Hi.. I am culturing RKO cells in DMEM media (10% FCS and 1%PEN/STREP).. I recently noticed when I was splitting the cells that the old media was very viscous.. Does it mean that my culture is contaminated or is it just a characteristic of RKO cells.. Other cell lines that I use have a perfectly normal media..
Does it look white and stringy? Look under the microscopy, does it look cloudly, white, and like spider weds? I have seen this with fungal contamination in RPMI-1640 based media, remember an incubator is a great place to grow things, even contaminants. If it is a fungal thing, then I would look to cleaning out your incubator and prep hood etc...you can buy a fungicide to put in fresh media if this continues to be an issue, just to prevent growth when starting a fresh culture, but It may be to late for this culture though. If it is a fungal contamination.
Hi Craig, thanks for your response. Yes I have looked under a microscope and the cells look pretty normal and there are nothing like what you mentioned. So I am not sure if it will be a fungal contamination. I have given it for DAPI and I hope I would get the results pretty soon. I just dont want another contamination again!!!
If it was a fungal contamination you'd know. Good luck and let us know what you find :-)
Hi Craig.. Mycoplasma results have come and it was negative for contamination.. Thank God!! Phew!!! :)
Do You have a lot of dead cells after splitting the culture? If You kill enough cells the released DNA increases viscosity of the cell culture and decreases viability of other cells through electrostatic interactions leading to the increased release of DNA. Add some DNAse to the supernatant from over the cell culture and check if the viscosity has decreased, then You will have an answer if it is the result of the DNA contamination. And try some other method of splitting cells, e.g., switch from trypsin to Accutase. Or use trypsin for a shorter time.
Hello Patrycja... I guess that would be the perfect explanation. Yes, I do have lot of dead cells after splitting but I thought it was because they grow very rapidly and become too confluent. I am not sure of the method of DNAse treatment.. Should I add the DNAse to the new media while splitting? and at what concentration?
I haven't heard of Accutase but I will have a look at them. I usually give 5-10 inucbation after addition of trypsin.
DNAse is added to the medium only during isolation of the primary cell culture, when a lot of cells die due to the harsh treatment. During the normal culture of the cel line it is better not to use DNAse, just try to passage cells without killing them so much. Trypsin treatment is quite often very harsh for the cells, especially when they are overtrypsinized. It is much better to try shorter time of trypsinization - 10 min. is a looong time to trypsinize cells. One can easily shorten time of trypsinization this way: throw away a medium from over the cells, wash them with sterile PBS, add trypsin, wash the cells with trypsin and throw away most of it leaving only a few drops to keep cells wet, put the cell culture bottle into incubator, take it out every minute and try to mechanically disperse the cells by banging the bottle in the side with Your hand, and when cells deattach add at once medium with FBS (quite often people leave trypsin over the cells and then addition of FBS with the medium does not stop trypsynization quickly enough, mechanical dispersion is also not often used as one can destroy the cell culture bottle but it helps quicken cell deattachement). Or You can try Accutase, it does not kill cells like trypsin does (but it is expensive, much, much more expensive than trypsin).
Thank you for the detailed explanation. I am not sure if I can afford Accutase considering your comment about its cost but I am sure I can follow the procedure you suggested.. I exactly do the way you mentioned in the last few lines and that might be the reason for the media becoming viscous. I am clear till the part where you discard most of the trypsin but if we try to detach them mechanically, will it be good for the cells? Of course we do it but that is after you notice the cells appearing as round and bright (like when they float). Won't detaching it mechanically stress the cells??
If You do mechanical detachment by sharply banging on the bottle, they will detach only if the trypsin made the connection between cells and cells and the plate already lose enough. You just encourage them to lose this connection fully without waiting for the full detachment by trypsin. It is not scrapping the cells from the plate so it is not so hard on the cells. And it is better than keeping them longer with trypsin.
And check the prices for Accutase as in Your country the price difference could be quite different than in mine as we have a very cheap local firm producing good quality trypsin and only one firm selling Accutase. ;-)
I get it now.. Thank you so much for the information.. I will do the same and hopefully the viscosity reduces. Haha, I am not sure about it but will check and if its cheap will ask my supervisor about buying it.. Not sure if he will accept ;) Thank you once again..
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