Cells dying in culture after 24 hours - (Oct/16/2013 )

Hi all,

 

If I could get some help here I would be very very grateful. I am having such massive problems with cell culture that it is seriously affecting the progress of my PhD, and it's starting to get very depressing as nobody seems to have any answers for me. 

 

I was using a cell line called 10T1/2 which is mesenchymal and grows in MEM supplemented with NEAA, 10% FBS, 2mM L-G and P/S. I cultured this line for about 6 weeks, passaging was fine the cells seemed reasonably easy to look after. When I froze down aliquots (LN2) and thawed them out I had no problems. All is well. Then suddenly it wasn't- the cells would not revive anymore, they would lay floating in the flask (this from batches which had previously worked) I obviously made new media up, cleaned the incubator, checked the temp (I cant check the CO2, other than believe what it says) but I used another one in the lab etc. Nothing made any difference. No one could tell me what I was doing wrong. 

 

I couldn't get hold of any more of these cells, so I switched to another line ATDC5 cells. We have a limited no of aliquots a previous lab member froze down and had done a lot of work with. I thawed these cells out, standard procedure 2 mins in a water bath, ethanol spray, into the hood, span 1 vial down (appropriate speed), the other I just seeded right away- T25 flasks, with warm complete media. The media I use is as stated on ATCC, UK cell repository etc etc DMEM/HAMSF12 1:1 mix, supplemented with 5% FBS and P/S mix.

 

I seeded the cells at 11am, I checked the cells at 5pm the same day they had attached and had taken on the characteristic fibroblast like apperance, the media looked good in colour and appearance. The next morning at 9am I checked the cells under the microscope (I checked each flask individually, each one was out for less than 1 minute) they looked great, fibroblast like attached and as though they had grown well- no problems so far. The media was also normal pH.

 

After 24hours you are supposed to give cells fresh media after thawing, so I planned to do that. However at 10:30am that day when I checked them again before changing the media- the cells had rounded up and become detached, completely useless to me and most definitely dying. Literally one hour previously they had looked fine.

 

By 1pm that afternoon you can see cell debris and all cells are rounded and floating.

 

The only things I can think of are:

 

Mycoplasma ( I have streaked some culture onto an agar plate to test ) although this does not explain why at 9:00am the cells were fine and by 10:30 am they were not. Also I dont believe they would outright kill the cells.

 

Incubator: Temp is at 37 (thermometer) cannot check CO2 but readout says 5%. Also cells were left in this incubator O/N and were fine, they were taken out for less than a minute to check under the microscope (very gently i will add) and then placed back in. 

 

Media: The FBS we buy is quite cheap I believe compared to some other products (£30 for 500mL from our University stores) it is cell culture sterile and filtered, FBS from south america. Other cell lines have been cultured using this, but perhaps this cell line doesn't like it? However I would imagine if this was a problem- the cells would not have attached and grown O/N in the first place?? Why would they just randomly die like they have done??

 

 

Please someone help me!!! I cannot think of a single logical explanation for the cells being ok at 9:00am the day after seeding and then not being ok at 10:30 am the same day. I don't have many aliquots so I can't just keep randomly trying. Plus with my absolute failure with the previous cell line, I am starting to think there is something wrong with me/something I am doing. 

 

Thanks very much,

 

K

I'm not suggesting that this is what's happening in your case, but if the cells are always fine at 9am and sick at 10.30 am it might be worth watching who is entering the cell culture in between...

http://www.nature.com/news/2010/100929/full/467516a.html

It could also be that there is some sort of shock happening when you take them out of the incubator, and that the cells are not dead, just weakly attached/detached. 

Could you make sure that the cells are dead by taking an aliquot and checking with trypan blue staining?

 

FBS can have a big impact on cell behaviour, it would be worth knowing which FBS the previous user used on these cells (and the medium they used too, just to make sure), as there can be big variations between batches even from the same supplier. 

Tabaluga- Thank you for sharing that article, it has crossed my mind (very very briefly) but I just can't imagine that being the case, I'm convinced its something I'm doing but I will give some cells to another lab/incubator set up right after seeding them and see if that helps.

