Passaging

The passaging is the process of sub-culturing animal cells. It is usually done to produce large number of cells from pre-existing ones. Instances where it is followed include vaccine production labs, 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 PBS (salt water), then 1ml of trypsin is added to make the cells unstick from the botton of the plate. Trypsin works best in the incubator, so the plate is incubated for 5 minutes. The plate is removed from the incubator, 9ml of PBS is added and the plate is mixed with a pipettor (triturated). 1ml of the mixture is then transferred each of 10 new plates, 10ml of fresh DMEM is added to each plate, the new plates are put in the incubator, and the cycle begins again. If large numbers of cells are not required, only one new plate needs to be made; the rest of the cells get dumped into 10% bleach or 70% ethanol to kill them.

For best results, keep cells less than 100% but more than 10% confluent. Cells die if they get too lonely or much too crowded.