Effects of different storage solutions on the viability of umbilical cord mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.2014.14.004

Abstract

Abstract:

BACKGROUND: Umbilical cord-mesenchymal stem cells (UC-MSCs) have been widely studied in clinics. Notably, UC-MSCs have to be properly preserved before clinical use and the effects of storage solution on viability of cells play critical roles in the effectiveness of cell-transplantation. However, there are few studies concerning this phenomenon.
OBJECTIVE: To evaluate the effects of various clinical common solutions on the activity of UC-MSCs.
METHODS: UC-MSCs were stored at 0.9% sodium chloride injection, 5% glucose injection and 1% human serum albumin respectively at 4 ℃ and room temperature (25 ℃) for 2, 4 or 6 hours. Viabilities, apoptosis/necrosis rate, multi-differentiation ability of preserved UC-MSCs and DNA damage were examined.
RESULTS AND CONCLUSION: The viabilities of UC-MSCs were rapidly decreased time-dependently in all of the storage solutions both at 4 ℃ and room temperature. Besides, the storage solutions resulted in cellular necrosis rather than apoptosis and the necrosis rate was greatly increased time-dependently. DNA damage was observed in cells after preserved in various storage solutions, but cell proliferation and differentiation were not affected, which demonstrated that DNA damage may be a key factor for the reduction of cell viabilities.

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

全文链接：

Key words: stem cells, mesenchymal stem cells, umbilical cord, stem cell transplantation, preservation, biological

CLC Number:

R394.2

Zhou Nan, Chen Yan, Lei Xin, Dong Yan-ting, Xu Wen-jing, Niu Yu-hu, Niu Bo, Cheng Niu-liang . Effects of different storage solutions on the viability of umbilical cord mesenchymal stem cells [J]. Chinese Journal of Tissue Engineering Research, 2014, 18(14): 2153-2158.

Figures/Tables 5

2.1 人脐带间充质干细胞形态学观察结果倒置显微镜下观察，改良酶消化法分离脐带间充质干细胞培养12 h即见散在的长梭形贴壁细胞。原代脐带间充质干细胞融合率达80%-90%后传代培养，细胞形态趋于一致，呈鱼群样或漩涡状生长，贴壁紧密(图1A)。 2.2 人脐带间充质干细胞表面标记检测结果流式细胞仪免疫表型鉴定结果显示(图2)，分离培养的脐带间充质干细胞第3代细胞高表达基质细胞与干细胞标记CD29、CD 44与CD105，阳性率不低于95%；低表达内皮与造血干细胞标记CD34与CD45，阳性率不高于2%。 2.3 人脐带间充质干细胞多向分化能力第3代脐带间充质干细胞在成骨细胞诱导培养基中培养23 d后，经茜素红S染色，胞浆中可见大量玫瑰红色钙结节形成(图1B)；在成脂细胞诱导培养基中培养16 d后，经油红O染色呈阳性，胞浆内可见红色脂滴形成(图1C)。 2.4 不同保存介质在不同温度及不同时间点对脐带间充质干细胞活性的影响将消化获得的脐带间充质干细胞重悬于完全培养基、生理盐水、5%葡萄糖和生理盐水+1%人血白蛋白，在4 ℃或25 ℃条件下，各组细胞活性均呈时间依赖性下降，较对照组相比，两组间差异有显著性意义(P < 0.05)。4 ℃条件下保存6 h，除对照组外仅生理盐水组可保持80%的细胞活性；室温组(25 ℃)保存6 h后，各组细胞活性均低于80%，远达不到临床应用的保存效果(图3)。 2.5 不同保存介质对脐带间充质干细胞多向分化能力的影响在4 ℃和25 ℃条件下，第3代脐带间充质干细胞经不同介质保存6 h后，各组仍可贴壁的细胞依旧存在向成骨细胞和成脂细胞方向分化的能力(图4)。 2.6 不同保存介质在不同温度及不同时间点对脐带间充质干细胞凋亡/死亡率的影响在4 ℃与25 ℃条件下保存6 h后，不同保存介质主要引起细胞不同程度的坏死而非凋亡(图5A，B)。各组间死亡率也呈时间依赖性，随时间延长死亡率增加，与对照组相比，差异有显著性意义(P < 0.05，图5C，D)，进一步验证了不同保存介质对脐带间充质干细胞活性的影响。 2.7 不同保存介质在不同温度条件下对脐带间充质干细胞 DNA损伤的影响对照组脐带间充质干细胞在4 ℃和25 ℃条件下保存6 h后细胞核均未出现明显拖尾现象，电泳后核DNA依然停留在核基质中，呈完整的圆形荧光团；其余各组细胞核DNA解旋，电泳时发生迁移，呈现不同程度的拖尾现象(图6)。数据分析结果显示各实验组拖尾细胞比例、彗尾长度及尾矩均高于对照组，且差异有显著性意义(P < 0.05，表1)。"

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