I am trying to do some experiments on peripheral blood lymphocytes. And for that purpose I need to keep these primary cells alive in culture medium for a day or two. Does anybody know if they will survive in the complete culture medium without any supplement for a day or so?
Yes, at room temperature .Keep the cells in dark, and not in refrigerator.
They can also survive and grow in a chemically defined medium. Please see my publications on this subject on my site.
NCTC 109 used to be the best medium; but that was years ago.
My experience is with human cells mostly.
Good luck, Alan Coulson
Most PBL are in survival in the circulating blood. Indeed they have an extremely short survival when they were purified.
It is estimated that 95% of B cells and T cells will have died in 24 hours, and that very few cells may be activated subsequently.
Only a few macrophages arrive to survive by feeding on other cells in poor condition.
However we can keep the PBLs purified for 24 hours at 4 ° C, without too much damage.
Furthermore cells can be stored in fairly good condition, if the PBL is kept in the blood sample stored at room temperature for 48 hours or at 4 ° C for one week: it works well enough for B cells (about 50% of activated cells with HuBBB).
For example: It was possible to develop 100,000 EBV B cell clones from 1,000,000 B cells that had been extracted from 10ml of blood stored at 4 ° C for 5 days in acetate buffer.
Yes, sure they survive. Talking about T cells, on day 2 you might observe a decline in effector functions (e.g. CTL cytotoxicitity) but on day 1 they are still perfectly fine. Keep the cells in complete RPMI in confluence (e.g. 4-6 mln per well in 24well plate), they like it.
We have kept human PBL at room temperature in defined RPMI+FCS media for 24 hours. The results in subsequent activation studies are much better than when the cells are kept at 4 deg C. The comments above that there is so much cell death are simply not true for human PBL under these conditions. However, there is always about 20% cell death by flow cytometry. I would not recommend going more than 24 hours before activation. The limitations of this approach is that human PBL will activate in these conditions, perhaps by cell stress or perhaps by reaction to FCS or perhaps by loss of some counter regulatory signals when taken from a very different environment in the blood stream. So any studies based on activation states should consider this limitation. And if molecular profiling by qPCR, DNA arrays or sequencing is planned, then I would strongly recommend against it.
I agree with Dr. Salomon, not more than 24 hours in culture would be the optimum. If you're interested in a particular cell subset, i would recommend to isolate it prior culturing.
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