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NK cell conditions - IL2

NK cell conditions - IL2 - Cell Biology and Cell Culture

NK cell conditions - IL2 - Cell Biology Forum. Cell Culture Forum. Post and ask questions about cell culturing, cell lysis, cell transfection, cell growth, and cell biology.


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  #1  
Old 11-14-2008, 01:49 AM
Pipette Filler
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Default NK cell conditions - IL2



Hi - I'm trying to find information about how to maintain isolated human NK cells in culture. I dont need to have them proliferate, but I'm trying to ascertain the correct conditions to keep them viable for functional assays over the course of about a week. The critical thing seems to be the amount of IL2 used, but I would like to use an approximation of physiological conditions, and not activate them so much with IL2 that they are activated regardless of what antigens they encounter. I've been using RPMI, 10% FBS, and 1% pen/strep and 50U/ml IL2...does that seem correct? There seems to be too much cell death. Thanks so much!
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Old 05-05-2009, 06:51 AM
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Default Re: NK cell conditions - IL2

Hi- I have a similar problem with NK cells which I isolate from cord blood. I have been culturing them in IL-2 and IL-15 but have a high level of apoptosis in the first few days. Where you successful with your cells? I have found that there is great variability between the different cord blood units. Do you find the same with your cells?
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Old 05-08-2009, 03:04 PM
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Default Re: NK cell conditions - IL2

I've been moderately successful - I tried using different concentrations of IL2, 12, and 15 to see how they affected NK phenotype (56/16 mainly) and cytotoxicity (against K562 cells) and found that with all the cytokines, the cells tend to skew toward a 16dim phenotype which is odd since that should be less cytotoxic. Viability has been better when I've kept them in a 6 well or 12 well plate instead of in a flask, and using 100U IL2. I think that's what I'm going to go with for now. What makes you use the IL15? I just cant seem to figure out what would be the best conditions for modeling the physiological conditions
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Old 12-02-2009, 08:32 PM
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Default Re: NK cell conditions - IL2

I personally culture human isolated NK cells all the time. The best way to culture this cells is indeed with 0.04U/ml of IL-2 in RPMI with 10%FBS, 1%Strep/Pen, 2% L-Glu. Very importantly, NK cells don't really like to hang around alone, so we always add irradiated 721 cell line and all-PBMCs. We irradiate these cells at 5000rads. Another thing is that NK cells seems to like round bottom plates, so we use 96 well plates round bottom. For one 96-well plate we add:
1 million NK cells
1.5 million 721
and 20 million allo PBMCs, but remember the 721 and the allo-PBMCs has to be irradiated.

culturing in IL-2 is going to make the cells proliferate so after a week or so in culture you will have to split those cells into 2 plates. I've personally tried culturing NKs without the feeders and most of the time they die. I hope this works, and if you have any questions you can contact me.
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Old 12-02-2009, 08:36 PM
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Default Re: NK cell conditions - IL2

IL-12 and IL-15 are both produce by mature DCs. NK and DCs are known to interact with each other resulting in reciprocal activation. IL-12 and IL-15 promotes NK proliferation and survival. So adding either one is indeed physiologically relevant.
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Old 04-01-2010, 04:32 AM
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Default Re: NK cell conditions - IL2

KACO, any luck with the NK cell culture?
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Old 04-01-2010, 04:39 AM
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Default Re: NK cell conditions - IL2

in more detail,
I stumbled over this post regarding NK cell culturing conditions.
I am going to try the same thing but we don;t really know with what to start.
1. we aim at culturing PBMC and check whether NK cells will survive in this context, however I do not wnat to activate them. Currently we are thinking about adding PHA and or Il-2 but I fear that both will activate them. I know CD15 and hi Il2 will lead to expansion, another thing I certainly do not want to happen.
I suggested to culture them in stimulant free medium for a week, just because we dont know what will happen.
2. I thought about culturing sorted NK alone - same thing here, keep them alive but don't activate.
anyone with good ideas?
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Old 06-16-2011, 08:00 PM
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Default Re: NK cell conditions - IL2

Quote:
Originally Posted by axv56 View Post
I personally culture human isolated NK cells all the time. The best way to culture this cells is indeed with 0.04U/ml of IL-2 in RPMI with 10%FBS, 1%Strep/Pen, 2% L-Glu. Very importantly, NK cells don't really like to hang around alone, so we always add irradiated 721 cell line and all-PBMCs. We irradiate these cells at 5000rads. Another thing is that NK cells seems to like round bottom plates, so we use 96 well plates round bottom. For one 96-well plate we add:
1 million NK cells
1.5 million 721
and 20 million allo PBMCs, but remember the 721 and the allo-PBMCs has to be irradiated.

culturing in IL-2 is going to make the cells proliferate so after a week or so in culture you will have to split those cells into 2 plates. I've personally tried culturing NKs without the feeders and most of the time they die. I hope this works, and if you have any questions you can contact me.
Hi Axv56, I am also working on NKs cells. Your post on the culturing conditions sounds reasonable and I will give it a try. Do you have a publication I can refer to?

-Victor
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