I am working with in-vitro and in-vivo T-cell proliferation assays and use the cells straight away after isolation. But we have a whole lot of OT-II mice which we might not be able to use due to a change in the course of work. So I was wondering if I can isolate the lymphocytes and freeze them down in freezing media containing DMSO, store them at -80 and use them for in-vitro and in-vitro proliferation assays in future. Has anyone tried anything like this before or should I just go ahead and try? Please let me know what you all think and if anyone has done this before, tips and advice regarding freezing would be much appreciated. Thanks!
I agree with Margit. Fresh is always better, but if you need to store the cells, especially for long-term storage, it should be in liquid nitrogen. At -80°C cell viability decreases to less than 50% within a month, at least in my experience
Hi,
Fresh is always better, well yes but sometimes it is important to do experiments on large number of different clinical samples and therefore you need to be able to work on samples that were prepared in the most optimal and standarised way over the longer periods of time. Doing it on every day based fresh sample would increase the data variability due to day to day variation of lab based experiments. So we need to be able to bank cells properly and therefore:
You need to keep cells in optimal conditions otherwise there is no point of doing any quantitaive experiment (last thing you want is the result-variability due to poor sample quality that means: waste of mice, waste of time, waste of money and false/meaningless data generation).
I would not use -80C to store lymphocytes even for couple of days.
Cells should be carefully frozen and once reach the -80 C temp (after ie over night incubation) should be transferred to the liquid nitrogen.
Doing experiments on poor quality samples is antiscience.
Also when we transfer frozen PBMCs between labs for an independent result replication but also just for a later (ie 1/2hour later) thawing in our lab we use small liquid nitrogen conttainers and not a dry ice. It is really important to keep conditions stable.
Of course you will see some cells being alive even after couple of months in -80 but this does not mean that you are going to later obtain reproducible data.
Lack of data reproducibility is a significant problem in life science.
Best wishes,
Marcin
If found it very different to use T cells generated after peptide-based vaccination vs. transgenic T cells after having made frozen aliquots. In my hands, I could thaw the former and maintain them in weekly restimulated cultures for several weeks. With the latter, I could only do short term assays and never managed to put them back in culture for more than a very fews days (3-4). Freezing was done in all case in 90% serum-10% DMSO. If you wish to make proliferation assays, I guess you would prefer to freeze them down in a naive state and stimulate them straight after thawing. This, I was unable to do it
Good luck!
Denis
Thanks everyone for your responses. I should probably mention I did mean to write liquid nitrogen in my post. -80 may have been a Freudian slip as we were moving labs. But thank you all again for the insight, I will try freezing them down in the mentioned freezing media and store in liquid nitrogen. I was also wondering if the lymphocytes need a recovery at 37deg for a day before use (remember coming across this for human PBMCs). Please let me know the thoughts.
I work with human lymphocytes and I suggest you to cryopreserve the cells in liquid nitrogen. You can freeze at -80°C (I use the cryobox with Isopropanol) and aftert 1 night I move the vials with the cells to liquid nitrogen. I suggest you 90% of serum and 10% of DMSO and be careful when you thaw the cells. I put the vial from liquid nitrogen directly at 37°C and immediately I wash the cells with medium to remove the DMSO in my experience it works well..Good luck
Paola
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