Glioblastoma stem cells (GSCs) are a unique subpopulation of cells within glioblastoma multiforme (GBM) brain tumors that possess the ability to self-renew and differentiate into bulk tumor cells. GSCs are resistant to currently available treatments and are the likely culprit behind tumor relapse in GBM patients. However, GSCs are currently inaccessible to the larger scientific community because obtaining a sufficient number of GSCs remains technically challenging and cost-prohibitive. Thus, the objective of this study was to develop a more efficient GSC culture strategy that results in a higher cell yield of GSCs at a lower cost. We observed that the basic fibroblast growth factor (bFGF) is indispensable in allowing GSCs to retain an optimal stem cell-like phenotype in vitro, but little change was seen in their sternness when grown with lower concentrations of bFGF than the established protocol. Interestingly, a dynamic fluctuation of GSC protein marker expression was observed that corresponded to the changes in the bFGF concentration during the culture period. This suggested that bFGF alone did not control stem cell-like phenotype; rather, it was linked to the fluctuations of both bFGF and media pH. We demonstrated that a high level of stem cell-like phenotype could be retained even when lowering bFGF to 8 ng/mL when the media pH was simultaneously lowered to 6.8. These results provide the proof-of-concept that GSC expansion costs could be lowered to a more economical level and warrant the use of pH- and bFGF-controlled bioprocessing methodologies to more optimally expand GSCs in the future. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.