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Is it necessary to pre-warm medium? - (Apr/02/2006 )

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Hi all
I've been following this string of responses about medium and thought i'd add my thoughts.

Ideally if you're growing monolayer cell lines its best to prewarm your medium to 37C before adding it to your cultures if you want them to grow optimally. We grow thousands of cell cultures each year and need to test them as rapidly as possible. We therefore leave our medium in the incubator permanently once it is prepared (5 x 100ml bottles) and use it within 3 or 4 days. This also ensures the pH is always correct (we use an open culture 5% CO2 in air system).

If usage is low, decant it aseptically into small lots and use it as required. Additional L-glutamine will need to be added if its kept at 37C for an extended period of time so its best to keep it refrigerated if usage is low. Its probably also best to keep the medium away from light (normal use is OK just don't leave it sitting out on the bench for extended periods of time). We prefer not to heat our medium in a water bath because of the risk of introducing water-born contaminants - we use the incubator. Same goes for PBS which sits in the incubator until the bottle (100ml aliquots) is used. We also preheat aliquots of trypsin in the incubator each morning prior to subculture.

With regard to color, if phenol red is used as a color indicator it should be a salmon pinkish color when the pH is correct (varies a little between different types of media). Most mammalian tissues should be in the pH range 7.2-7.4. As the culture becomes more acidic (because of increased cellular production of CO2) it will turn yellow. For example, when a cell culture becomes overgrown, or is contaminated with bacteria, the media will turn a characteristic yellow color indicating increased acidity due to CO2. By the same token, if you use an open culture system with 5% CO2, the medium will go a very deep pink color due to increased alkalinity if the lids on tissue culture flasks are left closed. Its the sodium bicarbonate in the media thats doing the buffering. Having said all this, many cell lines are extremely robust and you can "abuse" them in many ways and they'll still grow. But if your dealing with sensitive cell lines or you want your cells to be operating under "normal" physiological conditions prior to experiments then i'ts best to treat them kindly biggrin.gif


How about room temperature media? Sometimes I have to do things over a couple hours timespan. Would that affect my cells?


ph34r.gif In my admitttedly limited experience, it's the temperature difference that makes the difference, not the temperature itself....

Most of us "get" the idea of concentration diffusion, but there is temperature diffusion, too....
make part of the culture too cold and cells are going to woosh around in it.... as well as having conformational changes if the temperature is too extremely different....

I tend to let the cultures and the media come to ambient temperature if I'm working for a while... if I'm doing a quick feeding, I will use media that's been in a water bath that's the same temperature as the incubator.


QUOTE (piggy @ Apr 8 2006, 05:34 AM)
How about room temperature media? Sometimes I have to do things over a couple hours timespan. Would that affect my cells?

If your cells aren't too temperamental then probably not. However you will have both a temperature and pH differential after a couple of hours assuming you're using an open culture system. At the end of the day it'll depend on the cell type and cell line. I'd avoid the extremes eg. 4C -> 37C but it doesn't mean that many cell lines won't tolerate this - it must have a temporary effect on their metabolism though. Good practice would dicate leaving the cultures in the incubator until you need them and having the media prewarmed before use.


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