Hi,
I have been culturing Gc1-spg cells for a few years now and things were going fine till late last year. The cells started looking extremely unhealthy and dying in culture at early passage numbers (P5!). To get to the bottom of it, I changed a few things including (a) buying a new stock of cells from ATCC to be sure that the cells aren't dying because of late passage numbers, (b) switching to buying ready-made and filtered DMEM (ATCC recommended formula) from Gibco/ Sigma as opposed to reconstituting from powder media and filter sterilising in the lab since the quality of water might not have been good enough, (c) ensuring that the cells are not contaminated with mycoplasma since that was an issue at one point (Gc1 cells are very sensitive to mycoplasma), (d) splitting at a higher ratio (1:10 from 1:5 as I used to do initially), again, as recommended by ATCC and (e) switching from heat inactivated FBS to non-heat inactivated FBS to keep in line with ATCC recommendations.
At the time, the cells were growing better than before for upto 10 passages after thawing a stock. A lot of early passage stocks were made and then the lab was shut down for 4 months. I had stored the stocks at -80 (not the best way to store them) for various reasons. I have been told by colleagues that most cell lines revive well even after long term storage at -80
I have for the past two weeks been trying to revive the cells but they won't grow beyond 3 passages after reviving from stocks. They appear unhealthy (presence of projections and uneven cell membrane along with the presence of stress granules) and die in culture. I have ruled out contamination by yeast or bacteria including mycoplasma with tests. I can't figure out why they won't survive.
1. All reagents have been freshly prepared
2. Tissue culture room and incubators have been sterilised
3. Media hasn't expired, bottles have been procured fresh
I see in some papers that authors have used RPMI media but I can't change now since I don't know what the biological implications of such a switch might be. Can anyone think of a reason why the cells just die? Has anyone faced a similar issue? Any suggestions to overcome this problem would be much appreciated.
Thanks in advance!
Bhavana
Hi
The cells in culture not even die but changes their structure too. RPMI is best for culturing different cell lines as we are using it, it shows very good redult.
Let me share you one of my experience, before lock down their was shortage so we tried rest of different media's but it could not grow our cell lines except one, we have total of 10 plus. So, better to try RPMI Gibco and might you would have better results. (I am not asking to switch to RPMI Gibco, just make sure the quality of your current media, that is it ne enough to grow cells aor not, as the rest of conditions are looking fine sterilization of lab/infection of cells etc)
Second, make sure the cells were in -80 and there was no temperature up down in the mentioned time,because it was lockdown might electrecity and power backup issues, so if they have been for a day on high temp, they would have mutated/shape distorted, kindly check the shape at earliest when you thaw it and pour it into plate.
Hi Mudassir Khan ,
Thank you for the suggestions. The morphology of the cells looks normal when I thaw the cells and for upto 2 passages after thawing which is what I find strange. Would they cells not show signs of distress earlier?
As for the freezer, I have no way to check now if there was a temperature variation in the freezer unfortunately. But assuming there was some fluctuation, is there anyway to overcome this issue?
With regard to RPMI, I may have to make the change if nothing else works. Thanks again!
Respects to you Bhavana Kayyar
This is strange that morphology looks normal but changes later on after two passages, this shows there might be something in culture which influence the morphology change. That is what i mean contamination, and this shows the cells are in good position they are not resistant to the thing which alters its shape, better to sort out before the cells starts growing well again without any treatment (Resistance developed so it would most probably targets/alters your results). Are you adding Phen-strip in your media or not? DMSO has also proven as toxic to cells in culture, if you are adding DMSO, please lowers it concentration/amount/volume, and check might it would work. But i am writing again their is something in the culture which deviates the growth towards death/morphology change.
Assuming there was some fluctuations in temperature, that shows the cells are in shock, optimum temperature fluctuation shock. I guess it needs to be treated humbly so they can come out of the shock already disturbed with.
RPMI, if you can manage 50 ml from your colleagues and thaw cells in it, if it shows good results you can order yours.
Hope something better works for you
Regards
Mudassir
Thanks Samrat Das ,
That has already been done and the cells test negative as I have mentioned in the original question. Normally, that is the first test I set up since I have had issues with mycoplasma in this cell line in the past. It looks like the problem is something else this time.