I’m working with the Caco cell line in an animal cell culture lab. After 24 hours, the medium appears clear and orange, so I did not change the media or check the flask under microscope, However, after 48 hours, the medium turns yellow (but remains clear), I examined the flask under the microscope, the cells were detached from the flask surface, and the morphology of some cells changed, but, there are no visible signs of contamination (no turbidity). The confluency was around 90%. Could you help me understand what might be happening? Are the cells dead or stressed because of confluency? , or could there be another explanation?"
#animal_cell_culture #Caco_2 #Cell_Culture #confluency #contamination
Hello Yasmine Mohsen
You should never allow cell confluency to reach 90%. Always subculture Caco-2 cells when they reach 80% confluency. This is because cells at this stage are still in the log phase of growth and haven't reached the point where they may start to die off or exhibit adverse effects due to high cell density.
If you allow the cells to grow beyond 80% confluency, you will observe the following.
The cells will begin to compete with one another for nutrients in the media and space to grow and divide. Media acidification will set in namely, as the cells metabolize the nutrients, they will produce waste products which can lower the pH of the media turning it yellow. The stressful conditions generated like nutrient depletion and pH change due to overcrowding of cells, may cause detachment of cells from the surface of the culture vessel. Detached cells can be susceptible to cell death, in addition to cells dying due to other factors like nutrient deprivation and overcrowding.
So, I suggest you optimize the seeding density for 80% confluency in a set time. Decide how long you want the cells to take to reach 80% confluency. Start with a range of cell densities. Seed cells into culture vessels at different densities within the range you've chosen. This will help you to identify the cell density that results in approximately 80% confluency at your target time. As far as possible subculture cells before they reach 100% confluency to prevent stress and maintain cell health.
Best.
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