多孔明胶微载体旋转培养hMSC的研究

[目的]观察利用多孔微载体结合旋转细胞培养系统(rotary cell culture system,RCCS)培养人骨髓间充质干细胞(human mesenchymal stem cell,hMSC)对其增殖的影响。[方法]体外分离hMSC,扩增至第2代(P2)后分为两组。实验组采用多孔明胶微载体CultiSpher G在RCCS内进行动态培养,对照组则继续静止培养。应用倒置显微镜、扫描电镜对微载体表面的细胞粘附、生长情况进行观察,并在不同的时相点取样,行细胞计数、MTT检测,了解细胞的增殖情况。[结果]接种24 h后,大部分细胞贴附于微载体表面,随培养时间延长细胞数量增多并分泌大量基质。MTT检测及细胞计数提示细胞对数生长期较静止培养方法延长,达到生长高峰时旋转培养方法收获细胞总量为接种时10.75倍,而静止培养方法为3.19倍;细胞周期检测两种方法差别不大,大部细胞均处在S1期。[结论]Culti Spher G微载体旋转培养系统是体外扩增hMSC的有效方法,利用该系统可为工程化组织的构建提供大量特性稳定的种子细胞。

Study on hMSC by rotary cultivate with porosity gelatine microcarrier

[Objective] To observe the effect on proliferation of hMSC after cultivated with porosity microcarriers in rotary cell culture system.[Methods]The hMSC,isolated from mesenchymal in vitro,were divided into two groups when they were amplificated to P2.The experiment group was cultivated with a kind of porosity gelatin microcarriers,CultiSpher G,in RCCS.The control group kept stationary culture.The cell adhesion and growth on the surface of the microcarriers were observed with the inverted microscope and scanning electronic microscope,and the message of cell’s proliferation by sampled in different time points was got with the methods of cell count and MTT detection.Samples of two groups were taken after 5 and 7 days’culture,and detected their cell cycle with microcarrier in RCCS.[Results]Twenty-four hours later after the seed,most of the cells adhered to the surface of the microcarriers.The number of the cell increased with the extend of the culture time and a lot of matrix were excreted from the cells.Cell count and MTT detection showed that the exponential phase of the cells in RCCS had been prolonged.And the number of the cell at the peak ponint was 10.75 times of seeding time,while the times of the static cultivation was just 3.19.The cell circles determination of the two groups had no difference,most of the cells were in G1 period.[Conclusion]The method to cultivate the hMSC by using CultiSpher G in RCCS is an efficient way to considerably amplificate the hMSCs,which is capable to supply a large number of stalbilized seeding cells for the construction of tissue engineering.