Hi Mayada,

If the cells are grown in bicarbonate buffered media, CO2 incubator is definitely required to maintain the appropriate pH of the growth media. After reading all the posts I am in a point to comment something on the issue:

1. @Mandy:

If you tighten the unfiltered cap of the culture flask, I am sure you are culturing your cells in hypoxic condition, the cells can grow but the normal physiology and the protein expression levels will be significantly different.

2.@Prafull:

Your idea seems a better alternative. I have also cultured cells in a mixture of HEPES and bicarb buffered media for cells that secretes acid and had a good luck to keep a better pH control of the culture media. Though I never used 100% HEPES buffered media. If Mayada can culture her cells in media with HEPES (No bicarb) that would serve the purpose. But burning a candle inside incubator could create a toxic environment (with shoot and different byproducts coming form the fuel/wax and an elevated temperature) is definitely not a good idea.

3.@ Benjamin:

Using dry ice or in situ production of CO2 by reacting baking soda with acid could be a possibility for getting instant supply of CO2 but the incubator should be equipped with a controlled release mechanism to maintain the CO2 inside at 5%. But using dry ice inside incubator will significantly lower the temperature and for the other acid-base reaction it could be exothermic as well (may not be very significant though).

Best-

Option 1: use organic buffered media (eg. HEPES -buffered media) for 1-3 days of culture.

Option 2: use portable incubator.

Option 3: you can use chemically generated CO2 in a chamber and culture the flask in it.

CO2 is mainly required for pH balance of the culture medium. As suggested by Mukesh you can supplement your medium with HEPES organic buffer. Nevertheless, I have cultured a number of different cell lines (HeLa, Vero, MDCK, MDBK) in a non CO2 incubator without any problem in w/o filter cap flasks. Off course different cell line s have different growth requirements and if it is not working in your case and you dont have access to CO2 incubator, you can try keeping your cell in a dessicator with a candle inside. Please ensure that it is not opened frequently and off course safety is another issue. It worked brilliantly in my previous lab.

Hi Mayada,

If the cells are grown in bicarbonate buffered media, CO2 incubator is definitely required to maintain the appropriate pH of the growth media. After reading all the posts I am in a point to comment something on the issue:

1. @Mandy:

If you tighten the unfiltered cap of the culture flask, I am sure you are culturing your cells in hypoxic condition, the cells can grow but the normal physiology and the protein expression levels will be significantly different.

2.@Prafull:

Your idea seems a better alternative. I have also cultured cells in a mixture of HEPES and bicarb buffered media for cells that secretes acid and had a good luck to keep a better pH control of the culture media. Though I never used 100% HEPES buffered media. If Mayada can culture her cells in media with HEPES (No bicarb) that would serve the purpose. But burning a candle inside incubator could create a toxic environment (with shoot and different byproducts coming form the fuel/wax and an elevated temperature) is definitely not a good idea.

3.@ Benjamin:

Using dry ice or in situ production of CO2 by reacting baking soda with acid could be a possibility for getting instant supply of CO2 but the incubator should be equipped with a controlled release mechanism to maintain the CO2 inside at 5%. But using dry ice inside incubator will significantly lower the temperature and for the other acid-base reaction it could be exothermic as well (may not be very significant though).

Best-

I used use candel inside a jar where I put my cultures

It was very effective and I got good results.

As suggested earlier by me, I have similar experiences with Mona. Just take care that candle is small and should not glow longer just to ensure that the temperature inside the jar is not too high. Remember even a 2 degree high temp might be detrimental to the cells.

Sorry I forogt, you can also put small plate (culture plate) with water on it, inorder to keep humidity.As Parfull said the candel should be small. Also the jar should be well closed so air would not come in. I worked this way for years and I got just perfect results.

If you have no access to a CO2 incubator the alternatives suggested by others are OK provided you have a way to measure the concn. of CO2 in the incubator for reproducible results.

Thank you to all of you. Your high-quality answers were highly appreciated. But actually no body told me yet how much Sodium bicarbonates and how much acetic acid should I use to have only 5% of CO2 inside an incubator of 6 foot size. and how can I be sure about the percentage? Is the pH papers sufficient for this regard?

Thank you.

Hi,

Using a medium with a low concentration of Bicarbonate is possible to culture the cells for a long period the cell at 37°C in absence of CO2 atmosphere.

A.Notarangelo

Thanks Angelo, but how low should be the Sodium bicarbonate? I mean how much should I use /per 1L of media?

Tq.

Mayada

Hi,

A last year we have transfer the cell in lab space on Shuttle (NASA) and the same cell return after twenty days. In this perio the cell are grow in absence to Sodium Bicarbonate with leibovitz L-15 medium.

Regards

Hi,

.....Sorry, obviously without CO2

Angelo Notarangelo

Mayada, 

Though I've never tried it, bell jars and a lit candle do work. This is how the original CO2 incubators were developed. While I can't provide specifics on the technique, I would be careful of contamination if you do use this method.  I would suggest autoclaving any jars or external equipment (if possible) before bringing it into the incubator.  If you attempt this method, you can monitor the CO2 concentration externally using a FYRITE device (or something similar). The device reads CO2 levels via a probe that is inserted into the chamber. Devices like these can get pricey though, which might warrant using those funds towards a CO2 incubator with a precise sensor that automatically and accurately maintains 5% CO2.

I have a Question: 

To determine the Hypoxic induced protein expression in cancer cells, How to maintain Hypoxic environment inside a normal CO2 incubator.??

I have tried sodium bicarbonate and acetic acid trick. However I failed to get fruitful results. Color of the media quickly changed which I assume is because of the acedic fumes of  acetic acid. I will not recommend this method. 

All ad-hoc methods described in this thread will suffice for a amateur kind of research. If you are really serious that your data can be trusted, go ahead and grow cells in CO2 incubator. Now that you can't afford that, use these 2 alternatives":

1. C chamber (http://www.biospherix.com/cell-culture-equipment/hypoxia-chamber-c-chamber.html)

2.Modular chamber(http://www.brincubator.com/index.htm)

Ad hoc techniques will create enough background and  noise with variable errors that you will have to spends the rest of your available time in piecing together what is useful and what is trash

Best wishes

Subhradip