微载体cytodex3培养技术在大量扩增成人骨髓间充质干细胞中的应用

背景:间充质干细胞可以在体内或体外分化成肝样细胞,但是间充质干细胞数量少,在1 x 104-1×105个骨髓有核细胞中仅有个,肝细胞移植和生物人工肝的治疗都需要1×1010级的细胞数,利用常规的单层培养方法难以实现.目的:建立一种体外分离、大量扩增培养成人骨髓间充质干细胞的方法.设计、时间及地点:随机对照实验.实验于2005-09/2006-04在卜海市消化外科研究所(上海市教委重点实验室)及上海交通大学医学院附属瑞金医院普外科和器官移植中心完成.材料:骨髓标本收集于上海交通大学医学院附属瑞金医院血液科骨髓检查正常者,供者均为知情同意志愿者.方法:采用Percoll梯度离心结合贴壁法分离人骨髓间充质干细胞.应用微载体cytodex3培养人骨髓间允质干细胞,以普通单层聚苯乙烯(TCPS)培养作对照,用流式细胞仪(FCM)和MTT法对其细胞表型和增殖活性进行检测.主要观察指标:①hMSCs的分离及培养.②hMSCs的肜态学观察.③hMSCs在 cytodex 3表面生长时的增殖活性测定.④hMSCs在cytodex 3表面生长时FCM细胞周期测定.结果:①流式细胞仪检测表明,微载体cytodex3表面生长的人骨髓间充质干细胞表面标记与普通单层聚苯乙烯培养时一致,表达CD29、CD44和CD105,而不表达CD14、CD34、CD45、VLA-1和HLA-DR.②MTT检测显示,人骨髓间充质干细胞第1-3天处于适应期,3 d后进入对数生长期,人骨髓间充质干细胞此期间在TCPS和cytodex3表面的增殖活性无差异(P＞0.05),而存普通单层聚苯厶烯表面培养时,人骨髓间充质干细胞6 d后进入衰退期,而cytodex3表面生长第9天时仍然处于对数生长,差异显著(P＜0.05).③流式细胞仪分析表明,人骨髓间充质干细胞在TCPS和cytodex3表面培养细胞周期分布相同,差异不显著(P＞0.05).结论:微载体cvtodex3培养技术可以很好地大量扩增人骨髓间充质干细胞,并能保持其良好的增殖活性.

Microcarrier cytodex3 culture technique for amplification of a large amou nt of adult bone marrow mesenchymal stem CellS

OBJECTIVE:To create an in vitro harvesting method of culturing a large number of adult bone marrow MSCs(BMSCs) DESIGN,TIME AND SETTlNG:The randomized, controlled study was performed at the Shanghai Institute of Digestive Surgery (Key Laboratory of Education Committee of Shanghai City),as well as Department of General Surgery and Organ Transplantation Center,Ruijin Hospital,Medical College.Shanghai Jiao Tong University from September 2005 to April 2006.MATERIALS:Bone marrow samples were collected from normal persons.who did bone marrow examination at the Department of Hematology,Ruijin Hospital,Medical College.Shanghai Jiao Tong University.Donors were volunteers who signed the informed consent.METHODS:Human BMSCs were harvested using Pemoll gradient centrifugation and adherence method.and then incubated in microcarrier cytodex3.Common monolayer polystyrene was incubated as controls.Cell phenotype and proliferative activity were tested utilizing flow cytometry and MTT.MAIN OUTCOME MEASURES:Collection.incubation,morphology of human BMSCs.and prolireration and cell cycle of human BMSCs on the cytodex 3 were measured.RESULlTS:Flow cytometry detection showed that the surface marker of human BMSCs on the cytodex3 was ldentical to that on the common monolayer polystyrene;BMSCs were positive for CD29,CD44 and CD105.but negative for CD14,CD34,CD45,VLA-1 and HLA-DR.MTT detection demonstrated that human BMSCs were in the adaptive phase at days 1-3.and entered logarithmic phase frOm day 3.No significant difference was detected in human BMSCs on the monolayer polystyrene and cytodex3(P＞0.05).On the monolayer polystyrene,human BMSCs entered degenerating stage from day 6,whereas on the cytodex3,human BMSCs were still in the logarithmic growth phase at day 9(P＜0.05).Flow cytometry detection confirmed that the cell cycle of human BMSCs was the same both on the monolayer polystyrene and cytodex3 (P＞0.05). CONCLUSION:Using cytodex3 culture technique,a large amount of human BMSCs can be obtained,and the proliferative activity of these BMSCs is good.