Fresh tumor specimens (0.3-4 mL) obtained from GC patients (Table 1 ) were disaggregated into single cell suspension in DMEM/F12 medium (Biosera, France) containing collagenase type I (Gibco, USD)
(300 U.mL-1), penicillin (Biosera) (500 U.mL-1), streptomycin (Biosera) (500 mg.mL-1), and amphotericin B (Biosera) (1.25 mg.mL-1) at 37 °C for 2 hr. The cells (1 × 105 cell.mL-1) were cultivated in epidermal growth factor (EGF) 20 ng.mL-1 (Gibco), basic fibroblast growth factor (bFGF) 10 ng.mL-1 (Gibco), leukemia inhibitory factor (LIF) 10 ng.mL-1 (ProSpec, Israel), heparin 4 μg.mL-1 (Sigma-Aldrich, Germany), B-27 supplement 2% (Gibco), penicillin 100 U.mL-1, and streptomycin 100 μg.mL-1, and HEPES 8 mM (Biosera) in DMEM/F12 medium in T-25 ultra-low attachment flask (Corning, USA) at 37 °C for 1-2 months in a humidified 5% CO2 incubator to generate spheres.
(300 U.mL-1), penicillin (Biosera) (500 U.mL-1), streptomycin (Biosera) (500 mg.mL-1), and amphotericin B (Biosera) (1.25 mg.mL-1) at 37 °C for 2 hr. The cells (1 × 105 cell.mL-1) were cultivated in epidermal growth factor (EGF) 20 ng.mL-1 (Gibco), basic fibroblast growth factor (bFGF) 10 ng.mL-1 (Gibco), leukemia inhibitory factor (LIF) 10 ng.mL-1 (ProSpec, Israel), heparin 4 μg.mL-1 (Sigma-Aldrich, Germany), B-27 supplement 2% (Gibco), penicillin 100 U.mL-1, and streptomycin 100 μg.mL-1, and HEPES 8 mM (Biosera) in DMEM/F12 medium in T-25 ultra-low attachment flask (Corning, USA) at 37 °C for 1-2 months in a humidified 5% CO2 incubator to generate spheres.