I have been trying to freeze and thaw THP1 cells for quite some time now. I have been through different protocols suggested by colleagues as well as in different forums, but so far nothing has worked for me. I will give the details of the methods that I have tried so far.
For freezing, I use around 3-4 million cells/ml. I spin down the cells from media, resuspend the pellet in freshly made 90% FBS + 10% DMSO (cooled) and put in it a Mr. Frosty kind of box in -80 overnight. Next day I shift it to liquid nitrogen. Earlier I have performed cooling with 2 hours in -20, overnight in -80 and then liquid nitrogen too.
For thawing, I have tried the following:
1) Thawing the vial in 37C water bath, then spinning down at ~800rpm for 3 minutes. Then decant the FBS, resuspend in pre-warmed media and gave another spin. Then I resuspended the pellet in fresh 8-10 media with 20%FBS (to make around 0.25million cells/ml) in T25 flask.
2) Thawing the vial in 37C water bath and transferring the entire content into 10 ml pre-warmed media, spin down and resuspend the pellet in fresh media in T25 flask.
3) Thawing the vial in 37C water bath and slowly adding 10 ml media to it, spin down and resuspend the pellet in fresh media in T25 flask.
Everytime the cells look fine right after thawing but over the course of the next 2-3 days they die off. I am using the same media and FBS to grow THP1 borrowed from other labs, and they grow fine.
Any suggestions with the process will be immensely helpful.
Dear Ushashi
The cells following thawing looks fine but as they are not dividing, it tells that the cells are dead. Probably you lost cells during freezing. I suggest you to freeze cells again, store at -80 for 4h (instead overnight) and transfer to liquid nitrogen very same day. Also you can try replacing your freezing media to any commercial freezing media (till your problem resolves)
Conceptually, the cells need to to be exposed to liquid DMSO for as short period as possible.
You can achieve it by:
1. While freezing, re-suspending the cells first in FBS only and then add the equal amount of 20% DMSO+80%FBS solution. THis will result in final freezing solution consisting of 10% DMSO + 90% FBS.
This way you can minimize the time that is required to resuspend the cells in DMSO- because you already re-suspend the cells in FBS alone.
2. While thawing, directly add some warm media onto the frozen cells. The warm media will dissolve the ice of the solid icy surface of the frozen cells. Combine the dissolved cells with 20 ml warm media. And keep repeating this step till all the cells are dissolved.
This way as soon your cells are thawed it is diluted in the media and the percentage of DMSO is reduced drastically.
It is this reduced DMSO concentration that is required to keep the cells alive.
3. Then centrifuge the cells and resuspend the cells in fresh media.
4. Then freeze stepwise first in -80 ON and then liquid N2.
This should solve your problem if you started the freezing process with live cells.
S
I will try out the suggestions. Thank you, Harsh Panwar and Sudeep Kumar
Have you figured out the problem and solved it? Could you please share the procedure (protocol) to solve these problems? We are recently facing the same issues. Thank you Ushashi Banerjee .
Hi Sukumar Biswas,
I changed a couple of things in my protocol based on the suggestions. Now while freezing, I first suspend the cells (~3 million/vial) in FBS and then mix it with 20% DMSO + 80% FBS to reach the final 10% DMSO + 90% FBS concentration. Then I keep the vials inside a Mr. Frosty container and keep it and -80 C overnight. Next day vials are moved to liquid nitrogren.
For thawing, I keep the vials in 37C water bath until a little of the frozen part remains. Then I transfer the whole thing slowly to ~7-10 ml pre-warmed media. Spin in down, give one for media wash and finally resuspend in warm media with 20% FBS. It takes a couple of days to start growing. Once cells start dividing, I stepwise bring down the FBS to 10%.
This method is working much better for me, although not a 100% success. Hope it helps.
Thank you, Ushashi Banerjee , for replying and sharing your tricks to resolve this issue. We maintained four different solution conditions for preparing our last cryopreservation solution, including the final 10% DMSO + 90% FBS concentration and freezing them. We did follow the same procedure that you mentioned in your reply.
After returning from the holidays, we will try to recover them from freezing conditions and follow your suggestions and procedures. I hope it will work. Thanks.
Ushashi Banerjee could you kindly share the media that you use for the culture of THP1 cells.
Hi Abhishek Dutta, I use RPMI-1640 media with 10% FBS, 2mM L-glutamine, Pen-strep, and Nystatin. Also, I add beta-mercaptoethanol at a final concentration of 0.05 mM. It's not essential, but I found the cells aggregate less with it.
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