Hi everyone, I am trying to culture IL-2 independent NK-92 cell line for weeks now but they are still not proliferating.
Media used: MEM with 10% NU serum, 0.2 mM myo-inositol, 0.02 mM folic acid, 0.1 mM BME, 1 % Penstrep
After centrifugation, I resuspend the cells in 10 mL of media and placed in T25 flask. Every other day there is still no sign of proliferation nor high density clumping (Almost 3 weeks in culture now). All I see are crumpled cells but in a clump manner (I assume these are dead cells). This is my first time culturing NK92 cells and I'm still not familiar with how they should look like and troubleshooting them. Thanks in advance for any inputs you can provide .
Have these cells been grown without IL-2, before? According to ATCC, good quality IL-2 is very important. Also, they like to be clumped and will lose viability if dispersed to single cells. They also suggest using 12.5% horse serum or FBS in your media.
Hi Dominic,
In my experience NK-92 cells are quite challenging to keep in culture with a high viability. I want to add some things to Kerry's suggestions:
- Consult the culturing guidelines from the supplier. For the ATCC, you can follow this link: https://www.lgcstandards-atcc.org/Products/All/CRL-2408.aspx?geo_country=be#characteristics
- I am personally using alfaMEM Glutamax, no nucleosides (Gibco) + 12.5% FBS + 12.5% Horse serum as medium.
- Be gentle when dispersing the cell clumps, this will improve viability.
- If the viability is low (
Care should be taken when distributing cells to increase viability.
Complete Growth Medium
The base medium for this cell line is Alpha Minimum Essential medium without ribonucleosides and deoxyribonucleosides but with 2 mM L-glutamine and 1.5 g/L sodium bicarbonate . To make the complete growth medium, add the following components to the base medium: 0.2 mM inositol; 0.1 mM 2-mercaptoethanol; 0.02 mM folic acid; horse serum to a final concentration of 12.5%; fetal bovine serum to a final concentration of 12.5%.Culture
Conditions
Temperature: 37°C
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Subculturing
Cultures can be maintained by centrifuging cells and resuspending cell pellet in fresh medium at 2 - 3 x 10e5 viable cells/mL. Centrifugation and full replacement of culture medium may be performed for the first subcultures. Cultures can then be maintained by addition of fresh medium. These cells tend to grow in aggregates that may lose viability when they are dispersed. Accurate counts and viabilities may not be possible.
Maintain cell density between 2 x 10e5 and 1 x 10e6 viable cells/mL or use a 1:3 spilt ratio.
I hope it works.
Kerry S Vistisen I don't know if this has been cultured with IL-2 before, all I know is that this is the IL-2 independent NK-92 cell line. Regarding serum in my media- okay, I'll replace NU serum with 12.5% Horse serum and FBS and see if the cells will proliferate. One reason that I am seeing is that the cells may have produced IL-2 but too diluted (I used 10 mL media because I've cultured them in T-25 flasks)
Gils Roex alfa mem is currently not available in our lab that's why I tried using MEM with Earle's salts as substitute. I still have one vial for thawing and this time I'll set the pipettor to G or low to gently aspirate the cells, thank you. Should I thaw the last vial in 24-well plates first?
Dominic Cardoniga You can do that for sure. Thaw the cells in prewarmed medium, centrifuge and discard the supernatant completely to get rid of the DMSO. Resuspend in medium and let them rest for a while. Bring them to a concentration of 0.3x10e6/mL and divide them over the different wells. Once they start growing, pool them and transfer to an upright T75 (max 20-30 mL volume). I would recommend following the guidelines from the ATCC: for the first several passages, completely refresh the medium (i.e. centrifuge the cells and discard the supernatant).
Keep me posted!
Gils Roex Okay, I'll keep you posted. Thank you for your inputs sir!
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