Wang Xiao-Qing, Zhong Zhao-Dong, Chen Zhi-Chao, Zou Ping
Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, Hubei Province, China.
Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, Hubei Province, China. E-mail:
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2014 Apr;22(2):496-502. doi: 10.7534/j.issn.1009-2137.2014.02.042.
This study was aimed to investigate a more convenient and efficient method to cultivate the human bone marrow mesenchymal stem cells by means of natural erythrocyte sedimentation principle, based on the whole bone marrow adherent method. The bone marrow was cultured with a six-well plate instead of the flasks.Firstly, the bone marrow specimen was cultivated with the MSC complete medium for 48 h, then the upper RBC-free supernatant layer was drawn and placed into the new wells to isolate MSC. Inverted microscope was used to observe the cell morphology and to record the adherent time of first cell passage, first passaging time. The traditional whole bone marrow adherent method was used as the control. The cell cycle and cell surface markers were detected by flow cytometry,and the differentiative capacity of MSC into osteocyte and adipocyte was identified by alkaline phosphatase kit and oil red O, respectively. Besides, the proliferative curve of P1,P3,P5 of BMSC was depicted by counting method. The results showed that MSC cultured by the modified method highly expressed CD90, CD105, CD13, CD44 and lowly expressed CD14, CD45, CD34. Concerning the cell cycle feature, it was found that most of the cells were in G0/G1 phase (88.76%) , followed by G2/M phase (3.04%) and S phase (8.2%), which was in accordance with stem cell cycle characteristics. The proliferative curve showed a typical "S" type, and both the oil red O and alkaline phosphatase staining of MSC were positive. Compared with the traditional method, the modified method had the advantage of high adherence rate (P = 0.0001) and shorter passaging time for the first passage (P = 0.001), with the statistically significant difference. It is concluded that there is a large number of adherent, active and suspended MSC in the RBC-free supernatant layer after the culture of bone marrow for 48 h. Isolating MSC by the modified method is more convenient and efficient than the traditional whole bone marrow adherent method.
本研究旨在基于全骨髓贴壁法，借助自然红细胞沉降原理，探索一种更便捷高效的人骨髓间充质干细胞培养方法。采用六孔板而非培养瓶培养骨髓。首先，将骨髓标本用间充质干细胞完全培养基培养48小时，然后吸取上层无红细胞的上清液并置于新孔中以分离间充质干细胞。使用倒置显微镜观察细胞形态并记录首次细胞传代的贴壁时间、首次传代时间。以传统全骨髓贴壁法作为对照。通过流式细胞术检测细胞周期和细胞表面标志物，分别用碱性磷酸酶试剂盒和油红O鉴定间充质干细胞向骨细胞和脂肪细胞的分化能力。此外，通过计数法描绘骨髓间充质干细胞P1、P3、P5代的增殖曲线。结果显示，改良方法培养的间充质干细胞高表达CD90、CD105、CD13、CD44，低表达CD14、CD45、CD34。关于细胞周期特征，发现大多数细胞处于G0/G1期（88.76%），其次是G2/M期（3.04%）和S期（8.2%），这与干细胞周期特征相符。增殖曲线呈典型的“S”型曲线变化，间充质干细胞油红O染色和碱性磷酸酶染色均为阳性。与传统方法相比改良方法具有贴壁率高（P = 0.0001）和首次传代时间短（P = 0.001）的优势，差异具有统计学意义。结论是骨髓培养48小时后，无红细胞的上清液层中有大量贴壁、活跃且悬浮的间充质干细胞。采用改良方法分离间充质干细胞比传统全骨髓贴壁法更便捷高效。
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