2月至5月,取自上海市第六人民医院动物实验室雄性6月龄比格犬腹股沟处脂肪组织,采用胶原酶消化法分离培养ADSC,设置冻存组和非冻存组,其中冻存组,将犬ADSC传至第2代,予以-196℃液氮冻存。3个月后细胞复苏,倒置显微镜观察细胞形态,细胞计数试剂盒(CCK-8)绘制生长曲线,流式细胞仪检测干细胞标志CD45、CD90、CD105表达,体外经成骨成脂及成肌等多向诱导分化。比较两组犬ADSC上述生物学特性指标差异,组间均数比较采用t检验,细胞生长曲线分析采用重复测量方差分析。结果犬ADSC原代培养4~5 d便可达95%细胞融合,细胞贴壁生长,呈长梭形。与非冻存组比较,冻存组犬ADSC形态相似,CCK-8检测两组细胞增殖能力差异无统计意义(F=1.233,P>0.05);冻存组犬ADSC表面标志CD45、CD90、CD105表达分别为(1.53±0.11)%、(97.52±1.61)%和(96.86±1.88)%,非冻存组犬ADSC表面标志CD45、CD90、CD105表达分别为(1.44±0.12)%、(96.31±1.76)%和(97.90±1.25)%,组间比较差异无统计意义(tCD45=0.958,tCD90=0.877,tCD105=0.798,均P>0.05)。与非冻存组比较,冻存组犬ADSC同样具有强大的多向分化能力,包括成肌、成脂和成骨分化。结论冻存后犬ADSC一般生物学特性及多向分化潜能无明显改变,能作为犬ADSC长期储存和运输的保存条件。

Objective To study the effects of cell cryopreservation on the biological characteristics and induced differentiation ability of canine adipose-derived stem cells(ADSC)in vitro.Methods Canine ADSC were isolated and cultured by collagenase digestion from the inguinal adipose tissue of male six months old beagle in Shanghai Sixth People’s Hospital Animal Laboratory from February to May 2017.The cryopreserved group and non-cryopreserved group were set up,and for the cryopreserved group,the canine ADSC of second passage was frozen preservation in the liquid nitrogen(-196℃),the cells were recovery after 3 months,the cell morphology was observed by inverted phase contrast microscope,the growth curve was detected by cell counting kit-8(CCK-8)method,the stem cell antigen expression of CD45,CD90 and CD105 was determinated by flow cytometry and the differentiation potential to osteogenic,myogenic and adipogenic was analyzed by oil red staining,alizarin red staining and Desmin immunofluorescence in vitro,respectively.The differences of biological characteristics in both group were compared by t test and repeated measurement analysis of variance was used for the analysis of cell growth curve.Results After 4-5 days of collagenase digestion,cellular fusion rate reached 95%.The cell morphology of ADSC in the cryopreserved group was similar with the non-cryopreserved group,and on the 9th day of cell inoculation,the OD values of cryopreserved group and non-cryopreserved group detected by CCK-8 was 3.267±0.231 and 3.176±0.060,respectively,and there was no statistical difference in the proliferation ability(F=1.233,P>0.05);The expressions of CD45,CD90 and CD105 in the cryopreserved group were(1.53±0.11)%,(97.52±1.61)%and(96.86±1.88)%,respectively,and the expressions of CD45,CD90 and CD105 in the non-cryopreserved group were(1.44±0.12)%,(96.31±1.76)%and(97.90±1.25)%,respectively(tCD45=0.958,tCD90=0.877,tCD105=0.798,all P>0.05).Compared with the non-cryopreserved group,the ADSC in the cryopreserved group also had strong multidirectional differentiation ability,including myogenesis,lipogenesis and osteogenesis.Conclusion The general biological characteristics and multidirectional differentiation potential of canine ADSC do not significantly changed after cell cryopreservation,which can be used as long-term storage and transportation conditions for canine ADSC.