A CO2 incubator is used to culture cells to provide it with the optimum temperature, moisture (sterile environment) and to maintain optimum pH. When the media contains carbonate buffer, the CO2 gas from the cylinder is let into the incubator in such a way that the pH remains constant.. I hope you remember the equation where H2O + CO2 = H2CO3 exist in equilibrium ?? The pH is controlled on the same principle..

U can test this by keeping media inside the CO2 incubator and altering the CO2 levels..there will be frequent changes in the media color which indicates the change in pH (remember media contains an indicator which helps us detect the change in pH)..!!

I hope I have cleared your doubt?? Request refinement from the others participating in the discussion..

A CO2 incubator is used to culture cells to provide it with the optimum temperature, moisture (sterile environment) and to maintain optimum pH. When the media contains carbonate buffer, the CO2 gas from the cylinder is let into the incubator in such a way that the pH remains constant.. I hope you remember the equation where H2O + CO2 = H2CO3 exist in equilibrium ?? The pH is controlled on the same principle..

U can test this by keeping media inside the CO2 incubator and altering the CO2 levels..there will be frequent changes in the media color which indicates the change in pH (remember media contains an indicator which helps us detect the change in pH)..!!

I hope I have cleared your doubt?? Request refinement from the others participating in the discussion..

It is not mandatory. I did tissue culture with and without CO2 incubator. Cancer Cells are already adapted to hypoxia. Moreover, Dr. Gatenby's group has novel work that NaHCO3 has anti-tumor activity. So, most probably HCO3- might delay growth phase rather provide tissue culture by optimum conditions. In this regard, you can easily develop tissue culture in absence of FCS although its presence might be preferable.

Thanks to all

Dear Gael, Thank you for your comment,

Based on my simple knowledge, it is for Gases exchange. Presence of serum most probably enhances gases miscibility due to lecithin as surfactant...

Replacing of buffer: although it accelerates log Phase yet it accelerate declining phase too.

@ Gael Cristofari- I would like to add on your point.

HEPES is more effective buffer than bicarbonate and is independent of CO2 in its buffering action. Hence, a media with HEPES as the buffer need not be maintained in a CO2 incubator. Most labs however, use media with bicarbonate buffer systems which are relatively cheap and easy to use. Hence, the use of CO2 incubator becomes inevitable.

@saicharan - do we have any cell culture medium available with this HEPES buffer? 

@ Chandrashekaram Kans- Hyclone make DMEM media is available with HEPES Buffer

Any one here can have comment on new designed incubator with Tri gases; nitrogen, CO2 and Oxygen plus controlled %RH? As far as I know, N2 gas used to control oxygen level in incubator, by doing this one can set his incubator to hypoxic condition reducing oxygen level to less than 5%.

I wonder if old fashion incubator having only CO2 monitor will be good enough for cell growth comparing with new designed which have %RH and oxygen level monitored?

In order to culture cells under optimum conditions the media they grow in needs to be buffered so it stays at neutral pH (around pH 7). It is possible to add a buffering compound to the media, like sodium phosphates or Tris or Hepes, but often these compounds affect the cells' growth. A carbonate buffer system, of HCO3- and H2CO3 has been found to affect the growth of cells the least. H2CO3 in water is in equilibrium with H2O+CO2. To keep the CO2 in the buffer solution at the right level you have to supply CO2 in the atmosphere above the buffer, otherwise all the CO2 would dissipate and the H2CO3 would disappear and there would be no more buffering effect (and the medium would become alkaline).

So when you use a CO2 cell culture incubator, make sure the media contains bicarbonate buffer (usually from NaHCO3).

Of all the buffer system available to us and that can be safely used in tissue/cell culture system, CO2 -HCO3 have the extra advantage.... one of the component is gaseous(CO2). So just by adjusting the diffusion of this volatile component, the pH can be effectively regulated, without the need to re-dissolve any solute component. So CO2 finds its use is cell culture system (inspite of the fact that the buffer range of the CO2-HCO3 system is rather sleek and  removed  far away (9.2-10.8)from the physiological range(7.2-7.4)  . Interestingly even the human body adopts bicarbonate buffer system as primary  over other buffers, Respiration by lungs and alveolar exchange of CO2 can be adjusted to counter acidemia/alkalemia(in addition to the renal compensation). Imidazole buffer (buffer range of 6.2-7.8) is the next important one with His residue of the aa providing the necessary imidazole groups.

whats an acceptable CO2% range for growing cells in DMEM? if more than 5% means what will happened to cells?

Because the partial pressure of CO2 in arterial blood is 4.6–5.9% (35–45 mmHg). And the functions of CO2 in blood is, indeed, buffering. So if the function of CO2 was just buffering, you might not need to use CO2 incubator with HEPES. But I wonder whether there is only one function of CO2.

> whats an acceptable CO2% range for growing cells in DMEM?

> if more than 5% means what will happened to cells?

CO2% range to be set is depending on your experimental purpose. For example, CO2% in venous blood of healthy person is 5.4–6.6%.

Though higher CO2% might results in acidosis, I heard some cell lines require higher CO2% or grow better at higher CO2% (e.g. 8–10%).

Reference:

The blood gas pressures of healthy person is written in clinical books such as "Cardiovascular and Pulmonary Physical Therapy: A Clinical Manual" by Joanne Watchie (2009) p. 228.

Easily accessible information is listed below.

http://www.acu.edu.au/__data/assets/pdf_file/0020/54443/Arterial_blood_gases.pdf

http://www.aci.health.nsw.gov.au/__data/assets/pdf_file/0004/220675/ABG_poster_large.pdf

Just to follow up on Sittisak Pui-Ock's point about tri gas incubators. These incubators allow one to grow cells in a physiologically precise way as would be seen in their natural environment. Whereas physiological in vivo oxygen concentrations range from 1% to 13%, most cell cultures are maintained at ambient 21% oxygen.

One of the best ways I'veheard this described is we know that cells don't just show up the moment you begin your measurements. They all a metabolic history that is dependent upon their oxygen exposure and there is nothing physiologically relevant about room air.

In a similar fashion, CO2 also should be controlled in a way that limits what a cell would normally not face. As mentioned here CO2 also has the benefit that it helps stabilize pH.

There are many examples of these tri-gas incubators but the one that stands out for me in terms of all its features and price point is the HypoxyLab. Its a good sized incubator and workstation that should fit on any lab bench. Hope this helps.

https://www.scintica.com/products/oxford-optronix/hypoxia-workstation/

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