Hi, I am trying to perform an expansion of T-cells, purified (by miltenyi columns) from PBMCs and transduced with construct of interest.
I am wondering if there's a reliable and "working" protocol to do it in plates (6-well plates for ex.)? Is it a matter of splitting and changing media (+IL2) or there's more sophisticated process?
Thanking a lot in advance!
I am not sure if that's the answer you are looking for, but you can use this kit and follow instructions for T-cell expansion:
https://www.thermofisher.com/order/catalog/product/11131D
Advanced RPMI Medium 1640 with 2 mM L-Glutamine, 10% FCS/FBS, and 100 U/mL penicillin/streptomycin can be used. Alternatively, OpTmizer T Cell Expansion SFM with 100 U/mL penicillin/streptomycin.
Stimulate cells with anti-cd3 mAb and supplement the culture with Il-2 (10-20U/ml or more) on every third day. you can watch the growth of cells and split accordingly. Split cells by removing half of the suspension to other well and replace each well with fresh medium and Il-2
You can try to expand T cell in-vitro for 6-8 days, after which the T cells get exhausted and need re-stimulation.
You need to add IL2 (20 IU/mL) and also Anti- CD3 and CD28 antibody (1-2ug/ml) for a 24 well plate.
Add the cell suspension and your reagents to a suitable cell culture vessel at a density of 1×10⁶ cells per mL per cm² (e.g. 2×10⁶ cells in 2 mL/well of a 24-well plate).
Incubate at 37 °C and 5–10% CO₂ for up to 2 days. At day 2, gently pipette culture up and down to break up all cell clumps.
Split the cell culture every two days 1:4 or 1:2, depending on the proliferation of cells, and add fresh culture medium supplemented with 10% FBS and IL-2 (1×10⁶ cells per mL per cm²).
I hope it helps you and let me know how it worked.
You can also check this paper: http://onlinelibrary.wiley.com/doi/10.1111/j.1365-3083.2011.02564.x/pdf
Hello Mark,
One other option is to use STEMCELL's solube T cell activators (either CD3/CD28 or CD3/CD28/CD2) and our serum-free and xeno-free medium called ImmunoCult™ (linked below).
http://www.stemcell.com/en/Products/Popular-Product-Lines/ImmunoCult.aspx
What about mouse T cells? I would like to avoid FBS, but many "serum-free media" contains human serum-derived factors (transferrin, insulin, albumin...).
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Materials Science
- Plating