Cell culture technology is one of the most commonly used techniques in modern biological research, and cell subculture is one of the basic operations in cell culture. Through subculture, the number of cells can be continuously expanded, resulting in a large number of cells for experiments and research. The following are the steps to subculture cells:
1. Experimental preparation.
Before subculturing, you need to prepare the required laboratory equipment and reagents, including cell culture dishes, cell scrapers, trypsin, PBS buffer, culture media, etc. At the same time, it is also necessary to ensure a sterile environment in the laboratory, sterilize and ensure that the experimental operation is carried out under sterile conditions.
2. Cell seeding.
Primary or passaged cells are removed from liquid nitrogen or cell banks and growth resumes. Once the cells have grown to an appropriate density, the cells are digested with trypsin to detach them from the walls of the flask. Gently pat the flask so that the cells are detached from the walls of the flask and suspended in the culture medium. Transfer the cell suspension to a centrifuge tube, centrifuge to remove the supernatant, add the appropriate amount of fresh medium, and resuspend the cells. According to the needs of the experiment, seed the cell suspension into a new cell culture dish and gently shake the dish so that the cells are evenly distributed at the bottom of the dish.
3. Cell culture.
Place the seeded cell culture dish in a thermostatic incubator at a suitable temperature and humidity. During the culture process, the growth of cells should be observed regularly, and the morphology and density of the cells should be noted. If necessary, fresh medium can be replaced in a timely manner to ensure the normal growth and metabolism of cells.
4. Cell passaging.
When the cells have grown to a certain density, they are ready to be passaged. First, wash the cell culture dish with PBS buffer to remove residual medium. Then, the cells are digested with trypsin, and when the cells are rounded and suspended in the culture medium, gently pat the flask to make it easier for the cells to detach. Transfer the cell suspension to a centrifuge tube, centrifuge to remove the supernatant, add the appropriate amount of fresh medium, and resuspend the cells. According to the needs of the experiment, re-seeding the cell suspension into a new cell culture dish and gently shaking the dish so that the cells are evenly distributed at the bottom of the dish. Continue with cell culture.
5. Precautions.
During subculture, aseptic operation should be ensured to avoid contamination. At the same time, it is necessary to choose the appropriate digestion time and concentration to avoid over-digestion or insufficient digestion leading to cell death or contamination. In addition, it is also necessary to pay attention to the growth density and nutrient requirements of the cells, and replace the fresh medium in a timely manner to ensure the normal growth and metabolism of the cells.
In conclusion, cell subculture is one of the commonly used techniques in biological experiments, and by mastering the operation steps and precautions of subculture, a large number of high-quality cells can be obtained for experiments and research.