培养基对兔骨髓间充质干细胞扩增与分化的影响

Effects of culture mediums on the proliferation and differentiation of rabbit mesenchymal stem cells

目的:考察α-MEM,DMEM-HG,DMEM-LG3种培养基对兔骨髓间充质干细胞体外贴壁、增殖与分化的影响。方法:实验于2005-04/2005-12在华东理工大学生物反应器工程国家重点实验室完成。①兔骨髓间充质干细胞的获取:从1个月龄新西兰大白兔股骨中用全骨髓分离法分离得到兔骨髓间充质干细胞,体外培养,将第4代兔骨髓间充质干细胞分别在α-MEM,DMEM-HG,DMEM-LG三种培养基中以1×107L-1的密度培养于24孔板中,观察细胞形态,测生长曲线和贴壁率。②为消除贴壁差异,在α-MEM培养基中待接种的细胞贴附后,分别更换DMEM-HG或α-MEM培养基进行培养;在DMEM-HG培养基中贴附的细胞以同样方法处理,比较其生长速率和细胞密度。③以100个细胞/孔的密度将细胞接种于3种培养基的6孔板中,测克隆形成率。④细胞以0.5×107L-1的密度接种于24孔板,分别在3种培养基中扩增至50%汇合后,改用成骨诱导培养液进行诱导培养,茜素红染色测其矿化能力。结果:①3种培养基中细胞在α-MEM中贴壁率最高,达(41.7±1.4)%,第1天即进入对数生长期,平均比生长速率最大,为0.57d-1,平均倍增时间最短,为2d,培养期最大扩增倍数为27.9倍,克隆形成率最多,达(30.9±2.6)%。②消除贴壁差异后,细胞在不同培养基中生长无明显差别,两条生长曲线基本重合。③3种培养基中扩增的细胞经成骨诱导后,茜素红染色皆为阳性,其中DMEM-HG培养基中阳性最明显。结论:α-MEM培养基适合于骨髓间充质干细胞体外培养和扩增,引起细胞在不同培养基中生长差异的主要原因在于贴壁率的不同。

AIM： To investigate the effects of three media of α-MEM, DMEM-HG, and DMEM-LG on the adhesion, proliferation and differentiation of rabbit mesenchymal stem cells （rMSCs） cultured in vitro. METHODS： The experiment was carried out in the State Key Laboratory of Bioreactor Engineering of East China University of Science and Technology from April to December 2005. （1） rMSCs isolated from one-month-old New Zealand rabbits were cultured in vitro. The fourth passage cells were cultured in α-MEM, DMEM-HG, and DMEM-LG at a density of 1×10^7 cells/L in 24-well-plate. The morphologic features were observed and the growth curve and attachment efficiency of cultured rMSCs were measured. （2） In order to eliminate the difference of adhesion between the media, the medium was changed to DMEM-HG or α-MEM after cells were adhered in the α-MEM; the same operation was carded out after cells adhered in the DMEM-HG to compare the growth rate and density of cells. （3）The colony-forming efficiency was counted after the cells were cultured in three different media at a density of 100 cells per well in 6-well-plate. （4）After cultured cells reached 50% confluence at inoculation density of 0.5×10^7 cells/L in three media, the medium was replaced by osteogenic induced solution; the biological characteristics of the cells was detected with Alizarin Red staining. RESULTS： （1）Of the three media, cells cultured in α-MEM showed the highest attachment efficiency of （41.7±1.4）%, in which the cells entered logarithmic growth phase, and showed a highest average specific growth rate of 0,57 d^-1, a short average doubling time of 2 days and high colony-forming efficiency of （30.9±2.6）% with the highest proliferated times of 27.9 times. （2）After eliminating the difference of adhesion, there was no obvious difference in the growth condition among the cells cultured in different media, and the growth curves of rMSCs were similar. （3）After the rMSCs were cultured in different medium and induced through osteogenic differentiation, the result of Alizarin Red staining of them were all positive, especially in DMEM-HG. CONCLUSION： α-MEM is suitable for adhesion and proliferation of MSCs in vitro. The growth difference of cells in different media is mainly caused by the adhesion.