We've used HEPES in the past for exactly that purpose, just added to normal DMEM (with carbonate).

An alternative is to use a closed system with medium equilibrated in the incubator before. If you don't evaporate the CO2, the ph is stable. Cells acutally need very little Oxygen, so that is not a problem in most settings. In a students course we had HeLa cells in a closed chamber and cultivated them from Thursday to Monday on the microscope stage (37°C), they were still alive. Of course that will depend on cell density and cell type but may be worth a try.

With regard to your original question, I'm not sure if HEPES is sufficient by itself in DMEM.

However, I have used Leibowitz's L-15 medium in a humidified 37C incubator without CO2 to culture breast cancer cells. It required no additional buffers and we supplemented it with the usual 10% FBS and 1% antibiotics/glutamine.

We've used HEPES in the past for exactly that purpose, just added to normal DMEM (with carbonate).

An alternative is to use a closed system with medium equilibrated in the incubator before. If you don't evaporate the CO2, the ph is stable. Cells acutally need very little Oxygen, so that is not a problem in most settings. In a students course we had HeLa cells in a closed chamber and cultivated them from Thursday to Monday on the microscope stage (37°C), they were still alive. Of course that will depend on cell density and cell type but may be worth a try.

I cultivate my cells (HT22; mouse hippocampal neuronal cell line from David Schuberth at Salk Institute) in Leibovitz`s L-15 in a microtiter plate covered with "breathe easy" membrane for over a week (7-9 days) at temperatures from 25 to 37 degrees Celsius and they are over 95% alive afterwards. It also works with a 50% DMEM with cabonate buffer and 25 mM HEPES and 50% L-15. As Steffen Dietzel points out correctly this may depend on cell density and type. My HT22 are 10000/well initially and they slowly continue dividing under these conditions depending on the temperature.

If you need microaerophilic conditions, could you use the original CO2 incubator, namely a candle jar?: http://academic.pgcc.edu/~kroberts/Lecture/Chapter%206/special.html

Hi Nada,

Depending on the type of cells you are culturing, you may want to consider using CO2-independent medium, see following link:

https://www.lifetechnologies.com/order/catalog/product/18045088?ICID=search-product.

Some cell culture text books highlight the cytotoxicity associated with the use of synthetic buffers.

Good luck

Basil

Thank you all for your appreciable replies. But to make myslef more clear; I am directed to use this specific media DMEM besides my bacteria doesn't need CO2 as nutrinional supplement but 5% CO2 is needed only to balance the bicarbonate buffer. Conditions required were 5% co2 and 95% air.. That's why am afraid I wont be able to use the candel jar solution

Steffen. When you used the Hepes on Dmem, did you observe any changes in the pH and how did you use it; in a closed system or an open system? I think it has to be used in a closed non-ventilated flasks

Well.. If I decided to use the candle jar, what would be the composition of the air inside the jar? Would it be as I demand; 5%CO2 and 95% air?

Nada, we used medium with phenol red in an open chamber and the color was stable, so the pH was too. Our mammalian cells were happy for quite some time and they wouldn't be if pH changed. I seem to remember that the HEPES concentration was high, but I don't recall it (and am at home so can't look it up). In a closed chamber I don't use HEPES - no need for it. You just have to close the chamber fast enough after the medium comes out of the 5%CO2-incubator. 'Closed' in my case means a live cell chamber, that is to coverslips about a millimeter apart completely filled with medium. No air. But any other closed system should do as well.

Not sure about the sources for this laboratory manual, but there they mention the air composition inside a candle jar: http://delrio.dcccd.edu/jreynolds/microbiology/2421/lab_manual/oxygen_needs.pdf

Thanks a lot for your replies.. Which company can offer me that media DMEM with HEPES BUT NO BICARBONATE?

Hi, I am not sure if the question is still active after all this time, but nevertheless, since I have been performing quite a search on the topic lately, I thought that the info I found could be useful to anyone out there.

Here is a possibility of medium with HEPES W/O BICARBONATE :

https://www.sigmaaldrich.com/catalog/product/sigma/d1152?lang=en®ion=RO