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Some researchers use nitrogen or argon gas to blanket the vial headspace after each use, displacing oxygen entirely

The complete tumor stem medium (CTSM) was prepared by adding the following growth factor supplements to the TSM-basal: 50x-diluted B-27 supplement minus vitamin A (Gibco, Thermofisher Scientific, Reinach, Switzerland), Heparin solution (2 ng ml 1 , 0.2%, Stem Cell Technologies, Vancouver, BC, Canada), and human epidermal growth factor (20 ng ml 1 , H-EGF), human basic fibroblast growth factor (20 ng ml 1 , H-FGF-basic-154), human platelet-derived growth factor-aa (20 ng ml 1 , H-PDGF-AA), human platelet-derived growth factor-bb (20 ng ml 1 , H-PDGF-BB) were purchased from Shenandoah Biotechnology (Warwick, PA, USA)