Hello All,
I'm working with MDA-MB-231 cell line and I always perform same procedure while I'm passaging these cells. After unfreeze the cells ( at first I freeze them with 5% DMSO, 95% FBS solution and freeze them via mr.frosty at -80oC) they reproduced and there wasn't anything wrong. I followed the protocol that I mentioned below which I did all the time and they didn't die before but now after passaging they died second time;
1)getting rid of the growing media
2)washing the flask with PBS
3)adding 1ml 0.25% trypsin
4)incubate for 2 min at 37oC
5)add the cell solution (trypsin + cells into new growing media for deactivating the trypsin) and centrifuge it 3 min at 1200 rpm
6) dissolve the cell pellet with new media and divide it properly into new flasks
7)incubate them at 37oC
Can you please help me about solving the problem which caused cell dying ?
Have you checked the Flask (i.e. whether the cells detached) after point 4)? Two minutes of trypsin incubation seem rather short to me.
If the cells would not have detached you would have transferred only the dead cells floating in the flask.
Daniel has some good points above. Do you know you are getting culture growth? Are you using divalent cation free PBS wash? (Adding monovalent cations into the cell cultivation flask negates the EDTA effect of detachment.) Tryple ES is more consistent than frozen and thawed trypsin EDTA, which is autolytic. Check the RCF of your cell spin as well? A picture of the cells in flask might be helpful as well for advice. If none of the above are the issue. Did you freeze a log phase culture or not (which could have caused additional stress to the cells)? Bringing out problem cultures can be a problem.
So four issues with problem cultures from the freezer : 1). When frozen cells become prone to cyrogenic fracturing if not handled carefully. I have had several batchs lost do to the actions of others in a lab freezer because they did not care about anyone else's work besides their own. 2). Spinning down the thawed cells can move cryo-trauma (above) to cryo-necrosis, especially if issue 1 occurred above.
3). When thawing cells, I like to make my primary plating by diluting out the cells to where the DMSO is 0.5% or less. The dilution rate then influences freezing density. The first plating is allow to go for a couple day for attachment. Pulling off the first medium (i.e. the floaters) and transferring them to a second flask after spinning (the viable traumatized cells will then attach). The primary flask is fed fresh medium. Two days later, the medium in the second flask is removed, and fed with the medium from the first flask (i.e. it has been "preconditioned"). This helps prevent an apoptotic cascade, which is less prevalent in higher stage cancers. But badly damaged cells can take a
4). When the first flask needs split, add the contents of the second flask (if any) back to the first to try to better reconstruct the original population.
Hope this makes sense and good luck.
Dear Daniel,
I check flasks after 2 min incubation every time and if they don't detached I hit the flask with my hand slowly and I check again under the microscope usually they detached after that and I passage them quickly into the medium for centrifuge and for now every time this protocol worked.
Dear Raymond,
I used cation free PBS to wash all the time. Thank you for your comment there are lots of useful knowledge I'm appreciate. I started to concern about my DMEM because I prepared it about 2 month ago as a stock and I think everything in it spoil at 4 oC, also maybe cells need more FBS like 20% rather than 10% ?
Merve,
Glad that you use the divalent free PBS. Repeated freeze and thaws of aliquoted Trypsin changes time to detach.
You may have hit on the issue. Typically I only like to have medium 4 weeks older at most. If the FBS is in good shape (this also has aging issues, assume it is HI) the cells should easily stand 10%. If the 20% works, you might want to check growth on a new batch of FBS. I thaw 500 ml FBS stock (stored at -80), break it into 50 ml aliquots, and then store them at -20/-30 oC.
Regards,
Ray
Dear Merve
Yes I agree with Raymond. If they detach properly, and there is no sign of acute contamination, but still proceed to die there might be a problem with the medium.
Oh, forgot one thing. Unless you are using a stabilized glutamine form, that deteriorates as well in the medium over time.
Dear Raymond Orlo Fatig and Daniel Hauser,
Thank you for your recommendations and interest, all informations are really useful for my current and my other future research.
Best Regards
Remain calm and culture on (it's a cellists meme thing), Hope that the 231s are alive and giving you reliable data!
That's the good one I like that thanks ! I hope that too Raymond Orlo Fatig I will let you know !
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