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Cell culture - no CO2

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Cell culture - no CO2

Postby Biopolmonkey » Mon Aug 23, 2010 5:02 pm

Hi, I hope this is in the most appropriate forum.

My problem is that I came into the lab today to find that our incubator's CO2 cylinder had emptied over the weekend (faulty regulator) and - amazingly - even though I reside in a chemistry department, no-one else uses CO2 except another group who do cell work but their only cylinder also died this wkend. We have immediately ordered a new cylinder, and I imagine it will take about a week to arrive. What can I do to keep my cells in best health during this time? Also, they are due for passage .. Is it still worthwhile doing this?

The cells are osteoblasts.

Many thanks
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Re: Cell culture - no CO2

Postby biohazard » Tue Aug 24, 2010 8:15 am

Biopolmonkey wrote:Hi, I hope this is in the most appropriate forum.

My problem is that I came into the lab today to find that our incubator's CO2 cylinder had emptied over the weekend (faulty regulator) and - amazingly - even though I reside in a chemistry department, no-one else uses CO2 except another group who do cell work but their only cylinder also died this wkend. We have immediately ordered a new cylinder, and I imagine it will take about a week to arrive. What can I do to keep my cells in best health during this time? Also, they are due for passage .. Is it still worthwhile doing this?

The cells are osteoblasts.

Many thanks


Unfortunately I have experienced the same only with lymphocytes, but at least they didn't have any apparent problems in being without CO2 for several days. As far as I know it mostly affects the pH of your culture medium, so you could try changing the medium more often and perhaps even measure the pH of your stock medium and adjust it to whatever your cells like best, usually somewhere around pH 7.
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Postby JackBean » Tue Aug 24, 2010 8:24 am

do they really need CO2? Or is it just buffer? I think that human cells do not use CO2, so buffering your solution should be fine, shouldn't it?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Re:

Postby biohazard » Tue Aug 24, 2010 12:47 pm

JackBean wrote:do they really need CO2? Or is it just buffer? I think that human cells do not use CO2, so buffering your solution should be fine, shouldn't it?


CO2 in the incubator typically helps to ensure that the buffer works as intended; most cell culture solutions are buffered even if they are used in a CO2 atmosphere. The cells themselves do not use CO2.

The effect of CO2 is actually quickly seen if the cultures are left in the normal air and the medium contains some pH indicator, such as phenol red: the cultures quickly turn to pink/purplish indicating that the pH is increasing. Placing the cultures into the CO2 atmosphere quite rapidly turns the medium to bright red, indicating more neutral pH.

Apparently ~5% of CO2 in the air drives typical media towards neutral pH also if they are turning acidic (e.g. because of the cellular metabolism) - otherwise it would be quite contradictory to put something that lowers pH to a medium that is already turning acidic.

In my experience most dividing cells tend to turn the medium acidic even if CO2 is used, so in the absence of it I'd suggest replacing the medium more often with a neutral medium (whenever the pH starts to get too low). If the medium seems to turn too alcalic, maybe you can try to grow the cells in more dense cultures, so as to increase the acidfication of the medium (I don't think replacing alkaline medium helps much, because it turns back to alkaline soon in the air if the cells do not manage to keep the pH down).
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Postby shekar » Sat Sep 25, 2010 10:02 am

mainly co2 role is maintenance of pH for groing of cells if you wont suply the co2 the pH of the cell culture medium will chainge in to basic condition(means pH of the media increase ) maximum cells will grow at pH 6.0 to7.4 ,
so to salve your problem first you can adjust the pH of the medium in which pH your cells will grow active and you can culure your cells with that medium in closed cell culture flask (the flask without filter ) tarson company provide like that flasks and you can change the medium alternate days or with in 2or 3 days your cells will grow active
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Postby Traiani » Tue Jul 26, 2011 10:40 am

Hello. I need a clarification on Shekar's message. So, if cells are cultured in closed culture flasks, there is no need for specific humidity and CO2 conditions into the incubator?

Thank you
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Re:

Postby biohazard » Wed Jul 27, 2011 7:07 am

Traiani wrote:Hello. I need a clarification on Shekar's message. So, if cells are cultured in closed culture flasks, there is no need for specific humidity and CO2 conditions into the incubator?

Thank you


Well, naturally if you have a sealed container the composition of the outside air has little effect. If your cells grow well in a sealed flask and you can keep the pH ok by just replacing the medium I guess nothing prevents you from doing so.

However, the majority of cell cultures do best in non-sealed containers (most cell culture flasks have filter caps because of that) that allows gas exchange. If the container is sealed, cellular metabolism causes faster changes in the composition of the medium and the air inside the container, causing them to differ from the optimum sooner.
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Postby Traiani » Fri Jul 29, 2011 8:38 am

Thank you for your reply. It is really helpful.
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Postby Nadaezz » Sat Sep 21, 2013 12:41 pm

I have a question and I need your help badly,
I am having a cell culture without a cell line.. only the samples to be tested with DMEM suplemented with 10% FBS.. It has to be incubated in 5%CO2 and 95% air.. but there is no CO2 incubator available.. So how can I provide these conditions to my culture knowing that I am culturing in 25-ml tissue culture flasks with filter cap?
I need the reply urgently
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Postby biohazard » Mon Sep 23, 2013 9:01 am

The cells do not use the CO2 itself, it is for other purposes (see above posts). You may have success if you closely monitor the pH of your culture medium and adjust it when required during your culture. Normal cultures typically require maintenance every 2 to 3 days, but without a CO2 atmosphere you may have to check on your cells a couple of times per day, depending on their growth.
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Re: Cell culture - no CO2

Postby cheryl4ryl » Fri Nov 22, 2013 12:35 pm

Wow! this was so so helpful. I have been wondering why I use non-filter flasks for GMK and Vero and filter for all mammalian cell lines we grow in my Lab. I love you guys. Thanx alot.
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