Gastric Cancer Cell Primary Culture And Establishing Ming’s Infiltrative Type Gastric Cancer Cell Line

Objectives: Primary culturing of gastric cancer and establishing Ming’s infiltrativetype gastric cancer cell line.Methods: Human gastric cancer primary culture used tissues plant culture anddigestion culture. Primary culture system was established through optimizingextracellular matrix, mediums and promoting substance. Ming’s infiltrative typehuman gastric cancer cell line was established by repeating purification and passageculture. Morphology were observed by optical microscope and transmission electronmicroscope (TEM). The characters were analyzed through Periodic acid-schiffstaining (PAS), multiple time, karyotype analysis, Agarose clone experiment andanimal experiment in vivo.Results: The method of collagenase digestion got more number of cells in quantityand higher purity in quality than other methods. Culture dish covered with fibronectinpromoted cells attachment，and the effects had statistical significance(P<0.0001). Theeffects of medium in cell culture were significantly difference except mediumbetween L15and F12(P>0.05), and the medium DMEM/F12was best for primaryculture. The best effect in promoting cell attachment and growth were humanepidermal growth factor (hEGF) and basicfibroblast growth factor (bFGF) among thepromoting substances. It was not different among moderate FBS, high FBS and lowFBS which contained all promoting substances(P>0.05). By optical microscope, themorphology of Ming’s infiltrative type human gastric cancer cell line which wasestablished by primary culture and passages were spindle or oval, nucleus huge anddeeply stained. The ratio between nucleus and cytoplasm was high. All of these metedto the cancers character. By transmission microscope, the quantity of cytoplasm andorganelles was decreased. But, the number of ribosomes were increased. At the sametime, mitochondria swelled obviously. The morphological types did not change bypassage. The cell could grow like conglomeration and fell off when shortage innutrition, but, it adhered again when nutrition was enough. Cell immunohistochemistry showed that cytokeratin (CK) was positive and vimentin wasnegative, Ki-67≈75%. Periodic acid-schiff staining showed that cell secreted somematerial of glycogen. Multiple time was47.86hours. Karyotype analysis showed thatchromosome was triploid and amount was69, chromosome Y was lost. Phchromosome be found in karyotype. These gastric cancer cell line could grow in lowmelting agarose and the survive ratio was72%. Cells developed into a tumor in nudemouse.Conclusions:1. This experiment established the low serum gastric cancer primaryculture system can obtain a large number of purity cells, also can passage to be acell line.2. The Ming’s infiltrative type gastric cancer cell line showed many characterizations，such as highly vitality, severe malignant and metastatic tumor characteristics.