Does anyone know of a method for incubating CHO without a CO2 incubator? - (Aug/15/2006 )

Up to now we haven't had a need for a CO2 uncubator because we were culturing S. cerevasiae for GFP and we are trying to begin mammalian cell culture, but have no CO2 incubator or the funds to actually purchase one until the new budget comes through. Does anyone know of a way to incubate the cells without the use of a CO2 incubator. My only thought so far has been to somehow flush the cells with CO2 while in the regular incubator. Doesn't make for very sterile conditions though. Thanks

Use another PI's CO2 incubator? I have heard that some people will very tightly cap the cells to maintain the CO2 produced by the cells themselves and plate at a high density? I would still attempt to use another lab's CO2 incubator if possible.

Dear Chrisbean,

Years ago before the advent of 0.22uM filter top TC flasks, we had to loosen the lids of flasks/bottles to allow the entry of CO2 to buffer the media. On some ocassions we wanted to grow cells in closed systems but within the CO2 Incubator. We tightened the lids and used media buffered by 25mM HEPES.
In your case you can do the same. You will however need to change the media on a more regular basis. If you get anybody saying that they are not oxygenated, you can retort by asking them about tissue oxygenation.......i.e. everybody grows there cells in 21% Atmospheric oxygen. I am positive that tissues are not oxygenated at that level.

Hope this helps

Years ago, I was faced with this problem and I got round it by incubating my dishes/flasks in a sealed box that had been filled with 5% CO2 from a cylinder. A small plastic lunch box was used I seem to recall.


The CO2 was introduced via a flexible piece of tubing inserted between the lid and the box and left to gas for about 2mins. The box was then sealed with plastic tape and incubated in a 37 deg incubator.

It worked OK but naturally borrowing space in someone elses incubator would be by far the best solution!

Hope this helps

I read somewhere that the reason for CO2 in the incubator is to maintain the pH of the medium.

If you do not have a CO2 incubator, add HEPES to the medium and tighten the lid of the tissue culture flask.

I hope this may help.