Dear Cyrille,

1) 300 IU of IL-2 is huge concentration. I think, in 2 days after transduction you have got LAK cells, lysing nonspecifically other cells. To grow T cells, use no more than 5-10 IU/ml.

2) Even retroviral transduction does not result in 100% stable expression. After several days, decrease in the percent of cells, expressing the gene of interest is ordinary event. Therefore, you need to select required cells and, moreover, it would be nice to perform subsequent cloning of transfectants to obtain stable cell lines. Otherwise, you will deal with complex cell population changing characteristics due to selection processes. As a rule, cells not expressing the gene of interest will compete better and you will have opportunity to perform no more than 1-2 experiments with hardly reproducible results.

3) If you plan to perform subsequent experiments in vitro, you can use transformed cells (BW5147, 4G4, etc.) instead of splenocytes for retroviral transduction. It would be much easier.

Dear Cyrille,

1) 300 IU of IL-2 is huge concentration. I think, in 2 days after transduction you have got LAK cells, lysing nonspecifically other cells. To grow T cells, use no more than 5-10 IU/ml.

2) Even retroviral transduction does not result in 100% stable expression. After several days, decrease in the percent of cells, expressing the gene of interest is ordinary event. Therefore, you need to select required cells and, moreover, it would be nice to perform subsequent cloning of transfectants to obtain stable cell lines. Otherwise, you will deal with complex cell population changing characteristics due to selection processes. As a rule, cells not expressing the gene of interest will compete better and you will have opportunity to perform no more than 1-2 experiments with hardly reproducible results.

3) If you plan to perform subsequent experiments in vitro, you can use transformed cells (BW5147, 4G4, etc.) instead of splenocytes for retroviral transduction. It would be much easier.

Hi Cyrille,

we usually culture murine splenocytes in alpha-MEM supplemented with glutamine, beta-Me, 10% FCS, Pen/Strep, IL-2 and MMP. This way, we are able to culture splenocytes for at least 2 weeks. But I guess you need an additional in vitro stimulus to prevent the T-cells from dying.

Dear Cyrille

you may consider use of untransducted T cells for evaluation of cell survival in 2 weeks and then finding of transduction problems.

these articles may help you:

Cytotherapy. 2009;11(2):206-17

http://www.jove.com/video/2307/retroviral-transduction-of-t-cell-receptors-in-mouse-t-cells

Best Regards

Dmitry's answer is perfect. The only thing I can add is that optimal IL-2 concentration varies by batch and manufacturer, even though it is supposedly standardized. I assume you are using human rIL-2, but species source is also a factor. The optimal range for primary T cells is 5-20 IU. You may need to titrate to find the best concentration. You will also need to refresh the IL-2 even though the media itself has not become exhausted. When IL-2 dependent T cell lines use up available IL-2, they all will be healthy one day and dead the next. The media doesn't change color. If your experiments require IL-2 dependent T cells, there are clonal IL-2 dependent cell lines such as CTLL-2. BW5147 and other T-lymphoma lines are not IL-2 dependent, and are quite easy to grow. You didn't say which mouse strain you were using, so if it's important, select a T-lymphoma line that suits your needs. There are many around from different strains, and they are easy to transduce.

Hi Cyrille,

one other protocol you can use is to add in steadof IL2 add 5-10% Rat ConA sup. You can find protocols in literature or in protocols in Immunology.

Also add thymocytes that can help keep the cells feel better.

Best regards,

john

hi Cyrille,

you can use MEM+FCS 10% and glut strep. You have to use invitro t cell stimulant to prevent the cells from dying. lalitha kabilan

Please refer to our recent method paper

I want to thank all of you for your kind replies.

Your experience was typical since it has been difficult to keep in vitro growth of normal splenocytes i.e. With reducing survival from 70 % after 72 hr to 20 % for the next days, unless we provide any stimulus in addition to basic medium and supplements plus GF, cytokines or other ligands and allogen. IL2 is increasing its effectivity by the time, related to the generation of IL2R in the activated T cels. It will be interesting to check for the surviiving cells for the IL2R and/or CD25 marker and see the kinetic in relation to your purpose of characterizing the transduction exp. another consideration, whether the manipulation has influence on the apoptosis of any subset of the survivor spleen cells / aT cells, e.g Th1 and Th2' that might have a kind of downregulation of one and upregulation of the other. Such has been known in certain immunoregulation, including specific generation of cytotoxic T cells for allogenic stimulation that could cause prolonged T cell growth.

In addition I wonder whether you might consider any help of coculturing with dendritic cellls as recently used. The. Studies of Dr. Chen's group is good to be considered.