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背景:和骨髓间充质干细胞相比,脐血间充质干细胞取材方便,其免疫原性较低,能耐受更大程度上的HLA配型不符,分离出的间充质干细胞纯度更高。目的:分析新生儿脐血间充质干细胞体外分离、纯化、扩增,以及向成骨及脂肪细胞定向诱导分化的方法与条件。设计、时间及地点:观察性实验,于2004-09/2005-11在四川大学华西医院生物治疗国家重点实验室,干细胞与组织工程研究室完成。材料:胎龄37~40周的新生儿脐血。方法:以Ficoll-HyPaque淋巴细胞分离液密度梯度法、沉降红细胞后密度梯度法及CD34+免疫磁珠负选法分离脐血单个核细胞。将分离获得的单个核细胞采用L-DMEM培养基+体积分数0.1胎牛血清或MesencultTM培养基+体积分数0.1胎牛血清进行间充质干细胞培养传代,获得的第3代集落生长细胞进行流式细胞仪表面抗原测定,并诱导其向成骨、脂肪细胞分化。主要观察指标:①脐血间充质干细胞的鉴定。②经茜素红染色及油红染色证实其诱导分化。结果:经沉降红细胞后密度梯度离心分离的单个核细胞,使用MesencultTM培养基+体积分数0.1胎牛血清培养成功率高,第3代可出现明显的集落生长,而另两种方法分离培养的细胞则难以形成集落;集落细胞表面抗原测定表达CD29,CD59,D71,不表达CD34,CD45及HLA-DR等分子。成骨定向诱导分化的集落细胞经茜素红染色胞浆中出现有大量的钙沉积,成脂肪定向诱导分化的集落细胞油红染色示胞浆充满油滴空泡。结论:使用MesencultTM培养基+体积分数0.1胎牛血清培养由甲基纤维素沉降脐血红细胞后密度梯度离心分离的单个核细胞培养成功率高,第3代可出现明显的集落生长。经诱导后可分化为成骨细胞和脂肪细胞。
Background: Umbilical cord blood-derived mesenchymal stem cells (MSCs) are easier to harvest than BMSCs because of their low immunogenicity, tolerated a greater degree of HLA mismatch and higher purity of isolated mesenchymal stem cells . OBJECTIVE: To analyze the methods and conditions for the isolation, purification and expansion of neonatal umbilical cord blood mesenchymal stem cells (MSCs) in vitro and the direction-induced differentiation into osteoblasts and adipocytes. DESIGN, TIME AND SETTING: Observational experiments were performed at the State Key Laboratory of Biotherapy, Stem Cell and Tissue Engineering Laboratory, West China Hospital, Sichuan University from September 2004 to November 2005. Materials: neonatal umbilical cord blood from 37 to 40 weeks old. Methods: Cord blood mononuclear cells were isolated by Ficoll-HyPaque lymphocyte separation liquid density gradient method, sedimentation erythrocyte density gradient method and CD34 + immunomagnetic beads negative selection method. The isolated mononuclear cells were passaged with L-DMEM medium + 0.1 volume fraction fetal bovine serum or MesencultTM medium + 0.1 volume fraction fetal bovine serum for passage of mesenchymal stem cells, and the obtained third generation colonies were subjected to flow cytometry Cytometer surface antigen assay and induction of osteoblastic, adipocyte differentiation. MAIN OUTCOME MEASURES: ① Identification of umbilical cord blood mesenchymal stem cells. ② Alizarin red staining and oil red staining confirmed its differentiation. Results: Mononuclear cells isolated by density gradient centrifugation after sedimentation of erythrocytes were cultured successfully using MesencultTM medium + 0.1% fetal bovine serum. The third generation showed obvious colony growth while the other two methods isolated the cultured cells It is difficult to form colonies; CD29, CD59, D71, CD34, CD45, HLA-DR and other molecules are not expressed on the surface of colony cell surface antigen. Osteoblasts induced differentiation of colony cells by alizarin red staining of the cytoplasm in a large number of calcium deposition, adipose-oriented differentiation-induced colony cells oil red staining showed the cytoplasm filled with oil droplets vacuoles. CONCLUSION: Cultured mononuclear cells separated by density gradient centrifugation from methylcellulose after culturing mesenchymal stem cells with MesencultTM medium + 0.1% fetal calf serum have high success rate and obvious colony growth in the third generation. After induction can differentiate into osteoblasts and adipocytes.