Passaging

In cell culture, the passaging is the process of sub-culturing cells. It is usually done to produce large number of cells from pre-existing ones. Instances where it is followed include vaccine production labs and clonal expansion.

In the average lab, adherent (sticky) mammalian cells are grown in a 10cm-diameter petri dish (a plate), with 10ml of FBS + DMEM media (pink liquid food for cells), in an incubator at 37C with 5% CO2 and a tray of water in the bottom for humidity. In the case of RAW 264.3 or HeLa cells, a 10%-full (10% confluent) plate will reach 100% confluency in two or three days. If nothing is done, the food will run out and the cells will die shortly thereafter, so passaging is required. This is where the media is removed, the cells are washed with phosphate buffered saline (PBS) (salt water), then 1ml of trypsin is added to make the cells detach from the botton of the plate. Trypsin works best in the incubator, so the plate is incubated for five minutes. The plate is removed from the incubator, 9ml of PBS is added and the plate is mixed with a pipettor (triturated). An appropriate number of cells in suspension is then transferred new plates, fresh DMEM is added to each plate, the new plates are put in the incubator, and the cycle begins again.

For best results, cells are kept less than 100% (log phase of growth) but more than 10% confluent. Cells die if they get too lonely or much too crowded.

ee also

*Cell culture