Shu Xiao-chun, Zhu Dan-hua, Liu Jun-jing, Yin Dai-chan, Pang Tian-jiao, Lu Hong-yun, Sun Liao
Department of Endocrinology, the Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, China.
Zhonghua Yu Fang Yi Xue Za Zhi. 2010 Oct;44(10):923-7.
This study was to investigate the growth and proliferation characteristics of rat bone mesenchymal stem cells (BMSCs) isolated by the method of whole bone marrow culture and to explore the effect of cell inoculation density and incubation period on cell proliferation, with an aim to provide multipotential seed cells for preventing from degenerative disease.
Bone mesenchymal stem cells were isolated by the method of whole bone marrow culture and then cultured in vitro. The cell morphologic features were observed by inverted microscope. The cell surface antigens were identified by flow cytometry. The effect of cell inoculation density and culture period on cell growth and proliferation was explored by analyzing the characteristics of a ten-day cell growth curve in 96-well plates.
Flow cytometry results showed the detection rates for CD29, CD34 and CD45 were 97.68% (7607/7788), 7.93% (661/8340) and 2.76% (215/7788) respectively, which was consistent with the expression characteristics of BMSCs surface antigens. BMSCs became uniform after three cell passages, existing in a typical shape of whirlpool or radial colony. The senescent cells started to appear at 7(th) passage, and more senescent cells were found at 10(th) passage. The growth curve for moderate inoculation density was typically S-shaped. Lag phase was found during the first two days, and logarithm growth phase was in the following three days. Plateau phase started from the 6(th) day and cell numbers decreased slightly from the 8(th) day.
The whole bone marrow culture is an effective way to obtain BMSCs. A moderate inoculation density was more advantageous to cell proliferation, by which more seed cells could be obtained.
本研究旨在探讨采用全骨髓培养法分离的大鼠骨髓间充质干细胞（BMSCs）的生长和增殖特性，并探索细胞接种密度和培养时间对细胞增殖的影响，以期为预防退行性疾病提供多潜能种子细胞。
采用全骨髓培养法分离骨髓间充质干细胞并进行体外培养。通过倒置显微镜观察细胞形态特征。采用流式细胞术鉴定细胞表面抗原。通过分析96孔板中细胞10天生长曲线的特征，探索细胞接种密度和培养时间对细胞生长和增殖的影响。
流式细胞术结果显示，CD29、CD34和CD45的检测率分别为97.68%（7607/7788）、7.93%（661/8340）和2.76%（215/7788），与BMSCs表面抗原的表达特征相符。细胞传代3次后形态趋于一致，呈典型的漩涡状或放射状集落形态。第7代开始出现衰老细胞，第10代衰老细胞增多。中等接种密度的生长曲线呈典型的S形。前2天为潜伏期，随后3天为对数生长期。第6天开始进入平台期，第8天细胞数量略有下降。
全骨髓培养是获取BMSCs的有效方法。中等接种密度更有利于细胞增殖，可获得更多的种子细胞。
