Hi Alejandra,

For the expansion of your NK cell the easiest protocol is to cultured with IL-2. And yes you will need to isolate your NK cell first or else T cell will use all the IL-2. I will advise you to use freshly NK cells (more robust) but it should also work from frozen cells. How much time do you attend to culture your NK cells ?

Hi Olivier, I have been reading and some authors say IL-2 stim is not too good for the expansion. Many of them use cell lines, but I rather not to use cell lines.

The problem I have with expanding from NK cells is that my patients won´t have many NK cells so I don´t know if it will work that way...? what´s the minimun NK cell number I can use?

Length of expansion I guess whatever is needed, 1-2 weeks will be fine? not sure.

I have found two kits for NK cell expansion: one from miltenyi and another from R&D. Do you have any experience with them or know anyone that has used them?

Hi Alejandra,

We always used to culture the NK-cells with IL-2 and Beta-mercapto-ethanol.

For the phenotyping we always used pooled buffycoats so we did not have to expand NK-cells after purification (we used MACS from miltenyi and got about 95% purity)

Have a look at this paper: 2001 Blackwell Science Ltd, Scandinavian Journal of Immunology, 53, 103±110 by Jonges et al.

With kind regards,

Ronald

Hi Ronald, thanks but I cannot use buffycoats, I will have just 20ml of peripheral blood and from healthy donors purifing the NK cells and then expand will work but from patients might be too dificult as they won´t have much cells. That is why we want to expand them. But all protocols using PBMCs are done using cell lines, the ones I find using cytokines need to purify first.

I guess I will have to choose whether to use cell lines to stim or to try to purify first then the cytokines.

Thanks anyway!

Hi Alejandra,

20 ML is enough to isolated NK cells. You should check Rosettesep from stemcell. It works well to isolated form whole blood. Then you could start by culturing NK cell in 48 or 24 plates with IL-2 ( I think is 100-500 unit/ML) even if you don't have much (always keep them around 1.5 millions/mL). They are going to expanded eventually.

I never used NK expansion kit.

OK, I will try as you suggest then, the only problem will be if patients have very low numbers of cells. I will see how much I get from them.

Does it work with frozen samples then? project has started already and I have been incorporated to it last minute so part of the samples are already frozen.

Thanks

I never try on frozen sample. But I think stemcell also provide a specific NK isolation kit from PBMC (easysep). So if samples are already frozen you should continue isolate NK cell from frozen PBMC.

Hi Alejandra,

Yes you can still get enough NK cells for culture from 20mL whole blood. I used MACSexpress to isolate NK cells directly from whole blood and worked pretty well, I could get enough cells for culture from 30mL whole blood. Please have a look at this poster, I think it contains useful information for you: http://www.miltenyibiotec.com/~/media/Files/Navigation/Research/ApplicationSpotlight/Fast_NK_cell_isolation_poster.ashx

Also, as Olivier said, I would recommend to start the cultures in 48-24 well plates and plate the cells at no less than 300.000cells/mL. Optimal starting cell density would be 1million cells/mL in 24 well plates, but if you don´t get so many, then I would go for 3-500.000 cells/mL in 48 well plates. I would also recommend you to use NK MACS medium from Miltenyi supplemented with 5% human AB serum and 500 or 1000 U/mL of IL-2.

Regarding the NK cells from patients, I think the major problem may not be the numbers that you can get but rather that they won´t expand so well. For this, I would recommend to start the culture directly using PBMCs in the same cell densities and plates used for purified NK cells and NK MACS medium with same mentionned supplements. In 2 weeks NK cells should overgrow all other PBMC cell types and you can re-isolate them in case you need high purities to perform phenotypic or functional assays.

I hope this helps with your studies! Good luck!

Kind regards,

Maria

Hi Alejandra,

You already received great comments so far.

We recently published a review articel about NK cell expansion that could be inetersting for you as well (if you don't know it yet, because you seem to know already quite a bit of the different options): Article Shaping of Natural Killer Cell Antitumor Activity by Ex Vivo...

Expansion from freshly isolated cells is much more efficient.

There are several protocols that favor outgrowth of NK cells over T cells when starting with PBMC and the non-NK cells will support the NK cells to grow even better.

Isolation/Purification should be possible without any problems prior or after cultivation.

Let me know if you need more details for specific approaches.

Best wishes,

Markus

Dear Markus, thank you so much I will read it carefully and will contact you if I need further details.

Regards,

Alejandra.

Dear Maria, thanks for your input! I think with all this info received I can start planning experiments. Thanks to all for the help.