How to passage the cells in the cell culture flasks

 Subculture refers to the process of dividing the culture into small parts, re-inoculating them into another culture vessel (bottle), and then culturing. Cell culture flasks are commonly used consumables when cultivating cells. So, how to subculture the cells in the flask?

Generally, the cells in the cell culture flasks grow into a dense monolayer and are basically saturated. In order to enable the cells to continue to reproduce and expand the number of cells, it is necessary to carry out passage operations. Suspended cells can be separated directly, while adherent cells need to be digested before they can be separated. Generally, proteolytic enzymes (such as trypsin) are used to digest adherent cells into single cells. Ethylenediaminetetraacetic acid (EDTA) can also be added to improve digestion. Observe the cells until the cells become round and detach from the bottle wall. This process often takes 5-15 minutes. In order to avoid cell clumps, do not tap the cell culture flasks during the process of digesting the cells.

For particularly difficult-to-digest cells, you can add trypsin EDTA digestion solution and place the cells at 37°C to facilitate digestion. After the cells are separated from the bottle wall, immediately add a complete medium containing serum to stop the digestion. Certain proteins in the serum can inhibit the pancreas. Enzyme activity. In general, centrifugation is not necessary. If the cells require serum-free or low-serum medium, centrifuge at 125000g for 5 minutes, then discard the discontinued medium, add appropriate new medium and gently mix the cells before subculture.

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