Hi, everyone. I have some issues with THP-1 cell culture. I have little experience in cell culture and I hope some of you could share some experiences in culturing this type of cell.
I'm currently trying to grow non-differentiated THP-1 cells for MTT experiments. The chosen media is composed of RPMI-1640, glutamine, antibiotics, sodium bicarbonate and FBS (10%). These cells either don't grow enough or die over the weeks. So here are my questions:
1 - Should I replace the old media with a new one, or just add the new media to the old media? I've had success in the past by just adding new media to the flasks, but for some reason, it stopped working over time.
2 - How many times should I submit these cells to centrifugation? In order to dispose of the old media and add a new one, I need to submit the suspended cells to centrifugation. But in my experience, this process ends damaging cell structure of some of the cells and leaves cell fragments, which could be detrimental to the culture. I'm currently submitting them to 1200RPM, during 7 minutes at 4ºC, once a week.
3 - Is it normal for these cells to form clusters/clumps?
4 - Should I leave the culture flasks standing on a vertical position? I've seen some people recommending to leave the initial, post thaw, T-25 flasks on a vertical position instead of in a horizontal position. The reasoning behind this is to increase direct contact between cells in a smaller space, as these cells are dependant on each other to grow.
5 - At which confluence should I replace the T-25 flask with a T-75 one?
6 - Should I use 20% FBS for initial growth instead of 10%?
7 - Feel free to write any other tips you consider crucial while dealing with these cells, I really appreciate the help.
Thanks in advance!
Hello Vitor Cardoso Rocha
The following are the answers to your questions.
1 - Should I replace the old media with a new one, or just add the new media to the old media?
When THP-1 cells start to proliferate, they excrete growth factors that aid their growth. So, THP-1 cells grow better in conditioned media. Therefore, whenever you passage the cells, leave 1-2ml of old media and add fresh media to the culture.
2 - How many times should I submit these cells to centrifugation?
Spinning THP-1 cells out to split them can cause stress as the cells will be subjected to centrifugal force. Submitting the cells to 1200rpm for 5-7 minutes once a week is acceptable.
3 - Is it normal for these cells to form clusters/clumps?
Sometimes cells may clump. But if they are growing fine and have a normal morphology, it should not be a problem. Small clumps can be taken care of by just pipetting up and down a couple of times.
However, if they get denser than the required cell density, then they will be very unhappy, may slow down or stop dividing and may clump or have an irregular or blebbing appearance. THP-1 cells are sensitive to cell density. The recommended seeding density is between 3-7 x 10^5 cells/ml. Generally, 5 x10^5 cells /ml is used. Do not allow the cell concentration to exceed 1 X 10 ^6 cells/ml. Change media every 2 to 3 days. Dead cells if they are present should be removed as sometimes these dead cells could be the cause of clumping.
4 -Should I leave the culture flasks standing on a vertical position?
Yes, keep the culture flask in a vertical position until the cells reach the exponential phase of growth. This may take up to 6-7 days.
5 - At which confluence should I replace the T-25 flask with a T-75 one?
You may continue growing the cells in T-25 flask. If additional cells are required, larger flasks/culture volumes may be used.
6 - Should I use 20% FBS for initial growth instead of 10%?
Yes. You need to increase the FBS content to 20% for the first two to three passages, then you can switch over to 10% FBS for successive passaging.
7 - Feel free to write any other tips you consider crucial while dealing with these cells, I really appreciate the help.
a. The first propagation of cells should be for generating stocks for future use. This will ensure stability and performance of cells for subsequent experiments.
b. Growth medium for THP-1: RPMI 1640, 10% FBS, 2 mM L-glutamine.
c. As far as possible, avoid using antibiotics in the growth media.
d. Use THP1 cells with less than 20 passages for cell-based assays.
Good Luck!
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