Cell line died; unknown cause... - (Oct/03/2008 )

Hi there,

I hope you will be able to help me out here.

I've been culturing Molt-16 cells for 4-5 months now and I've got them from a collegae who already had this cell line in culture for quite a while.
We use RPMI 1640 medium containg 10% FCS supplemented with glutamine and antibiotics.
Last week all of a sudden 44% of my cells had died...I spinned my cells down to get rid of the dead cells and added fresh medium. When I look at them again almost 100% of the cells was death.
The medium looked fine (no signs of an infection) and also under de microscope there was no (visible sign) of any kind of infection. Two weeks ago one of my collegues had a combined yeast/molt infection and another one had a bacterial infection last week. We DON'T share media...

I use this medium for several cell lines and none of them suffers from sever cell death (so far)...of course I throwed the medium away and prepared fresh one...just to be sure.
Has anybody an idea what might have been the cause of the infection? Is it possible that there was an infection present that could not yet be detected, but worse enough to destroy all my cells. Most of the times I saw infections that were really severe and still the cells were alive...so I don't have a clue what happened here.

Please help me out here...Thanks!

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I'm not familar with that cell line. If you've ruled out infections, how about the natural life span of the cell line- some immortalized cell lines still have limits on how long you can maintain them in culture.
You said your colleague kept them for a while before you had them. Perhaps they have reached the end of their life. If you have a lower passage number stock of cells you can bring up and see how they grow or get from your colleague again.



Cool icon by the way (I had to reply with an icon like that- thought I'd been writing posts in my sleep!!)

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Thanks for your answer...However, I couldn't find anything about a maximum life span for this cell line.

I'm still wondering what might have caused this problem. Could it be that I've used the wrong split ratio and/or frequency for months...and that that eventely killed my cells???

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I suppose thats a possibility or your media was a little off- have you checked the incubator- the CO2 levels were fine? You mentioned that another person had an infection a couple of weeks ago, was the incubator cleaned out with something that may have been toxic to your cells?
I would still try and bring up an earlier passage of cells and see anyway, eventually after selecting (through passaging) sometimes your cell line can change its characteristics- what kind of ratios for splitting and frequency have you been using (and is it different to the other persons techniques?)?

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Sounds to me like there is either contamination with a mycoplasma (which is extremely difficult to see,) or the carbon dioxide is low. It is very possible that the original stock that you received from the other college was already contaminated with a mycoplasma, or that the yeast/mold infection from your colleague's cell culture cross-contaminated your cells in the incubator. Did anyone else's cells completely die off? If so, then like user LostintheLab said, maybe the incubator was cleaned with something that was toxic and left a trace amount behind. I for one really don't think that your splitting ratio could have caused this; you would have just had a much more dense amount of cells.

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Thanks for your answers.

First thing to mention...mycoplasm has been tested last month using PCR and all cell lines were free from mycoplasm at that point...I don't know at what rate mycoplasm affects cells?! And I don't think the strain was infected when I got it...because my collegues cells are still alive and happy (those cells are in a different incubator by the way). I've used a lower final concentration when splitting the cells than my collegue, because the medium turned really yellow over the weekend. I went back to the 'original' concentration however, because it did not sort any affect. The final concentration is 0.3 10^6 cells/ml (and 0.2 inbetween for testing).

Second there was really no trace of any infection...I had this checked by two other people.

Third none of my collegues had the same problems with dying cells...although I cultured the cell line Molt 3 for a week for a collegue and that cell line suffered also from extremely high percentage of dead cells only ones (the same time) and restored from that. The time before I've had these dramatic cell dead numbers I've splitted them and I'd just prepared fresh medium then. After these dramatic numbers I've again prepaired fresh medium!

Fourth I've cleaned those incubators myself (as I'm responsible) and we use a biocidal, rinse with water and than ethanol...dry and then we put everything back in...so I don't think there are really traces of the biocidal left at the end. Off course CO2 levels drop during the cleaning...Might these cells have suffered that seriously from the half hour of low CO2?!

For now I think that there might have been a problem with my medium and/or the low CO2 levels two times because of the cleaning twice within a short time...My cells might have been unable to restore from that...I also think Molt 3 and Molt 16 are more sensitive then my other cell lines.

Thanks again!

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That this has happened to you before sounds to me like a problem with passaging technique. Is this an adherent cell line? Do you use Trypsin on it?

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All my cell lines are suspension cell lines...so I definitely don't need trypsin.

I'm not sure wether you understand me correctly or not...I did not had this problem before...both cell lines suffered these losses at the same time...One restored, the other not.

Week 0 everything is fine
Week 1 cleaning of incubators because of yeast/molt infection collegue
Week 2 everything is fine
Week 3 cleaning of incubators because of bacterial infection collegue
Week 4 a] High percentage dead cells Molt3 (22%) and Molt16 (33%), spinned cells down and added fresh (newly prepared) medium
b] Molt16 died completely and Molt 3 has 43% dead cells, spinned Molt3 cells down and added fresh (again newly prepared) medium
Week 5 Molt 3 cells have restored with only 5% dead cells

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Hi Bindi,

Did you manage to find out why ur cells are dying. I am having the same problem. They were growing fine for months and suddenly the decide to die. My cells are suspension cells DT40, I don't see any infection at all, I havent changed the media or FCS batch....they are just too temperamental. Wud appreciate if u cud let me knw if have gotton around this problemn and how!!!

Thanks

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Oh dear,

Mycoplasma testing using PCR is a waste of time...The ATCC, the world's expert and largest supplier of commercial cell lines NO LONGER USE PCR ....because it is TOO UNRELIABLE....it gives too many FALSE NEGATIVES. I try to convince all the researchers in my Institute to use the Agar Growth test in combination with Hoescht staining....both methods approved by the FDA.....PCR is not FDA approved....what does that tell you.......SO MYCOPLASMA MAY STILL BE A POSSIBILITY.


Second...... FYRITE your CO2 Incubators regularly. If the displays says 5 % DO NOT BELIEVE IT.


Thirdly ....cell lines should be routinely grown WITHOUT ANTIBIOTICS. Why are you using Antibiotics for these cells......Cryptic contaminations could be present.......If you use Antibiotics, then you should regularly leave them out for a couple of passages, to see if Cryptic contaminations reveal themselves.

Fourthly, As lost in the lab CORRECTLY mentioned, some cell lines have a definite life span. You should regularly check your cells for markers that define them. You should then go back to your master bank and revive a LOW PASSAGE vial to continue your work

Fifthly, as these cells are suspension cells, what speed do you spin your cells at. THIS IS VERY IMPORTANT AS HIGH SPEEDS CAN DAMAGE CELLS.


Hope this may be useful

Rhombus

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