T 25 ultra low attachment flask

Cells were cultured as 3-dimensional spheres using a stem cell conditioned serum-free medium which is based on a neural stem cell medium[25 (link)]. Stem cell conditioned serum-free medium was prepared by adding 1:1 mixture of DMEM and HAM’s F12 medium (Lonza, Australia) supplemented with 4 μg/mL heparin sulfate (Sigma-Aldrich, United States), 1% penicillin/streptomycin (P/S) (ThermoFischer Scientific, Australia), 2% bovine serum albumin (BSA) (Sigma-Aldrich, United States), 20 ng/mL recombinant human epidermal growth factor (rhEGF) (Lonza, Australia) and 10 ng/mL recombinant human basic fibroblast growth factor (rhbFGF) (Lonza, Australia). Briefly, adherent cells were detached and collected following Trypsin-EDTA (ThermoFisher Scientific, Australia) treatment. Cells were washed three times with 50 mL 1 × PBS to remove serum. Cells were counted and seeded at 5000 cells/ml in a T-25 ultra-low-attachment flask (Corning Incorporated, United States) and cultured with stem cell medium at 37 °C in a humidified atmosphere of 5% CO₂ in air.

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.