Biological properties of human mesenchymal stem cells by the whole bone marrow culture method in vitro

Abstract

AIM：At the present stage, most researchers usually use animals as cultured materials to observe the growth characteristics of bone marrow mesenchymal stem cells. But the cultivation of human bone marrow mesenchymal stem cells is few. In this experiment, we cultured human bone marrow mesenchymal stem cells and established a stable cultural method.

METHODS：We performed the experiments at the Central Laboratory of Luzhou Medical College from March to September 2007. The Laboratory is province's key Laboratory. ①Aborted fetuses were six month and above provided by Luzhou Maternity Hospitals. Puerperants and their family members signed an informed consent. The experimental procedures were approved by the hospital ethics committee. ②We applied the whole bone marrow culture method to culture bone marrow mesenchymal stem cells and purified the cells through differential adherent time. ③Morphology and biological characteristics of bone marrow mesenchymal stem cells were observed under an inverted phase contrast microscope. Cell activity was indirectly detected by MTT method.

RESULTS: After 4-hour culture, mesenchymal stem cells had begun to adhere, the cell body into the circular-shaped or triangular irregular; In 2-4 days, bone marrow mesenchymal stem cells extended 2-3 short processes to the two ends; Cell morphology graded at this time of the spindle, a fibroblast-like growth, and accumulated colony. With the growth of cells, adherent cells were gradually showed backbone, vortex growth, cell neurite growth, enlargement, and issued a fine of more branches, intertwined processes could be inter-network. At day 7, cell were seen adherent 70% to 80% the wall of the culture flask; Cell were close and tight, and mesenchymal stem cells became monolayer. Haematoxylin-eosin showed that more longer spindle cells appeared, with a protruding branch, larger cell body, a larger nuclear was elliptical, to assume pale; many nucleoli were found, and cytoplasm was salmon pink. ②Mesenchymal stem cell surface marker CD44 immunohistochemical detection showed that the cell membrane appeared brown positive reaction. ③MTT assay showed that there was no significant difference in absorbance in 2, 3, 4, 5, 6 passages of cells in the 490 nm wavelength by enzyme-labeled instrument, but the absorbance was higher than 7 or 8 passage of cells.

CONCLUSION: We successfully cultivated human fetal mesenchymal stem cells by the whole bone marrow culture method, and found that the 3-6 passages of cells had high purity and good activity.

Han JN, Xu FC, Hou YQ, Wu SH.Biological properties of human mesenchymal stem cells by the whole bone marrow culture method in vitro.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(3):429-432(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-3/3k-429(ps).pdf]

摘要
目的：现阶段多以动物为材料培养观察骨髓间充质干细胞的生长特性，对人骨髓间充质干细胞培养和生长特性的研究相对较少。为此实验培养人骨髓间充质干细胞，拟建立稳定的培养方法。
方法：实验于2007-03/09在泸州医学院中心实验室省级重点实验室完成。①实验材料：6个月以上流产胎儿由泸州地区妇产医院提供，产妇及家属对实验知情同意，并经医院伦理委员会批准。②实验方法：应用全骨髓培养法培养人骨髓间充质干细胞，采用差速贴壁对细胞进行纯化。③实验评估：倒置相差显微镜下观察骨髓间充质干细胞形态及生物学特点，应用MTT法间接测定骨髓间充质干细胞活性。
结果：①培养4 h,见骨髓间充质干细胞已开始贴壁，胞体由圆形变为不规则形或三角型，培养第2~4 天，见间充质干细胞胞体向两端伸出二三个粗短突起，此时细胞形态渐变为梭型，呈成纤维细胞样生长，并聚集成集落；随着细胞的生长，贴壁细胞逐渐表现为脊梁状、旋涡状生长，细胞突起增长、增粗，并可发出更细的分支，突起间可相互交织成网。培养7 d时，见细胞铺满瓶壁面积的70%~80%，细胞排列紧密，此时骨髓间充质干细胞铺成单层。苏木精－伊红染色见细胞多为长梭型，有突起及分支，胞体较大，核一个，较大，呈椭圆，着色浅，可见多个的核仁，胞质浅红色。②骨髓间充质干细胞表面标志CD44免疫组织化学检测，可见胞膜出现棕黄色阳性反应。③MTT法分别检测结果显示，2~6代细胞于酶标仪490 nm波长处 吸光度值无明显差异，但均高于七八代细胞。
结论：利用全骨髓培养法成功地培养出人胎儿骨髓间充质干干细胞，且第3~6代细胞纯度高、活性好。
关键词：体外培养；骨髓间充质干细胞；纯度；活性

韩济南，徐富翠，侯艳秋，吴绍华.体外全骨髓法培养人骨髓间充质干细胞的生物学性状[J].中国组织工程研究与临床康复，2008,12 (3):429-432 [www.zglckf.com/zglckf/ejournal/upfiles/08-3/3k-429(ps).pdf]