Good day, I am having problems with culturing my THP-1 cells. I tried culturing them in several different volumes, I was recommended to seed 5,000,000 cells in 100mL to 150mL and let them sit their for around 1 week to 1 week and a half or so until concentration reaches 1,000,000 cells/mL. Now I have read these cells need cell to cell contact to grow. What I am doing now is seeding 5,000,000 cells in 25mL of RPMI 1640 with a vertical T-75 flask but they still grow slowly and sometimes they are not even viable. Some of my questions regarding to this matter are:
1) Why are my THP-1 cells growing so slowly? I seeded them on a friday at 200,000 cells/mL at 4pm and when I returned on monday at 10am, the medium has barely changed in pH color and I when I observed them through the ZOE microscope, they can be seen most in clusters but there do not appear to be much of them.
2) How do you count these cells or any suspension cells? Every time i try to count THP-1 cells through a hemacytometer, I do not see any cells present. It is very rare to see a cell in the hemacytometer, however, I know my flask has cells because I can see them through the microscope. I tried taking 10uL of cells directly from the flask, I tried taking +/- 100uL of cells and transferring them to an eppendorf tube and from there to count in a ratio of 1:1 and nothing. How exactly do you count suspension cells? Isn't it better to concentrate the cells into a pellet, resuspend the pellet in 1mL of culture media and then count them for a more accurate result rather than taking a small aliquot of culture media directly from the flask and count them?
3) How do you add media to these cells? I have read where people add media every 2 to 3 days to the old media. However, If I add more media to the cells with the pH color being intact and with little cells, aren't I diluting them further? I read these cells need cell to cell contact to grow. When adding even more media on top of the old one, doesn't that increase the passage number? Wouldn't it be better to subculture them to another flask with fresh media?
(1) Use this medium:
RPMI-1640 (+) L-glutamine + 10% FCS + Penicillin-Streptomycin + Non-essential amino acid + 10 mM HEPES + 50 uM beta-mercaptoethanol
because THP-1 cells like this pH environment and beta-mercapoethanol for proliferation
If you start from a frozen vial, resuspend cells and maintain them at a high density, give them 3-5 days. Check mycoplasma if necessary.
(2) If (1) is correct, you can see cells round and not in clusters. Pipet up and down for a couple of time before counting single-cell suspensions.
(3) THP-1 cells are a bit tolerant to acidic environment. Usually simply remove 50%-75% old culture and refill new medium to the original volume. Always retain HEPES and maintain the cells at a high density.
Good luck!
THP1 usually grow fast unless they are contaminated, differentiated or in bad quality media. From what you are saying they hardly if at all. Options: 1.Dispose and start new another batch -another source of cells. 2 . check versus another cell line as control or change media batch especially serum ( is it heat inactivated ?) 3. check for mycoplasma
Thawed total 9.2E5 THP-1 cells and mixed into total 15 ml of supplemented RPMI 1640. Actually experimented with variations on serum and other supplements (ferric pyrophosphate and/or beta-mercaptoethanol, the latter being recommended by ATCC), and found that iron-supplemented calf serum was better for initial THP-1 growth than FBS, although it seemed to cause some differentiation, and FBS eventually worked for growth without differentiation (but with a delay of a week after thawing). For growth, finally settled on FBS + supplement of 368 ug/l ferric pyrophosphate, equivalent to 6.6 uM Fe(3+) final concentration in the medium.
ATCC gives a dire warning not to go over 1.0E6 cells/ml, but I found that you can easily exceed this up to 2X without harm. They recommend 50 uM (= 3.9 mg/l = 3.5 ml/l) beta-mercaptoethanol (78.13 g/mole, density 1.114 g/ml), but in practice this seems to be too much and inhibits growth; smaller amounts seem that they may be stimulatory, but I only experimented a few times with this, and any beneficial effect is not huge, and isn't worth the trouble.
For differentiating them more fully than what constant phorbol myristate acetate or constant 1,25-dihydroxyvitamin-D[3] will do, see Article The Identification of Markers of Macrophage Differentiation ...
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Hello Eduardo
I agree with all the previous answers, however I don't use b-ME and the cells are fine. I also had a problem with culturing THP-1 cells - low viability, an addition of fresh L-glutamine did the trick. Does your medium contain regular glutamine or GlutaMax? In the first case I would suggest adding some fresh glutamine because it tends to degrade fast in the medium. I also never had any problems with counting THP-1 cells, even collecting 10 ul from the flask but I use much smaller volume (10 ml). I always seed them at 0,2 mln/ml and passage at around 0,6-0,7 mln/ml. Most of the time I don't even count then just visually inspect the flask ;)
Hope you'll get to a better terms with your cells soon.
All the Best
Agnieszka
Dear Edwardo,
THP-1 has a doubling time of less than 20 hours. The problem lies in the seeding area of cells. THP-1 cells should be grown at a density of 1 million to 2 million/mL .In a 24 well 1 million cells could be easily seeded and grown( no differentiation). In a 250ml culture flask the maximum media should be 50mL and between 20-30 million for seeding , after this the flask should be kept horizontally for proper balancing and aeration. you should check for l-gluatmine in media. L-glutamine decays quicker ( less than 25 days), use long acting l-Glutamine available in the market. L_gluatmine concentrations has greater impact on THP-1 growth. lesser concentrations can even screw up the cell morphology ,as well lead to cell death.also check co2 concentrations , it should always be 5%.
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