GC cell lines, AGS and MKN45, were purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. AGS cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Thermo Fisher Scientific, Inc.), while MKN45 cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS. All GC cells were incubated at 37°C and 5% CO2. GCSCs were cultured from GC cells at a density of 1×105 cells/ml in serum-free medium. EGF (20 g/l; Invitrogen; Thermo Fisher Scientific, Inc.), bFGF (20 g/l; Invitrogen; Thermo Fisher Scientific, Inc.), B27 (2%; Invitrogen; Thermo Fisher Scientific, Inc.), BSA (0.4%; Roche Diagnostics), insulin (4 mg/l; Invitrogen; Thermo Fisher Scientific, Inc.) and gentamicin (200 IU/ml;, Sangon Biotech, Co., Ltd.) were added to DMEM/F-12 (Gibco; Thermo Fisher Scientific, Inc.) in 4-well low adhesion culture plates in an incubator at 37°C with 5% CO2 for 7–10 days.

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