Primary culture in vitro

The cell line was established from a primary tumor, which was surgically obtained from above GBC patient. After rinsing thrice with sterile PBS containing antibiotics, the tumor was minced into small fragment having a diameter of 1 mm using a scalpel, and completely eliminated subcutaneous fat and submucosa. The fragment was rinsed with PBS for 3 min, wet with 20% FBS (Corning, USA), then seeded into 25 ml culture bottle (Costar, USA). And, the culture bottle was inversionally incubated in a humidified incubator (SANYO, Japan) at 37 °C in a 5% CO₂ atmosphere for 4 h, then was put in normal direction, and added 3–4 ml DMEM/F12 (Gibco, USA) containing 20% FBS and 100 U/ml antibiotics along the edge of culture bottle slowly. After 5-day incubating, a small amount of cells climbing out around the tissue fragment, and a large number of lymphocytes and other miscellaneous cells were observed. The growth medium was renewed and replaced every 3 days, and the bottles were regularly checked for epithelial cells and fibroblast outgrowth. If fibroblast growth was observed during primary cultures, differential trypsinisation was used to obtain a pure tumor-cell population. After 5–6 passages tumor cells were basically purified. The cell line was cultured for >60 passages.