Microcarrier cytodex3 culture technique for amplification of a large amou nt of adult bone marrow mesenchymal stem CellS / 中国组织工程研究

ABSTRACT

OBJECTIVE:To create an in vitro harvesting method of culturing a large number of adult bone marrow MSCs(BMSCs) DESIGN,TIME AND SETTlNGThe 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.MATERIALSBone 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.RESULlTSFlow 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.