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《Chinese Journal of Neuromedicine》 2006-10
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Isolation, cultivation and biological identification of human bone marrow mesenchymal stem cells

WANG Zhong, GAO Yi, WANG Yan, PAN Ming-xin. Department of General Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China  
Objective To establish a simple and feasible method to culture and proliferate human bone marrow mesenchymal stem cells (hBMSCs) in vitro, to analyze their biological characterizations such as cell phenotype, generation cycle, growth curve, ultrastructure and caryotype. Methods hBMSCs were isolated by combining density gradient centrifugation with plastic adherence and cultured in specified culture medium. Morphological observations were performed with phase contrast microscope; growth curves of the cells were drawn with help of MTT assay; cell phenotype and generation cycle were detected by flow cytometry; cell ultrastructures were determined by scanning electron microscope and transmission electron microscope, and cell caryotypes were measured by Giemsa staining. Results Higher purity of hBMSCs could be achieved by density gradient centrifugation. The cells displayed long spindle shaped and adherent to the wall. The survival rate was over 95%. The positive expression rates of cell phenotypes were various as followings respectively: CD29, 95.3%; CD34, 1.8%; CD44, 94.7%; CD45, 0.8%; CD71, 96.2%; CD105, 96.6%; CD166, 92.7%; HLA-ABC, 96.3%; HLA-DR, 1.1%; UEA-1, 98.7%. The growth curve of hBMSCs was "S" shaped, and the proliferation ability of hBMSCs was strong before 7th passage. The ratio of cells in S+G2+M stage of the 3rd and 6th passages was 17.1% and 22.8% respectively. Many evections and pores were observed on cell surface, while abundant cytoplasm, prosperous rough endoplasmic reticulum and plenty of protein excretion were found inside hBMSCs under electron microscopes. The chromosome number at the 3rd and 6th passages were both 46, same as the normal human being's. Conclusion Higher purity of hBMSCs can be isolated and cultured by combining density gradient centrifugation with plastic adherence, and cell phenotypes can be maintained as normal human being's. The chromosome number of hBMSCs cultured in vitro for a long time can be kept normal.
【CateGory Index】: R329
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