Hi everyone, I am currently culturing NK-92 cells in the ATCC recommended culture medium (Alpha-MEM without ribonucleosides and deoxyribonucleosides but with 2 mM L-glutamine and 1.5 g/L sodium bicarbonate. With: 0.2 mM inositol; 0.1 mM 2-mercaptoethanol; 0.02 mM folic acid; 100-200 U/ml recombinant IL-2; 12.5% horse serum and 12.5% fetal bovine serum)
The culturing has been successful before for a few weeks however now when using a thawed out sample (from liquid nitrogen) I see under the microscope that the cells are not forming the proper (grape-shaped) clumps. In fact they are quite far from globular shaped and are very oddly-shaped. Has anyone had this issue before? My coworker is culturing the same thawed-out cells in the same medium but hers form proper clumps, while mine are very strangely shaped. Any suggestions or ideas would be appreciated, thank you!
This is normal! NK-92 cells prefer to grow in aggregates and can lose viability when dispersed, making accurate cell counts close to impossible. If you got these cells from ATCC, you can confirm this with their Product Sheet and their Cell Micrograph (as other documentation). My lab likes to maintain them by adding media then dispersing them for passage (to start new aggregates) every 3 days or once they’ve reached a high density (over 50% confluence).
Hello Felipe
You can try thawing another fresh vial of NK-92 cells from liquid nitrogen. But this time you try seeding the cells not in T-25 or T-75 flasks but use culture petri dishes. Seed about 0.3 x10e6 cells/ml per petridish. Culture for at least 3-4 days. Then transfer the cells into T-75 flask.
You can also make use of 24-well plate besides petri dishes for initial culturing after thawing the cells.
All the best.
I agree with Malcolm Nobre but would seed them at a density of at least 0.5X10^6 cells/ml with freshly prepared media
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