 

Bob1- I thought about shock, that seems the most obvious answer, they were growing fine and then very suddenly they were not. However I was very gentle when handling the flasks (very cautious about it) I handled one at a time, and had them out for less than a minute- surely this would not be enough to shock the cells? Although it would seem it is! I haven't done trypan blue, I placed the cells back in the incubator and left them undisturbed in the hope they might re-attach if they were recoverable, if they cant they are as good as useless anyway. I'm pretty sure they were dying however as I have seen some cell debris. 

 

The media and FBS are things that I can try and change so they are exactly as the previous user- that will be my next step. However it is just driving me mad as a problem in itself- how would the cells attach and become fibroblast like, survive the night, look very well and then suddenly die, if media/fbs is an issue surely the cells would have just been unhappy and not attached in the first place? 

 

I just can't seem to get cells to survive in my care (even when the only thing my care includes is leaving them in an incubator and doing nothing to them) 

 

Anyone who has any idea if you have any suggestion whatsoever- it's the most frustrating thing in the world, I have this big block on stopping me getting experiments going and no one around me seems to be able to offer an explanation other than 'thats strange' but this is supposed to be the start of a whole thesis chapter!!! 

 

I had problems previously with the 10t1/2 cells which are quite similar, so this has been going on months and i just need a cell line to stay alive so I can do some work :(

KayW on Wed Oct 16 21:51:20 2013 said:

Bob1- I thought about shock, that seems the most obvious answer, they were growing fine and then very suddenly they were not. However I was very gentle when handling the flasks (very cautious about it) I handled one at a time, and had them out for less than a minute- surely this would not be enough to shock the cells? Although it would seem it is! I haven't done trypan blue, I placed the cells back in the incubator and left them undisturbed in the hope they might re-attach if they were recoverable, if they cant they are as good as useless anyway. I'm pretty sure they were dying however as I have seen some cell debris.

That sort of handling doesn't sound bad at all. I'm not familiar with these particular cells so I don't know what is good or bad for them unfortunately.
 

The media and FBS are things that I can try and change so they are exactly as the previous user- that will be my next step. However it is just driving me mad as a problem in itself- how would the cells attach and become fibroblast like, survive the night, look very well and then suddenly die, if media/fbs is an issue surely the cells would have just been unhappy and not attached in the first place? 
 
I just can't seem to get cells to survive in my care (even when the only thing my care includes is leaving them in an incubator and doing nothing to them) 
 
Anyone who has any idea if you have any suggestion whatsoever- it's the most frustrating thing in the world, I have this big block on stopping me getting experiments going and no one around me seems to be able to offer an explanation other than 'thats strange' but this is supposed to be the start of a whole thesis chapter!!! 
 
I had problems previously with the 10t1/2 cells which are quite similar, so this has been going on months and i just need a cell line to stay alive so I can do some work

I agree, the FBS thing shouldn't be the problem. It is possible that the cells were cultured in a different medium to the one that you are using, but this wouldn't have such dramatic affects as you are seeing, as I would expect the cells to not be happy at all right from the start.

Have you tried culturing other (easier) cells to ensure that it isn't something you are doing? Something like HeLa or 293, would be a good place to start. If these don't work, then there is a real problem.

Hi Bob

 

Yeah I have been culturing 293s and also a murine cell line called BCL1, without any difficulty. But these aren't any good for me as an experimental model unfortunately, I have just used them to optimise some of the knockdown conditions and qPCR confirmations of the knockdown that I eventually want to do in these ATDC5 cells which are mouse chondroprogenitors. 

 

I have looked at every resource I can find about these cells and the 10T1/2 to see if I am missing something, they are supposed to be relatively easy. 

 

Very very stuck Checked the cells today and they are most definitely dead, can't detect anything strange with my media (either in the flask or the bottle it came from)

am sorry about that kayw, not sure if will of any help but only time something like that happened to me was when i accidentally started using dmem knockout instead of dmem for my cells, cells grew fine for around 24 hrs and then detached and died, because of difference in the growth factors in the medium i beleive.  also check if anything is wrong with the liq n2 storage, is anyone else sucessfully growing any other cells from the same storage anywhere? hope you resolve it.