Question
Asked 14th Aug, 2015

Can anyone please explain how to culture T cells from PBMC ?

"T cell - primary culture from peripheral blood." by Raulf-Heimsoth explains it.but I cannot access the paper. The purpose is to study the T cell proliiferation .

Most recent answer

21st Aug, 2015
Francesco Nicoli
University of Ferrara
Dear Maneesha, it depends if you want to culture all T cells or just epitope specific T cells. In the latter case it be easy if you want to stimulate memory cells for some infection/vaccination that everyone for sure has, but you need to know HLA.
If you want to culture total T cells, you can activate wit coated CD3 and soluble CD28 and maybe keep in IL2 (but only after 5-6 days of culture). Here the concentration we used (http://www.ncbi.nlm.nih.gov/pubmed/24841128).  To avoid death after hyperactivation, I suggest you to use low doses of stimulation ....better to do a titration experiment before. For the medium, we used normal RPMI+10%FCS. In this way, T cells will be cultured together with other cells, that anyway will die (although not completly) during the course of culture. Replacing the media (and adding IL2) every 3-4 days may halp to culture cells up to 20 days or more.
If you want to culture purified total T cells, for sure you have to use some kits that separe it.
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Popular answers (1)

14th Aug, 2015
Sudipta Tripathi
Brigham and Women's Hospital
The paper is attached. Good luck.
4 Recommendations

All Answers (5)

14th Aug, 2015
Sudipta Tripathi
Brigham and Women's Hospital
The paper is attached. Good luck.
4 Recommendations
14th Aug, 2015
Benjamin von Bredow
Penn State Hershey Medical Center and Penn State College of Medicine
I separate them using the EasySep kit by Stemcell technologies. For culturing, I use RPMI + 20% FBS and stimulation with Life Technologies Human T-Activator CD3/CD28 Dynabeads. It works very well, and you can culture the cells for an extended period of time.
4 Recommendations
17th Aug, 2015
Eytan Breman
Celyad
Culturing T-cells is not that difficult you need to stimulate and thats about it. Depending on their enviorenment (in the presence of antigen presenting cells) you can use either PHA or CD3 alone. I would add IL-2 as a survival signal. To stimulate a pure T-cell population you can use PMA/Ionomycin or CD3/CD28 in the form of beads or antibodies, both methods work equally well. Culturing condtions should be IMDM or RPMI in the presence of 5% human serum. If a prolonged stimulation is needed you might want to consider adding feeder cells (irradiated PBMCs). 
3 Recommendations
18th Aug, 2015
Maneesha K Suresh
Amrita Institute of Medical Sciences and Research Centre
Thank you guys for the help. hope i get it right..
1 Recommendation
21st Aug, 2015
Francesco Nicoli
University of Ferrara
Dear Maneesha, it depends if you want to culture all T cells or just epitope specific T cells. In the latter case it be easy if you want to stimulate memory cells for some infection/vaccination that everyone for sure has, but you need to know HLA.
If you want to culture total T cells, you can activate wit coated CD3 and soluble CD28 and maybe keep in IL2 (but only after 5-6 days of culture). Here the concentration we used (http://www.ncbi.nlm.nih.gov/pubmed/24841128).  To avoid death after hyperactivation, I suggest you to use low doses of stimulation ....better to do a titration experiment before. For the medium, we used normal RPMI+10%FCS. In this way, T cells will be cultured together with other cells, that anyway will die (although not completly) during the course of culture. Replacing the media (and adding IL2) every 3-4 days may halp to culture cells up to 20 days or more.
If you want to culture purified total T cells, for sure you have to use some kits that separe it.
4 Recommendations

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