不同培养基培养人脐血间充质干细胞的差异

背景：脐血间充质干细胞为干细胞领域的研究热点,但目前传代及扩增此类细胞尚无简单、有效、完美培养方法。目的：采用不同的培养基分离培养融合状态的间充质干细胞,以筛选一种较好的体外培养人脐血间充质干细胞的方法。方法：无菌条件下取正常足月剖宫产新生儿的脐血,随机分为5组：低糖DMEM(Dulbecco改良的Eagle培养基)组、高糖DMEM组、α-DMEM组、低糖DMEM+干细胞因子组、低糖DMEM +骨髓间充质干细胞培养上清组。用淋巴细胞分离液分离脐血的单个核细胞。将脐血单个核细胞接种于含体积分数为10%胎牛血清的上述培养基中,放置于37 ℃、体积分数为5%的CO2培养箱内培养,倒置显微镜观察细胞数量和形态的变化并用流式细胞技术分析细胞的表面抗原。结果与结论：①各组间充质干细胞培养48 h后贴壁细胞数和细胞存活率的比较：低糖DMEM+干细胞因子组、低糖DMEM +骨髓间充质干细胞培养上清组的贴壁细胞数明显多于低糖DMEM组、高糖DMEM组、α-DMEM组(P < 0.05),细胞存活率亦明显高于低糖DMEM组、高糖DMEM组、α-DMEM组(P < 0.05)。②各组间充质干细胞在不同培养时间下生长状态的比较：培养第3,6,9,12,15,18,21天低糖DMEM+干细胞因子组、低糖DMEM+骨髓间充质干细胞培养上清组细胞增殖的速度均快于低糖DMEM组、高糖DMEM组、α-DMEM组(P < 0.05)。低糖DMEM+干细胞因子组与低糖DMEM +骨髓间充质干细胞培养上清组比较差异无显著性意义。结果可见人脐血间充质干细胞与骨髓间充质干细胞培养上清或干细胞因子共孵育,对脐血间充质干细胞体外分离培养及扩增有支持作用。

Differences of human umbilical cord blood-derived mesenchymal stem cells cultured in different media

BACKGROUND:The umbilical cord blood-derived mesenchymal stem cells are the hot spot in the field of stem cells, and there is no simple, effective culture method for the passage and amplification of umbilical cord blood-derived mesenchymal stem cells.OBJECTIVE:To explore a better culture method of human umbilical cord blood-derived mesenchymal stem cells in vitro with different media in the separation of mesenchymal stem cells at a confluent status. METHODS:Human umbilical cord blood was sterilely collected from full-term deliveries scheduled for cesarean section. They were assigned randomly into five groups: low-glucose culture medium group, high-glucose culture medium group, α-culture medium group, low-glucose culture medium + stem cell factor group, low-glucose culture medium + human marrow mesenchymal stem cells supernatant group. All culture medium used was Dulbecco’s modIfied Eagle’s medium. The cord blood mononuclear cells were isolated by lymphocyte separation medium. The monocytes of cord blood were inoculated into the culture medium containing 10% fetal bovine serum at 37 ℃ incubator with 0.05 volume fraction of CO2. Quantity and formation of cells were observed with invert microscope, and surface antigenic features were analyzed with flow cytometry. RESULTS AND CONCLUSION:(1) Comparison on number of adherent cells and survival rate of mesenchymal stem cells cultured for 48 hours: Number of adherent cells in the low-glucose culture medium + human marrow mesenchymal stem cells supernatant group, and low-glucose culture medium + stem cell factor group were significantly increased (P < 0.05), while the survival rate of cells was also increased compared with low-glucose culture medium group, high-glucose culture medium group and α-culture medium group (P < 0.05). (2) Comparison on growth status of mesenchymal stem cells at different culture time points: Cell proliferation in the low-glucose culture medium + human marrow mesenchymal stem cells supernatant group, and low-glucose culture medium + stem cell factor group was more rapid than that in low-glucose culture medium group, high-glucose culture medium group and α-culture medium group at 3, 6, 9, 12, 15, 18 and 21 days (P < 0.05). There was no significant difference between low-glucose culture medium + human marrow mesenchymal stem cells supernatant group and low-glucose culture medium + stem cell factor group. Experimental findings indicate that, co-culture with low-glucose culture medium + stem cell factor or human marrow mesenchymal stem cells supernatant can promote the in vitro isolation, culture and proliferation of human umbilical cord blood-derived mesenchymal stem cells.