U251 and U87 cell lines were acquired from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). LN229 cells were obtained from the American Type Culture Collection. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Grand Island, NY, USA), added with 10% fetal bovine serum (FBS) (Gibco), and incubated at 37 °C in a humidified condition supplemented with 5% CO2.
Patient-derived primary glioma cells were established instantly after separation of the primary patient tumor, and neurosphere cultures were conducted according to the previously published method [57 (link), 58 (link)]. Briefly, fresh GBM tissues were disaggregated into cells using both physical and enzymatic methods and then recovered in a stem cell medium (Neurobasal-A medium with 2% B27, 20 ng/ml rh-bFGF and rh-EGF). Magnetic cell sorting was used to separate CD15+ GSCs from primary glioma cells. Functional analysis of self-renewal and tumor propagation were conducted to verify the cancer stem cell phenotype of extracted GSCs as described previously [59 (link)]. All cells had passed mycoplasma and the short tandem repeat (STR) DNA profiling tests.
Patient-derived primary glioma cells were established instantly after separation of the primary patient tumor, and neurosphere cultures were conducted according to the previously published method [57 (link), 58 (link)]. Briefly, fresh GBM tissues were disaggregated into cells using both physical and enzymatic methods and then recovered in a stem cell medium (Neurobasal-A medium with 2% B27, 20 ng/ml rh-bFGF and rh-EGF). Magnetic cell sorting was used to separate CD15+ GSCs from primary glioma cells. Functional analysis of self-renewal and tumor propagation were conducted to verify the cancer stem cell phenotype of extracted GSCs as described previously [59 (link)]. All cells had passed mycoplasma and the short tandem repeat (STR) DNA profiling tests.
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