I'm new to T cell culture and would like to know how people generally culture antigen specific T cells. My plan is to isolate PBMCs from donor derived peripheral blood. Add specific peptides to activate the PBMC. After culturing for like 10 days (I'm not sure how long I should be culturing the T cells. This is a number that I saw most of the times in literature), do a flow cytometry to isolate the antigen specifc T cells I want. That is the plan and I would like to know as much as possible about the details in T cell culturing. If you could provide a protocol, that would be AWESOME. Thank you very much in advance.
For my T cell assays, I lyse the RBC before I plate the bulk PMBC. I add peptides at 1 ug/ml at the time of culturing PBMC. 2 days later, I add IL-2 and IL-7 (by replacing half of media with cytokine media) and I feed cells every 2-3 days with cytokines. I split cells only if the media is acidified. I look at CD8 T cell responses on Day 14 and CD4 T cell responses on Day 21 by re-stimulating cells with anti-CD28, anti-CD49d antibodies and peptides (negative control peptide and/or DMSO, test peptide, positive control peptide -flu etc.) and I include PMA/Ionomycin treated cells as an additional positive control. Then I look at IFN-g expression either by intracellular staining or ELISPOT.
Is this in humans or an animal model? What do you want to do with the cells after you isolate them? Also, do you mean FACS or flow? With flow you only collect the data for the cells, not the cells themselves.
Hi Jeff, Once you have isolated your PBMCs - usually by density separation eg Lymphoprep 1.077g/ml for human lymphhocytes - should be able to recover 1-2 million lymphocytes/ml of whole blood. I usually use a TC media eg RPMI-1640 supplemented with 10-20% FCS, pen/strep, 55mM 2ME, 1mM pyruvate, 2mM glutamine and non-essential amino acids for optimal proliferative support for 7 days of stimulation. For 10 days you may need to half change your medium, if it's starting to look yellow(acidified), every 3 days, this will depend on the metabolic demands of your stimulated cells(and remember to do the same with controls). Try not to disturb your cells when half replacing the media. After your period of culture you'll need to harvest your cells, wash, label with appropriate antibody/fluorophore, resuspend at ~5*million/ml and sort for your population(s) of interest. Check sort purity by reacquisition(record 500-1000 live cells), wash and count. Re-suspend as appropriate for down stream assay.
Thank you Martin. A couple follow-up questions:
1. Do you do RBC lysis after you have isolated PBMC from Ficoll centrifugation?
2. Do you see T cell proliferation after you have added specific peptides and say, IL-2 ( I don't know if you add IL-2 to your medium )?
3. Do your PBMCs clump massively in cell culture? If yes, what do you do with them? Thank you again. :D
morning jeff, For PBMC that have been density gradient separated no, they should be relatively free of rbc. Unless you have poor viability in your isolated PBMCs there should be minimal aggregation issues. Depending on the cells you are trying to expand you may supplement with IL2, to enhance/increase general activation ± secondary cytokines eg IL-7/-15 reported to support survival of CD8, or IL-4/-5 for CD4 expansion. Your specific assay end point will influence the optimal cytokine supplementation you require. Best wishes
Maybe my cells are dead already when they have clumped in the culture. After I pipet them up and down a couple times and then do a centrifugation @300g, 10mins, the cell pellet is almost invisible. Even after I resuspend the remaining cell pellet in culture, I see very little cells.
So for my second question, do you see PBMCs divide and proliferate after adding peptide antigens and cytokines? I don't see any... : ( Don't know what is wrong.
Jeff, Where do you get your PBMCs from and what is the cell viability of your isolated PBMCs initially, at set-up? I find viability above 95% with freshly isolated cells from fresh collection or buffy-coats up to a couple of days old, stored at 22°C. Clumping usually a result of free DNA, released after dead cell lysis, but human PBMCs, unlike murine do not default to apoptosis in culture without stimulation, so viability of controls in appropriate culture media should still be high(85-90%). Dead cells tend toward translucent, so instead of a white pellet(live) there is a near invisible one, as with fixed/permeabilised cells for cell cycle. As too proliferation, in antigen stimulated cultures I would expect to see foci of proliferating cells expanding from an antigen reactive progenitor. In flow cytometry, people track stimulation with cell proliferation dyes or induction of activation markers eg CD69(early) or CD45RO(late). It depends on what you are looking for?
I get my PBMCs fresh from donors. I usually separate PBMCs from peripheral blood within 8 hours. Viabiligy-wise, I think it's pretty high in the first couple of days, as there's minimal clumping issue in the culture in the first 3-4 days. It's after that period of time that I start seeing clumping get bigger and bigger.
The picture you attached, I take it as focal expansion of antigen reactive T cells? I don't see anything like that after I add my peptides to my PBMC culture. I dissolve my peptides to 1mg/ml in DMSO, and add 10ul to 1ml medium directly, so the final concentration is 10ug/ml. Is there anything unusual in what I do?
I'll get some pics of my PBMC culture and attach them in the thread later. They're usually in single cells in the first 3-4 days of culture and then start to clump, as I said earlier. Thanks again Martin.
Hello Jeff, Your final [DMSO] concentration could be a factor, reports would indicate cells can tolerate up to 0.1% in the final culture medium, but I use lower concentrations. What do the cells in your control wells(carrier without peptide) appear compared with experimental(carrier+peptide) with regard clumping.
