We have used the same isolation and expansion protocols for the generation of PMEFs for many years and never had any major issues. We recently changed our batch of FBS and now we are seeing massive cell death following the first passage. Most of the time its apparent the next day, but we have had two batches where they have looked fine the day after passaging only to have the majority detach the following day. If the surviving PMEFs are left to grow we don't see massive cell death with subsequent passages, so i'm thinking the survivors have adapted to the media (specifically the FBS).
The isolation media is pretty basic; high Glucose DMEM, 15% HI FBS, 0.1mM 2ME, 50U/mL Pen/Strep and 2mM Glutamine. We culture in this media for two days then passage into 10% HI FBS, all other components stay the same.
Historically our FBS testing has been focused on how our mES cells perform and they grow fine in this batch of FBS.
We would prefer not to have to use two different batches of FBS for culturing our cells to avoid mixups and additional expense, so before we start looking into new FBS does anyone have any suggestions/advice.
Thanks
Hi Natalie Daw . You could contact your FBS supplier and ask for samples from their lots for testing. If you are a steady customer, you can ask them to reserve lots that work well for you or buy up certain whole lots.
Hello Natalie Daw
FBS may vary in quality in terms of growth factors, amino acids and other components from batch to batch. Initially the cells may not show the effect of the new batch of FBS because they already have stored the necessary growth requirements for their growth from the previous batch of FBS. Once these stores are depleted the detrimental effect of the new batch of FBS if any becomes visible because the cells will be utilizing the new batch of FBS.
So I suggest you culture your cells in the new FBS batch alongside the present usual batch of FBS in which the cells are growing fine. Compare various parameters such as cell viability, morphology, etc. just to see that the new batch of FBS does not have any detrimental effect on the cells. If everything works fine you can grow your cells in the new batch of FBS.
I hope this helps.
Best.
As long as you buy from a reputable manufacturer, it should never make a life or death difference. Even unknown, half-decent manufacturers do these tests for you. If a batch kills all the MEFs, they will not sell it. 99% it's some other problem that just happened to coincide with your new batch. You'll need to test everything systematically.
Clearly you have differences from one batch of FBS to another in terms of growth potential and survival. It is necessary to test each batch before starting series of experiments and if possible to buy or reserve a large quantity of serum. Before blaming the serum you have also to test other potential factors leading to cell death as mentionned by John
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