Isolation,Culture And Identification In Primary Cells And Its Stem Cells Of Human Brain Glioma

Objective: To isolate,culture the human primary glioma cells and it's stem cells,and to provide an model in vitro for investigating the biological behavior properties of human primary glioma.Methods: The samples of fresh brain tissue cut from 20 cases of patients were prepared by trypsin digestion into single cell suspension.One group cells were cultured primarily.The morphology was inspected by a inverted phase contrast microscope,the expressions of glial fibrillary acidic protein(GFAP)and Ki-67 were detected by immunohistochemistry,and the growth curves were analyzed by cell proliferation assay(CCK-8 method)to investigate the growth character of cells in vitro culture.Another group of cell suspensions were sorted CD133~+ cells by immunomagnetic beads,and cells were cultured by neural stem cell culture medium.The expression of Nestin,GFAP and ?-Tubulin were detected using immunofluorescence method.Results:1.The primary human glioma cells were cultured successfully in 17 cases and failed in 3 cases.The cultured cells were adherent to grow with different morphology,good condition and significant logarithmic growth phase,and could be cultured continuously and continuously.Moreover,the logarithmic growth phase of WHO ? and ?glioma cells appeared in fourth day and tenth day respectively,and WHO ? grade was located between them.2.Cell proliferation experiments showed that the cultured cells in vitro were active,and the difference of absorbance between primary culture cells was statistically significant(P<0.05),the higher the degree of malignancy,the stronger the cell proliferation ability.HE staining and immunohistochemical staining showed that the cultured cells were glioma cells,and immunofluorescence assay showed that GFAP was positive in cultured cells.3.The CD133~+ cells sorted by immunomagnetic beads were suspension growth and formed neural stem cell-like spheres,which have strong proliferation ability with positive expression of glioma stem cells marker nestin and CD133.After added into fetal bovine serum,the cells adhered to the cell wall,the expression of GFAP and ?-Tubulinwas was positive by immunohistochemical staining.20 cases of human glioma were successfully cultured 10 cases of glioma stem cells,the success rate of culture was 75%.4.There is a small amount of CD133~+ cells with stem cell properties in human glioma.The higher the pathological grade of the tumor,and the shorter the formation time,the faster the growth rate,the more the number,the stronger the proliferation and the higher proportion of the cell spheres.Conclusion: The human glioma primary cells were successfully isolated and cultured from different pathological grades of glioma specimens by enzyme digestion in vitro with higher success rate,it is suitable for the establishment of brain glioma in vitro culture model.CD133~+ glioma cells with stem cell properties were sorted out from human glioma tissue by immunomagnetic beads.This further illustrates that the higher the pathological grade of glioma,the higher the proportion of glioma stem cells and the stronger the proliferation ability.