Objective To determine the ideal culture method for SHG44 human brain glioma stem cells.Methods SHG44 human brain glioma stem cells were cultured in the serum-free medium supplemented with epidermal growth factor, fibroblast growth factor, and B27. According to different culture instruments, they were divided into three groups:culture dish group, culture flask group, and suspension cell culture plate group. The SHG44 human brain glioma stem cells were identified by the immunofluorescence staining of CD133, Nestin, and Bcl-2. The morphology, size, and counts of stem cells and stem cell spheres under an inverted microscope were compared between the three groups.Results In the culture dish group and the culture flask group, the first enriched SHG44 glioma stem cell spheres were large and not regularly shaped. After the second or third purification, most of the glioma stem cells were irregular, and most of the glioma stem cell spheres were scattered, small, and irregular. Compared with the first and second purification, after the third purification, there were fewer glioma stem cell spheres, but which were regularly spherical. In the suspension cell culture plate group, the glioma stem cells were round, and almost all glioma stem cells formed glioma stem cell spheres. And compared with the first and second purification, after the third purification, the stem cell spheres were larger and more regularly spherical. The statistical analysis showed that the suspension cell culture plate group had significantly larger and more glioma stem cell spheres than the culture dish group and the culture flask group (P<0.05). For the suspension culture plate group, the glioma stem cell spheres were larger and more numerous (P<0.05) and more regular with the increase in times of purification.Conclusions It is an ideal method to purify glioma stem cells using suspension cell culture plate.