Media doesn't change colour? - (Feb/27/2013 )

Hi, this is my first post!

I've been working in a molecular/cellular neuroscience lab for the past couple months working with hiPSCs from patients with autism.

Everything has been fine so far but we have met with something a bit odd in the last week. Normally the media we use is quite pink (or red depending on the batch), and after a few days of being with the cultures it starts to turn more yellow, we change it every 3/4 days. However a fair amount of the media has been staying quite pink in the last week. We have some glia which have been in the same media for over a week now (they aren't needed and we wanted to see if it would eventually change) and are still pink.

It's not the batch, as we've used the same batch on other samples and it's gone yellow. We are wondering if it could be the incubator? We just autoclaved it to get rid of a fungal infection and have recently received a low CO2 reading

We feed our neurons/progenitors/glia with DMEM+Glucose+penstrep (solution = 2 mls penstrep, 0.32g Glucose, 20mls horse serum and then made up to 200ml with DMEM).

Has anyone got any ideas?

Thanks!

Nick.

There is usually phenol red in tissue media that acts as a pH indicator, which in turn tells you how quickly your cells are consuming nutrients. If you have a low CO2 reading in your incubator, it is possible that your cells are not in an optimal environment, which is indicated by your media maintaining a constant pH. I would increase the CO2 to the recommendations provided by published literature (optimal % CO2 for your cells) and try again. It is probably because you have insufficient amounts of CO2. Also check your water bath that is at the bottom of the incubator (especially since your autoclaved it). Sometimes people forget to replace the water and the cells do not grow properly.

I do not know much about viable of the cells you are using. Are your cells immortal? Have your cells reached a high passage which is causing there growth to be hindered?

Good luck

The pink/red/yellow colour is most likely a pH indicator called phenol red. This is pinkish purple at high pH, red at about pH 7 and yellow at low pH. The colour change is dependent to some extent on the amount of CO2 in the incubator as dissolved CO2 forms carbonic acid, which along with the bicarbonate in the medium causes a buffering to about pH 7 with 5-10% CO2 (depends on the exact amount of bicarb in the medium). A low CO2 reading could easily give you a purple colour - the same thing happens when you leave the medium open on the bench as there is about 0.1% CO2 in normal air. Also note that the humidity of the air in the incubator can affect the CO2 reading from the incubator - make sure the humidifier in the bottom has some water in it.

The first thing you should do is culture some of the cells without antibiotics (if your sterile technique is any good you shouldn't need them for culturing at all) and see if you get a contaminant growing - these will often cause medium colour changes, especially towards basic pH's. The presence of antibiotics in the medium can suppress but not eliminate low level bacterial contaminants, which can still have an effect on the cells.

Thanks for the advice!

I'll pass it onto the lab and we'll do some more CO2 checking. The issue with the incubator is that it's CO2 monitor seems to be off, it registers as the correct CO2, but the centre tech measured with his own monitor and came up with a different reading. We'll do a third test to make sure it's not the problem. the incubator is meant to be at about 6% CO2, how low do you think it would have to be until the media fails to change colour?

In terms of the cells. They are not immortal and don't replicate a much if at all (past a certain stage). The Glia have been passaged twice, and won't be passaged again (although it is possible, I wouldn't do more than 3/4 times as past that they won't grow at all). The Neurons on the other hand, if they have lost their pluripotency and are truly turning into neurons, passaging tends to tear them apart so can't be done much at all!

Considering the work we are doing (hiPSC derived neurons from patients) the cells are generally difficult to work with and extremely valuable so can't really do any side-testing. May be able to do something with a different sample.

Your cells will change color depending on the growth rate and nutrient requirements of your cells. It is hard to say what will happen at 6%. 6% should be more than enough CO2 for your cells.