1. Primary cell culture of gastric cancer

The obtained human gastric cancer primary cells were inoculated into appropriate culture vessels according to the cell number specified in the instructions, and NIH-3T3 cells irradiated by gamma rays were added (gamma-NIH-3T3 was used as trophoblast cells, and the number of trophoblast cells was specified in the instructions), and cultured in an incubator at 37℃ and 5%CO2. Do not move the primary cells for 2-3 days after the initial inoculation (conducive to cell adhesion). During the culture process, if the medium color turns yellow but the cells are not overgrown, half of the liquid can be changed. When the cells are not overgrown but the γ-NIH-3T3 cells are insufficient under the microscope, the appropriate amount of γ-NIH-3T3 is added (generally half of the initial number is added).

2. Primary cell passage of gastric cancer

The clones were observed under the microscope, and the confluent degree of 85-95% could be passed. The cells were removed from the incubator, the old culture medium was discarded, 0.05% pancreatic enzyme was washed for 5 seconds, then sucked up, appropriate amount of 0.05% pancreatic enzyme was added, incubated at 37℃ for 2 to 5 minutes, and the cell digestion was observed under a microscope. When the cells became round and began to fall off, the digestion was terminated with primary cell medium, and the cells were transferred to a centrifuge tube at 1500 rpm for 3 min. The supernatant was abandoned, the cells were re-suspended with the corresponding primary cell medium of 1-5 mL, the live cells were counted, the cell suspension was inoculated on the culture bottle/plate according to the appropriate cell density, and the NIH-3T3 cells irradiated by gamma rays were added (see the instructions for different sizes of the bottom area of the culture bottle/plate, the number of primary cells inoculated, and the number of trophoblast cells added). The culture medium of gastric cancer was supplemented to the required volume, mixed in a cross way, 75% of the culture container was disinfected and then cultured in an incubator at 37 ℃ and 5% CO2. Do the same for other steps.