CAJ | 학술논문

背景:脐血中存在间充质干细胞,目前国内外尚未见到对脐血间充质干细胞体外分离、培养及扩增较统一且有效的方法.目的:探讨影响人脐血间充质干细胞成功分离培养的相关因素.方法:分别从不同胎龄(≥40周,37周和≤32周),脐血中单个核细胞数量(≥2.5× 109 L-1,< 2.5×109 L-1),不同细胞接种浓度(1×107,1×109,1×1011 L-1),不同体积分数胎牛血清(5%,10%,15%,20%)以及培养瓶是否被胎牛血清包被等方面对人脐血间充质干细胞分离培养成功率进行比较.结果与结论:脐血间充质干细胞的分离培养成功率为58.3%,且随胎龄的增高而培养成功率降低(P < 0.01);脐血中单个核细胞浓度≥2.5×109 L-1组培养成功率高于< 2.5×109 L-1组(P < 0.01);相同容量脐血中单个核细胞数量与胎龄呈负相关(r = -0.95,P < 0.01);1×1011 L-1组原代及传代培养的间充质干细胞生长及扩增情况高于1×107,1×109 L-1;体积分数5%FBS组间充质干细胞贴壁速度较其他3组略慢,但细胞纯度较高,且细胞传代速度与其他3组无明显差别;胎牛血清包被组脐血间充质干细胞原代和传代后的纯度及扩增能力均高于未包被组.提示脐血间充质干细胞分离培养成功率受多种因素的影响:通过选择较低胎龄的胎儿,采集足够量的脐血,以较高的细胞密度接种,培养基中添加较低浓度的胎牛血清,并将培养瓶预先用胎牛血清进行包被,能在体外建立稳定的脐血间充质干细胞培养体系.
배경:제혈중존재간충질간세포,목전국내외상미견도대제혈간충질간세포체외분리、배양급확증교통일차유효적방법.목적:탐토영향인제혈간충질간세포성공분리배양적상관인소.방법:분별종불동태령(≥40주,37주화≤32주),제혈중단개핵세포수량(≥2.5× 109 L-1,< 2.5×109 L-1),불동세포접충농도(1×107,1×109,1×1011 L-1),불동체적분수태우혈청(5%,10%,15%,20%)이급배양병시부피태우혈청포피등방면대인제혈간충질간세포분리배양성공솔진행비교.결과여결론:제혈간충질간세포적분리배양성공솔위58.3%,차수태령적증고이배양성공솔강저(P < 0.01);제혈중단개핵세포농도≥2.5×109 L-1조배양성공솔고우< 2.5×109 L-1조(P < 0.01);상동용량제혈중단개핵세포수량여태령정부상관(r = -0.95,P < 0.01);1×1011 L-1조원대급전대배양적간충질간세포생장급확증정황고우1×107,1×109 L-1;체적분수5%FBS조간충질간세포첩벽속도교기타3조략만,단세포순도교고,차세포전대속도여기타3조무명현차별;태우혈청포피조제혈간충질간세포원대화전대후적순도급확증능력균고우미포피조.제시제혈간충질간세포분리배양성공솔수다충인소적영향:통과선택교저태령적태인,채집족구량적제혈,이교고적세포밀도접충,배양기중첨가교저농도적태우혈청,병장배양병예선용태우혈청진행포피,능재체외건립은정적제혈간충질간세포배양체계.
BACKGROUND: Mesenchymal stem cells (MSCs) exist in umbilical cord blood (UCB), currently, there is not a method to in vitro separate, culture and amplificate human UCB-MSCs effectively. OBJECTIVE: To explore factors that influence yields of UCB-MSCs. METHODS: The relationship between the success rate of yielding UCB-MSCs and several factors, such as gestational ages (≥40 weeks, 37 weeks and ≤32 weeks), the number of mononuclear cells (MNCs) in UCB (≥2.5×109/L, <2.5×109/L), the inoculum density of MNCs (1×107, 1×109, 1×1011/L), the concentration of fetal bovine serum (FBS, 5%, 10%, 15%, 20%) in culture medium, and whether the culture flask being coated with FBS or not beforehand, as well as relationships among these factors were investigated. RESULTS AND CONCLUSION: The success rate of yielding UCB-MSCs was up to 58.3%. The success rate decreased as the gestational ages increasing (P < 0.01). The success rate could be enhanced to 76.9% when the MNCs count was more than 2.5×109/L, and there was significant difference when comparing to that of the group (36.4%) with MNCs count less than 2.5×109/L (x2=8.07, P=0.005). There was a negative correlation between the MNCs count and the gestational ages in the specimens with the same volume of UCB (r=-0.95, P < 0.01). In the group with the cell inoculum density of 1×1011/L, the growth and proliferation of primary and subculturing MSCs were better than that of the groups with the cell inoculum density lower than 1×1011/L. The adherence of MSCs in the group with the culture medium containing 5% FBS happened much later than other 3 groups, while the purity of MSCs in this group was much higher. When comparing the passage rate, there was no significant difference among the 4 groups with different concentration of FBS. In the group of culture flask being coated with FBS beforehand, the purity and proliferation ability of MSCs was higher than that in the groups with culture flask not being coated. It is suggested that culture of UCB-MSCs was influenced by several factors. The success rate could be increased by choosing the fetus with relative lower gestational ages, collecting enough volume of UCB, inoculating cells with a higher density, choosing the medium with lower concentration of FBS, and coating the culture flask with FBS beforehand.