How to Passage Cells in the Cell Culture Flask

 Subculture refers to a process in which the culture needs to be divided into small parts, re-inoculated into another culture vessel (bottle), and then cultured. Cell culture flasks are commonly used consumables when culturing cells. So, how to passage cells in the cell culture flasks?

Generally, the cells in the cell culture flask are basically saturated after they grow into a dense monolayer. In order to enable the cells to continue to reproduce and expand the number of cells at the same time, a passaging operation is required. Suspension cells can be directly divided into flasks, while adherent cells need to be digested before they can be divided into flasks. Adherent cells are generally digested into single cells using proteolytic enzymes (such as trypsin), and ethylenediaminetetraacetic acid (EDTA) can also be added to improve digestion. Observe the cells until the cells become round and detach from the flask wall. This process usually takes 5-15 minutes. To avoid cell clumping, do not tap the cell culture flask during the digestion process.

For particularly difficult-to-digest cells, trypsin EDTA digestion solution can be added and the cells can be placed at 37°C to facilitate digestion. After the cells are separated from the wall of the flask, complete medium containing serum can be added to terminate the digestion. Certain proteins in serum can inhibit pancreatic secretion. enzyme activity. In general, centrifugation is not necessary. If the cells need serum-free or low-serum medium, centrifuge at 125,000g for 5 minutes, then discard the medium used for suspension, add suitable new medium, and mix the cells gently before passage.

The ratio of cell passaging is generally 1:2-1:20, depending on the type of cells. During the passaging process, trypsin will damage the cell membrane of some cells. You can gently scrape the cells with a cell scraper, add an appropriate amount of medium, mix the cells gently by pipetting, and transfer them to a new culture flask.