Freezing culture media - (Jul/27/2011 )

I am using Invitrogen MCDB-131 medium (catalog no: 10372-019) for cultuing HUVEC cells (Primary endothelial cells from human umbilical cords).

When I open a new 500ml bottle (doesn't come in smaller sizes) I add L-Glutamine from a frozen aliquot, and when I use it I tend to make 50ml aliquotes of 20% FCS in MCDB (i.e. 10ml FCS and 40ml MCDB). I make 1 50ml aliquot at a time, so when I finish using the 50ml I decant out a new 40ml MCDB and 10ml FCS.

The problem I am having is that I don't use it especially fast, perhaps a 500ml bottle every 40-50 days, and use it gradually and slowly within that period. However by the time I get to the last 1/3 or so of the bottle the media has started to go a pinkish colour indicating it is going off, and the cells don't grow anywhere near as well as they do when the media is fresh. When I open a new bottle I add L-Glutamine and I assume this is also going off within that time.

I am wondering if it would be a good idea to make my 50ml aliquots at the start of opening the bottle then freeze them down, to preserve the L-Glut and other componants of the media. I hve not heard of anyone freezing media before but just wondering if it is possible or if it would damage various media componants.

it is not going off. the pH changes, that's normal. still usable. don't freeze, you might hurt the components.

yes I know the colour change is due to pH, but if the pH is going up as indicated by the pink colour then surely that will hurt the cells more then fresh media? It coudl just be the L-Glut going off I suppose.

I agree that freezing the media is a bad idea. I wouldn't be concerned about the pH change, but if you're concerned about the L-glut, instead of adding it to the stock media, pull out 50 ml aliquots of media as you use it and add the L-glut to that.

Another thing that could be causing your cells to not grow as well near the end of your bottle is L-Glut breaking down and forming ammonia. You could either do what Kfunk106 suggested and aliquot your media and only add L-glut to the one you are using, or you could replace some or all of your L-glut with Glutamax from Invitrogen--it is L-glut in a dipeptide form that is stable even at room temp. You would have to test it on your cells to see if they grow as well on it--I am doing CHO-S and DG44-CHO work and have found a 50:50 mix keeps my cells happy and reduces ammonia toxicity. As for the pink color--it shouldn't really effect your cells. Pretty soon after you have that media on the cells at 37C in the CO2 incubator the CO2 will start to buffer it back to normal. It is when the media starts looking more orange/yellow that you need to worry=contamination