Growth and proliferation of human urine derived stem cells in three kinds of culture media

doi:10.3969/j.issn.2095-4344.0442

Abstract

Abstract:

BACKGROUND: Little is reported on human urine derived stem cells, and there is still no stable and efficient culture method until now.
OBJECTIVE: To compare the effects of three kinds of culture media on the growth and proliferation of human urine derived stem cells, and to optimize the culture methods.
METHODS: Human urine derived stem cells were isolated and purified using centrifugation method, and then cultured using adherent method in keratinocyte serum-free medium, progenitor cell culture medium, and urinary
cell culture medium (equal-proportion mixture of keratinocyte serum-free medium and progenitor cell culture medium). Cell morphology was observed, and cell proliferation was detected by MTT method to draw a cell growth curve.
RESULTS AND CONCLUSION: Human urine derived stem cells could be successfully cultured in these three kinds of culture media, which grew slowly in the keratinocyte serum-free medium, grew rapidly in the progenitor cell culture medium, and grew best in the urinary cell culture medium. To conclude, the urinary cell culture medium which can rapidly and efficiently culture target cells is the best choice for the growth of human urine derived stem cells.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Stem Cells, Urine, Culture Media, Cell Proliferation, Tissue Engineering

CLC Number:

R394.2

Hamulati Tusong, Zhao Chang-hui, Maimaitiaili Maituoheti, Ma Jun, Wang Feng, Mulati Rexiati. Growth and proliferation of human urine derived stem cells in three kinds of culture media[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(5): 723-728.

Figures/Tables 1

2.1 人尿源性干细胞呈贴壁生长状态用角质细胞无血清培养基培养的干细胞数量较少，视野中可见部分干细胞贴壁，部分没有贴壁，透明度较高，细胞呈短梭形或圆形，排列紧密，仅在第3，4天开始细胞数量略有升高，见图1A，B。用祖细胞培养基培养1-3 d的尿源性干细胞与角质细胞无血清培养基无明显差别，仅有少量短梭形细胞，折光性较好，生长缓慢，细胞活力不强，第4，5天，大部分细胞贴壁生长，只有少量细胞悬浮，细胞开始增殖明显，速度较快，集落缓慢形成并增多，透光性也逐渐变低，见图1C，D。在尿源性细胞培养基中培养的干细胞形态均一，轮廓清晰，呈短梭形或圆形，贴壁生长，3 d后细胞开始缓慢增殖，散落在6孔板内，细胞透亮度较好，排列相对紧密，随着培养时间的增加，培养基内集落数量也随之增多，在培养第3，4天，细胞开始迅速增殖，呈纺锤形，到第8，9天，培养基内细胞明显增多，漩涡状生长，占培养孔80%-90%，此时对其进行传代培养，得到第1代人尿源性干细胞，细胞增殖迅速。经多代增殖，细胞形态未见明显变化，见图1E，F。 2.2 三种培养基培养的尿源性干细胞的生长曲线见图2。角质细胞无血清培养基组细胞在第1，2天生长缓慢，第3-5天开始加速生长，第6-8天后生长渐趋缓慢。祖细胞培养基组细胞生长较角质细胞无血清培养基组好，在第1，2天生长缓慢，第3，4天开始进入对数生长阶段，第5，6天生长较快，第7，8天呈现平台生长。尿源性细胞培养基组较前两组生长快而好，曲线大致呈“S”形，分别从培养开始经历缓慢生长、对数生长、平台生长与衰退4个阶段，细胞在最初1-3 d缓慢生长，在4-6 d后细胞开始大量增殖，呈对数生长状态。生长至第6天后，细胞生长与衰亡的细胞大致相等，此时细胞生长进入平台期。 2.3 人尿源性干细胞的鉴定结果尿源性细胞培养基培养尿液离心得到的沉渣，六七天开始出现细胞，细胞生长状态良好，生长迅速，形态大小相对均一，呈梭状，排列紧密。培养11 d后，细胞排列紧密，见图3。第4代尿源性干细胞经尿源性细胞培养基培养后加入荧光标记的CD29、CD44单克隆抗体，进行流式细胞仪检测，结果显示CD29、CD44抗体呈强阳性，阳性表达率分别为99.2%、98.4%，见图4。经成脂诱导后细胞内逐渐出现脂滴，脂滴散在分布，部分脂滴融合成较大脂滴，油红O染色后逐渐呈现红色；经成骨诱导后逐渐出现颗粒状分布的钙化结节，茜素红染色后呈红色。证明人尿源性干细胞有向成脂、成骨诱导分化的能力，见图5。"

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