Hello everybody
I am working on expanding and activating CD8 T cells from mouse spleens for my experimental work, but i have some troubles with the number of cells I got after 3 days culture. I am appreciating any suggestions for trouble shooting.
I purify CD8 t cells from mouse spleen single cell suspension using magnetic beads, and culture them in anti-CD3 coated wells, soluble anti-CD28 with or without IL-2 and IL-7. My cells form clusters in wells but the number of the cells is always less than the starting number. They look bigger and nice under the microscope but they don't expand. what could be the problem with my cells?
I used anti-CD3 1ug/ml, anti-CD28 1ug/ml, IL-2 0.2ug/ml, and IL-7 10ng/ml.
I'll have to let others answer this question about the concentration of hte cytokines. but normally expansion of CD4 t cells takes 7 to 10 days. I'm going to assume that it's the same for CD8s?
Hello Shada,
from your questions and your experiment design, I see that you are trying to stimulate CD8 T cells to induce a certain profile. So why are you using IL-2, low frequency of CD8 T cells express IL-2RA (CD25) receptor and IL-7R (CD127) is even lower: Is it related to Treg profile cytokine stimulation in vitro? If not you should re-establish your experiments design with the proper cytonies. CD8 T cells are quiet different from CD4 T cells. Instead of beads you can try to expand them with DCs cells in presence of GM-CSF from same spleen or with irradiated splenocyte ( to test).
As Shen-An Hwang mentioned, 3 days is too early to see expansion. Not all CD8+ T cells you plate would survive and proliferate. You need to leave the surviving cells for at least 5-6 days to see an appreciable increase in numbers. As to the cytokines, IL-2 and IL-7 work equally well in humans while IL-2 is more often used when expanding mouse cells. I do not see a problem in using the combo. I used to use 1-10 ng/mL IL-7 and/or 1-10 ng/mL of IL-2. Try reducing the amount of IL-2 and leave the cultures for 7-10 days.
Fadi Jebbawi Yes I am trying to get antigen-independent Cytotoxic T cells (CTL). I used anti-CD28 ab and antiCD3e Ab to stimulate T cell expansion and to activate them. IL-2 is a growth factor for T cells and it induces CD8+ T cells activation and expansion (as in the lit.), IL-7 maintains the viability of CD8 T cells. That's why I tried adding them to the culture.
I would try expanding them with DC, but isn't it in this way becomes Antigen specific?
Julie Joseph I tried combining IL-7 (10ng/ml) with IL-2 (0.1ug/ml), but the cells did not form any clusters after 3 days culture, and they even did not expand. So I would try lower concentrations of IL-2 with IL-7. Don't you think that letting them in culture for 7 days is too long time? usually CD8+ T cells don't survive that long.
As long as your cells are stimulated, they shouldn't die off. So leaving the cells for 7 days shouldn't be a problem. You may have some AICD but I doubt it. You could always take a look at the cells every day and terminate the cultures earlier if you notice anything.
If all you want are a lot of T cells, you could try stimulating them with PMA+ionomycin. This should be a super strong stimulation and you may have a lot of expansion by day 3. You just have to make sure that you are not adding too much and inducing a lot of AICD.
I recommend you to :
-Most importantly , sort your cells instead of magnetics procedure as you might have contamination with other cell type as CD4 T cells.
-Check your medium, FBS, cytokines and beads on other T cells population as CD4 T cells taken as control.
-Check the expression of IL-2RA and IL-7R on CD8 T cells surface
-Maintain your cell culture for 2-3 weeks and change your medium each time you see it necessary without stimulating the cells again with beads .
-To make sure that your cells are activated (check activating markers or cytokines secretion in the supernatant of your cell culture)
-As Julie suggest you can also use unspecific stimulation by PMA/ionomycin as control
and hopefully you might get a better expansion...
hi dear Shada,
you should have stimulated T cell with CD3/CD28 and then fed them with IL-2 supplementation "every 2 to 3 days" until about 9 days . then you can removed anti-CD28 and anti-CD3 but you should keep in culture with IL-2/IL-15 supplementation every 2 to 3 days. so you can get the amount you want.
why don't you use of IL-15 instead of IL-7?
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