This is done in new media, in a new flask and at a lower cell density or cell count. Cell passaging ensures that cells can continue to grow for future experiments and do not run out of nutrients. Like you and me, cells need to eat and breathe to be happy and healthy!
The frequency of cell passaging needed depends on the growth rate of the cells, the size of the culture vessel, and the seeding density of the cells. When I passage the two cancer cell lines, I seed every new passage at 10% of the previous cell count in a T75 (75 cm2) flask. With these conditions, I passage the cells once or twice a week. On the other hand, I rarely need to passage the macrophage or monocytes that I culture!
Although I typically have at least two flasks of 2.D. cells in the incubator at any time, most of the cellular work that I do in the lab is in 3.D. Growing cells in 3.D. is much different than 2.D! When performing experiments with spheroids, I culture cells in 96-well ultra low adherence plates. By seeding cells in 96 separate wells, I am able to test several different conditions at a time. The ultra low adherence plates are used so that the cells are not attracted to the plate, and instead group and adhere together in a sphere. I seed cells in each well at a cell density of at least 10,000x less than what I seed in flasks. For this reason, cell growth and proliferation happen at slower rates in a well compared to in a flask. I do not have to perform typical “cell passaging” for my 3.D. cells. Instead, I just remove the old media and exchange it to replenish nutrients. I exchange media every other day to every three days, depending on the length of the experiment.
