Isolation, Incubation And Preliminary Identification Of Cancer Stem Cell In Human Gliomas

Objective: Isolation, culture and identification of brain tumor stem calls (BTSCs) within glicoma tissue in vitro. Observe the growth pattern of BTSCs, and to investigate their expression of specific markers. To establish a Isolation, culture and identification method for BTSCs. To establish groundwork for the further research.Methods: Gliomas samples were got from twenty-one patients. Tumor from patients undergoing tumor resections were acutely dissociated, triturated into single calls in sterile DMEM-F12 medium and then filtered. The tumor cells were seeded at a concentration of 1000000 viable cells per mL into serum free DMEM-F12 medium simply supplemented with B27 (1:50) , human basic fibroblast growth factor (20ug/L), human epidermal growth factor(20ug/L), leukaemia inhibitory factor(10ng/L), insulin (4uL), L-Glutamine, penicillin and streptomycin. After the primary brain tumor spheres (BTs) generated, they were separated again and passaged in fresh medium. Monoclonal culture were progressed simultaneously. Brain tumor spheres were seeded in serum DMEM-F12 medium, then the differentiation capability was detected by immunocytochemistry and immunofluorescence. At last we performed immunocytochemistry of BTSs for CD133 and ATP-binding cassette superfamily G member 2 (ABCG2 ) expression and immunohistochemistry of tumor specimen sections for CD133~+ ABCG2~+cells. The stem cells were detected and to count percentage in all tumor tissue by immunohistochemistry technology.Results: In ten samples, only a few tumor cells survived in serum-free medium and suspendedly proliferated into the cell sphere. The cell sphere still kept the capability of self-renewing and proliferation after subculture presenting CD133 and ABCG2 positive products. In serum medium, cells adhered and specific protein of neurons and glial cells could detected after they differentatiated. In tissue slice, the CD133+ and ABCG2+ cells diffused distribution, and a few distributed with nest form. Furthermore, in the hypso-hierarchy pathology tissue, tumor stem cell sphere were more and proliferated prosperity in the primary culture, the percentage of CD133+ and ABCG2+ cells was high also.Conclusion:A few of tumor stem cells are present in human gliomas, which can be isolated, incubate and induced to differentiate in vitro.