Hi Martin, Attached please find my PBMC culture pics. This is not fresh PBMC culture. I thawed them yesterday. This is what they look like in culture on the second day of thawing. I didn't do a RBC lysis step, so I think there's a lot of RBCs in my culture.
With regard to peptide stimulation, I didn't do a DMSO w/o peptide control. So I don't know what my cells will look like under DMSO only condition. But I add peptide/DMSO on the second day of culture, and supplement fresh peptide/DMSO every time I change my medium. If DMSO is causing cell death, I should start seeing clumping from the second day on.
Another note, how do you stimulate your t cells? do you only pulse the cells for couple hours then change medium or keep them in culture throughout the process? Thank you.
Jeff, I don't know what your assay format is but I would expect a carrier alone control to be included, I wouldn't think media alone would not be enough, as DMSO is reported to have effects on cell differentiation in many systems. A carrier control would also help answer where your stimulation issue resides. Do you completely replace your medium? I would set-up the assay, add DMSO±peptide in minimal volume and if I needed to refresh the media, I would half change to maintain paracrine and autocribe levels. Freezing will usually lyse any contaminating rbcs, you may see pyknotic-like bodies, little dark spotty material. Are all your cells Isolated fresh, but stored frozen before use?If so, It may be a freezing/thawing protocol
I'd add a DMSO w/o peptide control. When I change the medium, I resuspend the cells and basically split them into 2 wells. Then replenish with fresh medium to original volume (total volume is 1ml per 24-well). So I guess if my cells are expanding like a cell line, cell density wise it's OK. But if my cells are staying stagnant, i.e. not dividing. I'm halving my cell density every time I expand my cells. The initial concern is I don't want to lose any PBMCs.
Not all my cells are stored frozen before use. The reason I use frozen cells is because I've used up all my fresh ones. When I isolate the PBMCs, I keep 1*10^7 cells for experiments right away and freeze the rest of the cells for later use. Thanks.
Hi Jef,These are probably are small clumps of live cells as they look bright and shiny, though a bit mis-shapen, are they floating above the floor of the plate...rather than stuck down? You might want to add in a mitogen control too. This would confirm the proliferative potential of the cells independently. Here are mitogen(PHA) stimulated PBMC @ day 6 of culture.
Hi Martin, I did a Ficoll Paque Gradient Separation of PBMCs again, fresh from the peripheral blood of a donor. Attached please find the pic of my cells. I realized I had been using 400rpm to separate my PBMCs when I thought that was 400g. The centrifuge isn't a very advanced one. How stupid of me.
Anyway, so my PBMCs are much more dense in culture than previous ones, as you can see now. From my hemocytometer counting, the culture density is about 2*10^6 cells/ml/well. I'm planning to add my peptides to stimulate my cells. I will pulse stimulate some wells and leave the peptides in culture in other wells. My purpose is to get as many antigen specific T cells as possible, for down-stream applications.
Do you think cell culture density plays a huge role in stimulating T cell proliferation, based on your experiences? Thanks.
Oh, as for your previous questions, the cells are float, but attaches a bit to the plate. When I shake the plates, the cell clump fluctuates a bit, but some of the clump attaches to the plate.
I'm completely new to T cell culture or primary cell culture, and my lab doesn't have PHA. I'll look around to see any lab has that and probably borrow some before my own PHA arrives.
For my T cell assays, I lyse the RBC before I plate the bulk PMBC. I add peptides at 1 ug/ml at the time of culturing PBMC. 2 days later, I add IL-2 and IL-7 (by replacing half of media with cytokine media) and I feed cells every 2-3 days with cytokines. I split cells only if the media is acidified. I look at CD8 T cell responses on Day 14 and CD4 T cell responses on Day 21 by re-stimulating cells with anti-CD28, anti-CD49d antibodies and peptides (negative control peptide and/or DMSO, test peptide, positive control peptide -flu etc.) and I include PMA/Ionomycin treated cells as an additional positive control. Then I look at IFN-g expression either by intracellular staining or ELISPOT.
Hi Cansu, I guess it's better to keep the peptides in PBMC culture if you're looking at IFN-g expression, to provide persistent stimulation? Do you know much about TCR stimulation? I heard TCR expression down-regulates if you keep stimulating the T cells?
Also, what conc. of IL-2 do you use to stimulate your T cells? Thanks.
Afternoon Jeff, Sorry for the delay in response but this is looking good. These are myeloid cells in your the PBMC mix and is what you would expect to see in a good culture. Cansu's protocol looks good for a re-stimulation assay scenario that matches your requirements, and if she is actively performing these assays, will generally provide better value information, than a literature survey. The only source of knowledge is experience(albert einstein). best wishes for your future re-stimulation assays
Jeff, I use IL-2 and IL-7 at 10 IU/ml and 10 ng/ml final concentrations, respectively.
Also, the peptides I add would not be stimulating the T cells throughout 2-3 weeks of culture. I don't know the exact degradation kinetics but I'm pretty sure the peptides degrade rather quickly (by peptidases in human serum etc.). In fact I found out that if I add peptide multiple times during the culture instead of just adding once at the beginning, I increase the T cell response.
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