Passaging Cells

Passaging Cells

Summary

Passaging cells is the process of removing a medium and transferring cells from a previous culture into a new, fresh growth medium (ThermoFisher). Passaging, also called subculturing, is necessary because this ensures that growing and proliferating cells will not use up all the nutrients provided from the medium, which will lead to a decrease in proliferation and the cells will die. When cells are growing in a medium, a typical cell growth pattern is observed. Cells that have just been introduced into new medium enter a "lag phase" where they are adjusting to the medium and preparing for growth; this phase is relatively short, lasting about one day. Then the cells enter the "log phase", where they proliferate fast and cell density increases exponentially. It is during this phase where the nutrients in the medium are consumed, and once the medium has no nutrients left, proliferation stops and the cell density levels off. The "log phase" can last ~6 or 7 days, and cells should be passaged during this phase to ensure the growing cells don't use all the nutrients. When you passage your cells depends on the cell line you work with, but with our LM45 or LM45-CD47 cells, we typically passage them every 6-7 days.

In our Lab

Currently, we only passage and culture two lines of cells: LM45 and LM45-CD47. They were both types of melanoma, with the LM45-CD47 cells permanently expressing GFP (and the cells were a gift from Dr. Baer in the ATSU Physiology Department). Although we passage these cells typically once every ~ 7 days, when we passage depends largely on the confluence of the cells in each dish. Confluency refers to the percent of the bottom of the dish is covered by the cells, with 100% being used to describe a dish who's surface is completely covered. Ideal passaging is around ~90% confluence (or when you believe the nutrients in the media are running low). When passaging, we take only a small amount of cells from the highly confluent dish (usually one small drop) and place it a new dish with fresh media. This is then repeated every week or so. The protocol can be seen here.

Protocol