I am doing some cell work which requires me to incubate some cells with a few compounds of interest. I have read from a few articles claiming to remove the complete DMEM (growth media) and adding the serum-free media (i.e. DMEM without Fetal Bovine / Calf Serum and the compounds. What is the reason behind this? For your kind advice, thank you.
Hi Justin,
I think that it should also be taken into consideration that in complete medium your test substance likely binds to albumin and other serum components whereby its effective concentration is lowered
Hello, it is likely for synchronization of cell cycle, and preventing cells from enter into proliferation just before drug treatment -- so that the experiment can start with a relatively homogeneous population.
However, depending on cell type and serum starvation duration, cells may respond differently: either go into autophagy, apoptosis or even differentiation. So the experiment should be carefully planned.
See some references.
https://www.google.com.hk/webhp?sourceid=chrome-instant&ion=1&espv=2&ie=UTF-8#q=serum%20starvation%20synchronization%20protocol
I agree with my colleague: all cells will be synchronised and there fore physiologically similar leading to a more consistent normalised response
in addition however done substances in serum can inhibit uptake or drug and/or antagonise the response complicating the effects of the drug on cell populations
In addition, growth factors in the serum can activate some kinases, such as ERK, or other kinases involved in transcription
Hi,
It is generally considered to incubate cells in serum free media to get a homogenous cell population as the researchers have said above. Replacing dmem with serum,free dmem just stops cell division. And it depends on the assay you are gonna do and the type of cells you are using.
hope this helps
Hi Justin,
I think that it should also be taken into consideration that in complete medium your test substance likely binds to albumin and other serum components whereby its effective concentration is lowered
Simonetta's answer is perfect. That's probably the most significant problem with leaving the serum in the medium.
Just a few comments on previous replies. Removing serum will stop cell division, but not immediately. It's not unusual to have to leave cells 24 -72hrs before this has ceased. Also the points about albumen and other 'carriers' are well made and for this reason it's very common to deliberately include around 1mg/ml BSA or HSA in serum free medium. This often improves cell health due to improved uptake of certain nutrients and buffering of waste product. For this reason the addition of albumen will actually improve the ED-50 of certain moieties, (although it may of course produce the opposite effect on others).
Dear all researchers,
Many thanks for your kind responses. It has really enlightened my understanding. Really blessed to have you guys in the science community.
Best wishes,
Justin
In addition to the other comments: Since the composition of FBS is unknown and can vary between batches it is scientifically advisable to use a serum-free defined medium.
See also attached publications.
Dear Sir, if I need to incubate the cell samples for more than 24h , such as 48h to 72h, would it be better to have them cultured in FBS supplemented complete media so that the cells would not die off?
It really depends on the cell type and what you need to achieve. If you are working with recombinant CHO.K1 or HEK cells, these will have no trouble surviving 48-72 hrs in serum free media. I have used this option for decades and have never seen any reduction in viability in the control cells. However not all cells are as robust as these, (especially non-transformed). Even with robust cells though you should take certain precautions. If they are adherent then remove the serum when they are about 50% confluent. They will complete the divisions they are already engaged in or have already been prompted to begin by FBS growth factors, thus they will still reach 80-90% confluence 2-3 days after its removal. If the cells are over confluent with no serum present then layers may peel off and die or at least give inconsistent results. I would also include 1mg/ml BSA or HSA to help buffer waste product, (as I mentioned earlier). This also reduces the chances of compounds adhering to the cell culture plastic, (in the absence of FBS). You should also bear in mind that although results from serum free cultures are in general easier to reproduce and generally allow compounds to display greater activity, (outside of cell division based parameters), they are also one step further from a real life environment. You should certainly be more alert to the toxic effects of any compounds in a serum free environment. If any test materials show toxicity when FBS is present, they may be 10 fold more so in its absence. The presence of BSA may reduce that issue a little, but often not by much.
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