Cell Growth - Help! (Apr/27/2006 )
Hello Everybody,
This is my second post. I have been culturing rat hepatoma cells. We recently moved to new setup and my cells stopped growing. I never had such problem. I cultured the cells again but despite having a fairly good cell density, cells are not growing.
Any help or suggestion would be appreciated. 
Could you be more specific when you say "new setup"?
We moved from one building to adjacent one. Something bad happened to my cells and all the cells got contaminated. I threw all the cells and cultured the cells again but now the cells are not growing.
Nobody else faced any kind of problem (well their cell lines were different) buy hepatoma cell line are pretty much stable.
I dont know what to do.
Rohit
Can you give more details on how your cells are set up. For instance did you switch to a new type of media? Have you cleaned your incubator and made sure it is fully functional (I know you said your labmate's cell lines are doing fine ... but are they all in the same incubator)? Are you doing anything differently? Did your batch of cells come from frozen ... maybe this batch just wasn't frozen properly (in that case there wouldn't be much you could do to remedy the problem)? You need to give us a bit more detail and maybe we can offer you some more advice.
Earlier my cells were growing fine in DMEM. I switched to DMEM - phenol red free subsituted with glutamine and sod. pyruvate...even then cells were fine. We moved to new building. The incubators are new and sterilized. But due to some reasons my media in the flask seemed turbid. I chnaged the media and after that i found that cells stopped growing. I threw all the cells and took new vial (same batch as earlier) and then cultured in DMEM with phenol red. There are more than 2 million cells in t25 flask and its second day. Cells are looking dormant..circular and not seemed to grow further.
Well if it is only the second day it may take the cells some time to fully start growing ... especially if you just thawed them. How confluent are the cells? You may want to just give them some time. I find sometimes it takes some cell lines a while to get growing really well. You may also want to move the cells to a larger flask or to split them to provide more surface area this may encourage them to grow more (if they look too crowded that is). IF the cells are too confluent (90-100%) they put off a chemical that to each other essentialy says hey we are really happy we don't need to grow any more. You should also check your incubators to ensure they are reading temperature and CO2 levels properly. When I moved labs ... my incubators were reading a 5% C02 level, but when we checked them with the fyrite it was a level more around 10%! Sometimes things get messed up with electrical devices when they are moved or are set up for the first time. So you could possibly look into this. You could also just have a bad batch of cells that happens every now and again even to the best of us.
Agree with jamie... check your temperature, humidity and CO2 levels regularly, don't just trust your incubator readings... Some cells are more sensitive to CO2 level changings than others..;
Also: for this particular problem, culture a bit longer, split them and give them time to recover from thawing.
Also: for this particular problem, culture a bit longer, split them and give them time to recover from thawing.
Also agree with above mentioned issues. C02 level also infuences the PH of your Medium (different for different mediums as well). Though a big change should be visable in Phenol-red mediums, because it is petty much changing to blueish/pinkish or yellowish if PH changes away from physiological values. So if you don't noticed a colorchange in your flasks it should be close to normal. Most immortilized cell lines (of course their are tricky once too) are quite rubust.
Have you considered a infection with mycoplasma? other infections?
You might want to check for this. Some institutes offer this as a core facility service, if not you can buy test kits for this. Make sure there is no antibiootics in your Medium, because it will give false negative results in the test. Comply with core facility protcols or as given by the kit you are using.
Oliver