I Started culturing cd8+ t cells. I used t cells transact to activate them during 3 days. My culture medium is texMacs supplemented with il-2 (20 IU/ml). during activation they formed clusters (which i think its normal) . However after I remove the activator my t cells don't grow. What should I do if they continue like this?
Dear Rafaela!
Please You look at the following articles:
Article Optimization and validation of a robust human T-cell culture...
Media and cytokines
For in vitro T cell cultures, we used complete RPMI 1640 medium containing 1% penicillin/streptomycin (MediaTech, Herndon, VA), 50 μM β-mercaptoethanol (Gibco, Invitrogen, Grand Island, NY), and 1% L-glutamine (Gibco, Carlsbad, CA). The medium was supplemented with 10% pooled human serum (PHS; Gemini Bio-Products, Woodland CA), both heat-inactivated for 30 min at 56 °C. Sterile, lipopolysaccharide-, pyrogen-, and mycoplasma-free recombinant human cytokines IL-2 (Chiron, California, USA), IL-7 and IL-15 (R&D Systems, Minneapolis, MN) were used at concentrations of 10 IU/ml, 10 ng/ml and 10 ng/ml, respectively. All cytokines were supplied carrier-free by the manufacturer and were reconstituted in 1% human serum albumin (HSA) (25% HSA, NDC 63546-251-05, pharmaceutical grade, manufactured by Swiss Red Cross, distributed by Alpine Biologics, Orangeburg, NY) in phosphate buffered saline (PBS).
Figure 1
Autologous PBMCs function as potent stimulators to expand antigen-specific CD8+ T cells during short term in vitro culture
PBMCs were stimulated with corresponding peptide-pulsed and irradiated autologous PBMCs at an effector:APC ratio of 1:1 with addition of cytokines IL-2 (10 IU/ml) and IL-15 (10 ng/ml). (A) Representative dot plots of flu, Melan-A and NY-ESO-1 antigen specific tetramers are shown at baseline (Day 0) and after in vitro stimulation (Day 10). (B) The stimulation index of tetramer-positive cells, calculated as the absolute number of tetramer-positive cells at the end of each stimulation over the number of tetramer-positive cells in the initial culture. The results represent three independent experiments and the error bars represent the standard error of the mean.
https://www.nature.com/articles/nprot.2014.064
Figure 2: Experimental outline.
Hello Rafaela,
This paper: Optimizing Manufacturing Protocols of Chimeric Antigen Receptor T Cells for Improved Anticancer Immunotherapy (mdpi.com/1422-0067/20/24/6223/htm) talks about T-cell transact and that it was reported that this activation strategy mediated lower expansion, supported the expansion of CD4+ T cells with a CD4:CD8 ratio of average 2:1. This might be the reason you are seeing lower T-cell expansion.
Hope this helps.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line