Asked 25th Jan, 2022

How to calculate the amount of cell culturing media needed for my fluid-flow cell culturing chamber?

I am building a fluid-flow chamber, However, how can I calculate the amount of culture media needed for the flow experiment? does this depend on the size of the flow chamber? is there any method to calculate that?

Similar questions and discussions

Problems with THP-1 cells. How to culture them?
8 answers
  • Eduardo AlvarezEduardo Alvarez
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?

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