We cultured antigen - specific CD8+ T cells for 3 passages with IL2 supplement (also stimulated once with CD3/CD28/CD2); we noticed a lot of elongated cells floating (not attached) within the culture (see the image attached - one view presented lots of those irregular - shaped cells). We have never seen these type of floating cells before by using the same parental T cells and the same culture condition.
Normally, after stimulating by CD3/CD28/CD2, we see lots of "spheres (clusters)" plus small round cells, not this type of elongated big floating cells. These cells are also grow super fast. It also seems that this CD8+ T cells lose its specificity by killing all type of cells regardless of antigen of HLA type.
Is it possible a contamination? or our culture agents are no longer good? Too much stimulation? Has anybody seen those big, elongated T cells in the culture? if they are activated CD8+ T cells, they should be round withing a cluster, instead of floating individually?
Thanks!
These elongated floating cells called cytotoxicity , may be due to your culture agents were no longer good .
pls. try to repeat your work with other new cultures agents.
These look like macrophages. Is there any chance that your culture has monocytes/macrophages? If yes, try isolating T cells and then putting them in culture. If you are working with a pure T cell line, your culture may have gotten contaminated somewhere along the way. The third possibility is that these are dead cells. You could harvest the cells, stain them with CD3 (for T cells), CD11b/F4/80/CD14/CD16 (based on the species the cells were derived from) and cell death markers and then do flow cytometry to identify which scenario it is. Or you could just chuck these and start fresh.
You are correct, activated T cells should form clusters and the cluster sizes increase. If you have AICD, cells would die and most often they are round and small. I do see some spindly cells when doing T cell culture from mixed populations (PBMC/spleen and as such) as these cultures have some macrophages/monocytes/DCs. The non-specific killing could be coming from whatever contaminant you have in your culture.
You see normal long cultured CD8+ T cells. Usually they are shaped like worms. They're mobile. Big size and the loss of specificity may be due to too high concentration of IL-2.
We usually see cells like this late in culture, after they have undergone the blast phase (clusters).
