Isolation and Culture of Gastric Cancer Cells

Human gastric cancer cell lines HGC-27, MKN-28, SGC-7901, MGC-803, BGC-823, NCI-N87 and immortalized gastric epithelial cell line GES-1 were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). Adherent cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO, NY, USA) with 10% fetal bovine serum (FBS) and 1% P/S in a standard condition at 37 °C with 5% CO₂. Gastric cancer stem cells (GCSCs) were obtained and cultured as previously described [13 (link), 14 (link)]. Briefly, GCSCs were extracted from fresh GC tissues resected in Chinese PLA General Hospital (PLAGH) and cultured in ultra-low-attachment 6-well plates (Corning, NY, USA) with modified DMEM/F12, 2% B27 supplements (Invitrogen, CA, USA), 1% ITS (insulin–transferrin–selenous acid, Corning, NY, USA), 20 ng/ml epidermal growth factor (Peprotech, Hartford, CT, USA), 10 ng/ml basic fibroblast growth factor (Peprotech), 10 ng/ml LIF (Peprotech) and 10 ng/ml gastrin I (Peprotech).