Isolation, Culture And Identification Of Human Gliom Stem Cells In Serum-free Medium In Vitro

OBJECTIVE:To explore the feasibility about isolating and culturing gliom stem cells in serum-free medium from primary glioblastoma.DESIGN,TIME AND SETTING:The cytology in vitro experiments were performed at the Brain Research Institute,Qingdao City,Shandong Province in August 2008.MATERIALS:The glioma tissues were removed during operation at the Department of Neurosurgery of Affiliated Hospital of Medical College,Qingdao University.DMEM/F12 medium and B27(GIBICO),basic fibroblast growth factor (bFGF) and epidemal growth factor(EGF)(Peprotech) were used in this study.METHODS:The tissue without necrosis and electric coagulation in deep part of the tumor was sterilely removed.The tissue formed single cell suspension after shearing and digesting,and then incubated in DMEM/F12 medium with bFGF,EGF and B27 into single cell suspension.Glioma stem cells were obtained in vitro.While the quantity of cell group was increased and the culture solution's color was changed,the cell group was taken suction to transfer of culture.The third passage of glioma stem cells were detected by immunoflurescence after adding Nestin and CD133.Cells were incubated in the DMEM/F12 medium with 0.1 volume fraction of fetal bovine serum in culture solution for 5 days.Cell differentiation was observed by glial fibrillary acidic protein immunofluorescence staining.Innoculated different desenty cell suspension with glioma stem cells and gliom cells into oxter of 4 weeks Balb/c athymic mouse.Observe the tumor formation in athymic mouse.After 6 weeks,the tumor were resected.By a series of fixation,dehydration,immerse into wax,embedding.The tumor were staining by HE methods.MAIN OUTCOME MEASURES:The form and growth of primary culture and passage of glioma stem cells,the expression of Nestin and CD133 and the differentiation of glioma stem cells were measured.RESULTS:The cell group was found under primary culture of 7-10 days.The cell group could grow with suspension or semi-suspension.The stem sells were observed with uniform shape and good refraction.The secondary cell groups formed after transfer of culture 24 hours,and there was no obvious difference in cell size and form compared with primary culture.The cell groups had an active proliferation after five times of serial passage.The third passage of glioma stem cells expressed Nestin and CD133,and they could adhere and differentiate after adding fetal bovine serum.After differentiation,they expressed GFAP.After transplantation into athymic mouse,they can form tumor.The tumor were conformed was glioma by HE staining.CONCLUSION:Human glioma stem cells exist in human brain glioma,and keep undifferentiated suspension form under serum-free medium in vitro.These cells present a glioma cell appearance or neuron appearance after adherence and differentiation in medium with serum.The cells were accordance with the character of stem cells in self-reproduction and multiple directional differentiation.