Multiple biomedical research fields require the use of living cells in order to do multiple assays. These experimental analyses are performed using mammalian cell cultures, as they grow and proliferate rapidly.
Although in order to work with the mammalian cell lines, working in controlled conditions is a must. In addition to that, they also require their own specific growth medium. So monitoring their growth at regular intervals is important for consistency in the experiment.
It is essential for the cells to be subcultured after the cell line reaches about 80% confluence to ensure the health and growth of the cells. This essentially means that the cells should be covering 80% of the cell culture plates.
So here is how you subculture an adherent cell line in a typical workflow.
Why Do I Need To Split My Cells?
Before we get into the experimentation process, we first need to understand why this process is important. Once you start a cell culture, it cannot be grown indefinitely due to nutrient consumption, increased toxic metabolites, and expanding cell numbers. This can eventually lead to the death of cells in the experiments. Moreover, the cells are used several times for experimentation purposes, which is why using them all at once is not practical. So subculturing, or splitting the cells, is the best way to produce new cultures. Granted the new culture would have lower cell density than the original culture.
After you seed the cells, there is an initial lag phase before the cells go into the log phase, where the cells proliferate exponentially. This is followed by a stationary phase where the growth rate of the cells and death rate are uniform. In the death phase, the cells’ poor living conditions and lack of nutrients leads to their end.
So it is necessary to renew the growth mediums and subculture them at regular intervals, in order to keep the cells in the growth phase. Based on the cell type, change in the culture or growth medium can be done multiple times in the log phase. The prime time to subculture these cells is before they reach confluence, in between the log and stationary phase. (Straube and Müller)
How To Subculture Adherent Cells?
You can prepare the cells for subculture by breaking the cell-to-substrate and inter-cell connections with proteolytic enzymes. Trypsin combined with Ethylenediaminetetraacetic acid acts as a calcium chelator and results in cells detaching from the growth surface.
The removal of calcium from cells causes the cadherins to break and separate cells from one another. Once they are separated from the surrounding cells and growth surface, cells can be easily separated and grown in new cell culture plates.
Here is how you can perform a subculture routine on Madin Darby Canine Kidney cells (MDCK) in a 90mm Petri Dish.
You would require a 37° C pre-warmed medium, PBS, and 0.05% Trypsin. The experiment would also require Trypan Blue and 70% Ethanol.
A cell counting chamber, pipette, and inverted microscope are essential to the experiment. Apart from that, you should also have personal protective equipment, a water bath, Erlenmeyer Flask, incubator, centrifuge, and pre-labeled dishes.
Step 1: Examination
Place the cells inside the microscope for a quick check. Cells should be healthy with no contamination and growing as expected. You would notice that the cells are mainly attached to the bottom of the dish and would have a pink-orangish tint to them. They will turn yellow upon acidification as a result of contamination or metabolism.
Step 2: Harvesting
Pipette the medium from the cell into the waste container and store the cells in a clean petri dish. Wash them carefully with 5ml of PBS to get rid of fetal bovine serum from the culture. Add 3ml of trypsin and swirl gently to cover every cell. Incubate at 37°C to detach them from the dish and each other. Make sure to check them every once in a while to avoid damaging the cells. In the end, you can see the detached cells floating in the trypsin solution. You also need to inactivate the trypsin by adding 5ml of the culture medium in the cells.
All you need to do next is transfer the required amount of cells into a new petri dish and let them subculture. You should keep a check on them from time to time so that you can keep an eye on the progress of the experiment.
Just let the cells grow until they are confluent and ready for the subsequent subculture.
Straube, Tamara, and Claudia Müller. “How to do a Proper Cell Culture Quick Check.” Leica Microsystems, 24 March 2016, https://www.leica-microsystems.com/science-lab/how-to-do-a-proper-cell-culture-quick-check/. Accessed 29 September 2021.