A simplified and modified procedure to culture brain glioma stem cells from clinical specimens

Abstract

In recent years, the theory of brain glioma stem cells (BGSCs) has facilitated the study of gliomas. BGSCs have been accepted as the origin of gliomas and determine their biological features. Numerous efforts have been made to probe into the biological characteristics and behaviors of BGSCs. However, the culturing of target cells remains the essential first step for research on BGSCs. In this study, we established a simplified procedure to culture and isolate BGSCs from samples of clinical glioma patients. Samples of 17 glioma patients were included in the study, and the processed glioma cells were grown in serum-free stem cell media. After the tumor spheres appeared, a proliferation assay, a single-cell-derived colonies formation assay and an induced differentiation assay were carried out, followed by an immunocytochemistry assay. Serial passage was used to purify the target cells, whereas neither animal experiments nor sorting techniques were included. As a result, CD133 + BGSCs from 8 out of 17 patients were grown and maintained in a serum-free condition combined with EGF, FGF and B-27 supplements. The tumor sphere cells were serially passaged and showed pluripotency in an induced differentiation assay. Immunocytochemistry identified the committed markers (CD133, GFAP and TU-20) and confirmed the cells were BGSCs and their progeny. The results proved that CD133+ BGSCs from resected glioma tissue may be cultured in serum-free stem cell media, and may also be purified by conditioned culture combining serial passage, which is time-saving and cost-effective, and allows the cells to be used for subsequent research. The cell sorting techniques and animal experiments of tumorigenecity are optional. Thus, this modified procedure is more practical and feasible than other available procedures.