人脐带血间充质干细胞分离培养体系的优化筛选

背景：目前所报道的脐带血间充质干细胞体外培养条件不尽相同，尚缺乏统一标准，且培养效率较低。目的：拟对人脐带血间充质干细胞的分离方法、培养条件进行优化筛选。设计、时间及地点：细胞学体外对照观察，于2007-05/2008-04在中国医学科学院北京协和医学院北京协和医院中心实验室完成。材料：35份脐带血标本来源于北京协和医院妇产科顺产或剖宫产胎儿。方法：应用淋巴细胞分离液法、羟乙基淀粉沉降法、羟乙基淀粉沉降与淋巴细胞分离液两步分离法分别从脐带血中获得单个核细胞。细胞接种后，应用含体积分数为10%胎牛血清的DMEM/F12、L-DMEM和MesencultTM不同培养基，在不同体积分数胎牛血清（5%，10%，20%）、不同细胞接种密度（5×106，1×107，5×107，1×108）下进行细胞培养。细胞传至第3代诱导成骨，行Von Kossa染色。主要观察指标：不同分离方法、培养基、胎牛血清浓度、种植密度分离培养细胞的效果，脐带血间充质干细胞表面标记的表达及成骨情况。结果：与淋巴细胞分离液法比较，羟乙基淀粉沉降法、羟乙基淀粉沉降与淋巴细胞分离液两步分离法获得的单个核细胞数量明显增多（P < 0.05，P < 0.01），羟乙基淀粉沉降与淋巴细胞分离液两步分离法细胞原代培养时间显著短于另2种方法（P < 0.01）。应用含体积分数为10%胎牛血清的DMEM/F12或MesencultTM培养基，T25培养瓶中细胞接种密度为5×107时，培养效率高于其余各种条件组合（P < 0.01）。贴壁细胞表达CD29，CD105，不表达CD34，CD45，CD90及CD133，经成骨诱导培养21 d后Von Kossa染色可见黑色矿化结节。结论：应用羟乙基淀粉沉降与淋巴细胞分离液两步分离法可以获得较多数量的脐带血单个核细胞，加入含体积分数为10%胎牛血清的DMEM/F12或MesencultTM培养基、细胞接种密度为5×107时可以提高脐带血间充质干细胞的培养效率。

Optimal selection of culture systems for human umbilical cord blood mesenchymal stem cells

BACKGROUND: There is no standard culture system of umbilical cord blood mesenchymal stem cells (MSCs), with a low culture efficiency. OBJECTIVE: To optimize the cultural conditions and isolation method of human umbilical cord blood MSCs. DESING, TIME AND SETTING: The cytology, in vitro, controlled study was conducted at the Central Laboratory, Peking Union Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences from May 2007 to April 2008. MATERIALS: A total of 35 samples of umbilical cord blood were obtained from fetuses of spontaneous delivery or uterine-incision delivery at the Department of Gynaecology and Obstetrics, Peking Union Hospital. METHODS: Mononuclear cells were isolated from umbilical cord blood using the lymphocyte isolation method, hydroxyethyl starch sedimentation, hydroxyethyl starch sedimentation + lymphocyte isolation method. Following incubation, cells were separately cultured in DMEM/F12 containing 10% fetal bovine serum, L-DMEM and MesencultTM, at various fetal bovine serum (5%, 10% and 20%) and various cell densities (5×106, 1×107, 5×107, 1×108). At the third passage, osteoblasts were identified by Von Kossa staining. MAIN OUTCOME MEASURES: Cell isolation outcomes at various methods, media, fetal bovine serum concentrations and densities; umbilical cord blood MSC surface marker expression and osteoblasts. RESULTS: Compared with the lymphocyte isolation method, the number of mononuclear cells significantly increased using the hydroxyethyl starch sedimentation and hydroxyethyl starch sedimentation + lymphocyte isolation method (P < 0.05, P < 0.01). Primary culture time of cells was significantly shorter using the hydroxyethyl starch sedimentation + lymphocyte isolation method compared with the hydroxyethyl starch sedimentation, and the lymphocyte isolation method (P < 0.01). Cells at 5×107 were incubated in DMEM/F12 or MesencultTM medium, containing 10% fetal bovine serum in the T25 culture flask. Culture efficiency was higher than other condition media (P < 0.01). Adherent cells were positively for CD29 and CD105, but negatively for CD34, CD45, CD90 and CD133. After 21 days osteogenic inductive culture, black mineralization nodules appeared following Von Kossa staining. CONCLUSION: Plenty of umbilical cord blood mononuclear cells are obtained using the hydroxyethyl starch sedimentation + lymphocyte isolation method. Cells at 5×107 in DMEM/F12 or MesencultTM medium, containing 10% fetal bovine serum can elevate the culture efficiency of umbilical cord blood MSCs.