Separation, culture and identification of rabbit bone marrow mesenchymal stem cells by iliac puncture: operation details and techniques

doi:10.3969/j.issn.2095-4344.2014.23.006

Abstract

Abstract:

BACKGROUND: Bone marrow mesenchymal stem cells are considered as commonly used seed cells to construct tissue-engineered for repair of bone and cartilage defects. It is of great significance for cytology and tissue engineering experiments to study the common problems existing in the basic operation and how to avoid these problems in a timely manner.

OBJECTIVE: To summarize the common problems existing in the process of operation in order to provide reliable methods about separation, culture and identification of bone marrow mesenchymal stem cells for beginners and researchers. These can reduce or avoid some errors and problems during operation.

METHODS: Sixteen New Zealand white rabbits were selected as experiment objects, and bone marrow mesenchymal stem cells were separated from rabbits by iliac puncture, purified and augmented by using density gradient centrifugation combined with adherent culture method. Then cell morphology was observed by inverted phase contrast microscope, growth curve detected by MTT method and cell phenotype identified by flow cytometry.

RESULTS AND CONCLUSION: We encountered some problems in the process of separation and culture, when we operated the first five rabbits. After carefully summarizing and analysis of the reasons, the operation was successfully completed on the rest 11 rabbits. Bacteria pollution and cell aging were not found in the process of cell culture. What is more, the cells at passage 3 appeared with high-expression of CD29, and CD44, but low expression of CD14 and CD34. The cell growth curve showed that the proliferation activity of cells at passages 3 and 5 was higher than that at passage 10. Although the technology of separation, culture and identification of bone marrow mesenchymal stem cells is mature, the failure will be happen if we do not pay attention to the details of operation. By strictly carrying out normal operations, we can get high purity of bone marrow mesenchymal stem cells, which lays a good foundation for cell and animal experiments in the future.

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, bone marrow, antigens, surface, cell proliferation

CLC Number:

stem cells|mesenchymal stem cells|bone marrow|antigens|surface|cell proliferation

Zhang Cong, Liu Hong-mei, Li Qing-wei, Chen Guo-wu, Liang Xiao, Meng Chun-yang. Separation, culture and identification of rabbit bone marrow mesenchymal stem cells by iliac puncture: operation details and techniques[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(23): 3639-3644.

Figures/Tables 2

2.1 实验动物数量分析实验纳入健康幼年新西兰大白兔16只，骨髓髂骨穿刺分离骨髓液成功11只，失败5只。失败原因为髂穿、分离、培养过程中遇到的常规问题。 2.2 骨髓间充质干细胞分离及形态学观察实验过程中在前5只失败问题总结之上，后11只兔骨髓抽吸、骨髓间充质干细胞分离均获成功，髂骨穿刺抽吸骨髓液，离心后可见管内液体分4层，其中第2层为白色细胞层，内含有间充质干细胞(图1A)。倒置相差显微镜下观察，原代细胞在24 h后已有部分细胞开始贴壁生长，呈多边形或小梭形，胞核清晰，48 h 后首次换液，随培养时间延长，细胞呈梭形，集落开始出现且逐渐增大，细胞群呈漩涡状或人字状排列，细胞6-8 d即可达到80%汇合。传代细胞的生长较原代要快些，多于接种后四五天即可铺满整个培养瓶的底面。传代后的细胞(P3-P10)仍呈梭形(图1B-D)，形态大小较一致，但是P10细胞生长速度略慢于P3，一般需6-8 d可长满培养瓶。 2.3 不同代数骨髓间充质干细胞生长曲线分析比较选择MTT法对P3、P5、P10传代细胞培养的生长曲线进行观察比较，发现3代细胞培养具有以下共性特征：传代培养潜伏期为24-36 h；传代培养对数增殖期为4-6 d，第7天达到高峰；对数增殖期结束后骨髓间充质干细胞生长减慢，进入下降期(图2)。P3与P5骨髓间充质干细胞生长曲线大致重叠，而P10骨髓间充质干细胞吸光度略低于前两组，说明P3与P5 骨髓间充质干细胞增殖速度大致一致，没有明显变化，P10骨髓间充质干细胞生长速度略低于P3和P5。 2.4 骨髓间充质干细胞表面标记物的表达流式细胞仪检测结果显示，第3代的骨髓间充质干细胞表面表达CD29(99.82%)，CD44(94.14%)不表达CD14(3.11%)，CD34(0.34%)(图3)。表明分离培养获得的细胞符合骨髓间充质干细胞基本特征，即高表达骨髓间充质干细胞表面抗原CD29、CD44，低不表达造血干细胞的一些特异细胞表面抗原如CD14、CD34。"

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