I use the following composition for NK-92 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,

2.2 gram/Litre NaHCO3 ,

100 IU/ml each of Human recombinant Interleukin-2,

100 IU/ml Interleukin-15,

1X Penicilin /Streptomycin solution

50 µg/ml chloramphenicol

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 20-25 mL media in T175 flask)

but since the cell number is not growing, I use all the cells in new media, it is as if I am just changing the media keeping the same number of cells everytime. I have not been able to cryopreserve the cells as the cells are very less in number.

I have been doing the culture for months now, its 10thpassage now since I got the cell. I cannot succeed in making the cell proliferate. Can anyone suggest what is the problem?? The cells are alive but its not increasing in number. Can anyone suggest what can I do to further increase the proliferation???