I have access to peripheral blood mononuclear cells that have been cryoprepreserved and stored in liquid nitrogen for 10 years. They belong to an interesting cohort.
Cells that have been cryopreserved for a relative short time can be used for immunophenotyping by flow cytometry, despite some changes in extracellular markers. However the cells I refer to have been kept for a very lengthy time. They havenot been subject to any procedure other than the initial ficoll separation, and DMSO preparation followed by slow freezing to -80° before storage in liquid Nitrogen.
Any experience with a similar situation will be appreciated.
I think that it depends on what you want to obtain from such cells but, as far as I understand, most features are preserved in spite of the time in cryostorage. In other words, features are lost mostly during freezing and hence, once frozen, it does not matter if they are thawed within a week or after a century.
Hello Enrique
I have never worked with cryopreserved cells, but maybe this publication will be usefull. By the way 10 years is a long time.
Good luck
Article A Method for Generation of Bone Marrow-Derived Macrophages f...
If they have been correctly maintained without temperature fluctuation during the storage in liquid nitrogen, the cells should be as Ok as if they were in liquid nitrogen 10 days. Once cryopreserved they will only be affected by background radiation or human mishandle (Glenister et al. 1984, campos et al 2011, Germann et al. 2013). Make sure you have the correct thawing protocol.
Seems doubtful. You can be sure that isolation of DNA, PCR, DNA sequencing will be successful. Also, I would hope on success to restore transformed or hybridoma cells after so long storage. To restore PBMC after 10 years, seems too doubtful. In any case, you can try.
It depends intensively what You want to use them for, and how they were preserved. In case of DMSO based cell culture medium freeze, it is important to remove DMSO as fast as you can [thaw under warm water, as fast as You can, centrifuge, and add fresh medium (RPMI with 10 or more percent FCS), and wash with that twice and see what happens. You can expect to lose 30-60%, if the freezer was fine, and the preserving protocol was done well. If not, more.
If they were frozen correctly and smoothly: no problem.
Use a small sample for thawing and check viability by staining, for example with Giemsa smear or functional assay for proliferation, for example after stimulation with PMA/ionomycin or Concanavalin A.
Good luck.
It depends on the type of the experiment you want to run. For cell immunophenotyping you can get quite a good result but you will loose some markers such as integrin or lectin makers. For PBMCs culturing or stimulation, it will be more tricky (25-50% of the thawed cells will undergo apoptosis)
Hi Enrique.
I've resurrected hybridoma cell lines that have been frozen in a similar fashion for over 10 years with reasonable success. So long as the storage has been consistent over that time, it's really more the preparation for freezing that is most important.
Assuming that has been done correctly, you should be able to resurrect the cells just as you would cells that have been stored for a shorter period. I know many are partial to DMEM with their own concoction of supplements, but I've just used the standard recipe for RPMI-10% FBS with success.
What I tend to do with any cells I resurrect is partially thaw them in a water bath, keeping an eye on them so that there is just a bit of ice left. Then, if I have room in the vial, I pipet in cold media, so that it melts the small amount of ice left. I then transfer the cells to a 15ml conical, raise the volume to about 12-14 mLs with cold media, pellet the cells in a centrifuge, and resuspend the pellet in warm media to transfer to a flask or plate. All along, any pipetting I do is very gentle.
I hope that helps.
Take care.
We regularly use PBMC to develop human monoclonal antibodies from samples frozen under identical conditions, with a fairly good efficiency, and that with frozen cells for over 25 years.
Yields are lower than with fresh cells, but this is entirely acceptable.
I noticed that chemotaxis ability of frozen BMDMs was reduced when compared to fresh ones, but as said above, it depends of your objectives.
It depends on what you want to use them for. We have successfully managed to EBV transform PBMCs cryopreserved over twenty years ago. If your PBMC's were cryopreserved by someone with relevant experience, the issue will be whether their long term storage remained below the glass transition point. Some vapour phase storage systems have documented temperature gradients climbing above this eg Rowley S. and D. Byrne. 1992. Low-temperature storage of bone marrow in nitrogen vapor-phase refrigerators: Decreased temperature gradients with an aluminum racking system. Transfusion 32: 750-754.
You don't say what markers you are interested in so my answer is somwehat qualified, but if you achieve good viability after careful thawing, I would expect you should have a reasonable chance of success.
Have fun!
We often use cells 5-10 y of age to look at stimulated cytokine secretion. Importantly, you should rest the cells after thawing, preferably over night but at least 1-2h in the incubator, before using them for experimentation. As said above, you will loose a portion of the cells. When you rest them in the incubator, take a look after 1-2h, resuspend the cells carefully and let cell clumps sediment to the bottom, then transfer to new tubes.
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