I use the following composition for 5o ml NK cells culture media (alpha MEM) :
12.5 % Horse serum,
12.5% FBS,
2mM L-glutamine,
0.2mM-inositol,
20µM Folic acid
10 ^ (-4) M β-Mercapto Ethanol,
0.2% NaHco3 (2gram/Litre),
I further use 100 IU/ml each of Human recombinant Interleukin-2 and Interleukin-15 for expansion.
I Change Media 3 times per week. (every 2 days) [spin cells 1000 G for 5 mins, resuspend in 1 ml media, and use 1:1 i.e 500 µL cell suspension for growth in 25-35 mL media in T175 flask)
1X Penicilin /Streptomycin solution (as NK cells media is very prone to mycoplasma contamination)
50 µg/ml chloramphenicol
I have been doing the culture for months now, its 10th passage now since I got the cell. I cannot succeed in making the cell proliferate. Can anyone suggest what is the problem ??
Try doing half media changes rather than full media changes? Perhaps they might require conditioned media?
I am assuming these are primary mouse NK cells.
- eliminate the chloramphenicol
- culture1–3 × 106 NK cells in 6 well plates
- 10,000 IU/ml human rIL-2 (Proleukin; Chiron)
never needed IL15.
Thank you for the responses Annemarie Laumaea and Lora Benoit . They are Human Natural Killer cell lines. But I am little worried if increasing the concentration of Interleukin by 100 times will increase the cost very high. I am still not sure if the concentration of the beta-mercapto-ethanol is doing the bad thing, is it too high for immune cells? I am planning to see decreasing it first hand. changing only the half media seems good idea. I will try that, thanks!
Human NK cells should be cultured in RMPI 1640 not alpha mem.
plate 3 mL at 2 × 106/mL in six-well plates in complete medium with 1000 IU/ml recombinant human IL-2, exchange 1/2 the media every 3 days using a lower dose of IL2 (200-500 IU/mL). You need higher IL2 to begin with because resting NK cells do not normally express CD25. Using the 6 well plate means far less IL-2 and the cells density of the cells is important.
Lora Benoit Thank you so much, I really appreciate. I will try this, and ask if any further confusion.
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