MEF culture - (Apr/24/2008 )
Hi - I want to know for basic MEF culture, approximately how many cells do you typically get out of a T150 flask after they are confluent enough to inactivate and freeze down or use? I seem to be having some problems expanding MEFs efficiently.
I would guess about 1-1.5*10^7 cells. Most MEFs have a limited life span of about 20-30 generations before reaching the Hayflick limit and aren't really stem cells.
Try using a 20% instead of 10% FCS to get the cells to grow a bit better,
When we make MEF, we expand them in expanded surface roller bottles, using media with no antibiotics and with 10% FCS. Works beautifully.
And we don't use MEF after passage 4, counting thawing as P1. We split them between 1 in 4 and 1 in 6, so we can get as many as 60 or so P150 tissue culture plates out of 1 vial of cells (1x 10^6), if its a good batch. There is some variation between batches, depending on the size of the embryos we've used.
Thanks. I think it's more cost effective for me to just purchase them, rather than growing them up like this...
lauralee - what are expanded surface roller bottles? I've seen the Corning hyperflask system (it's like 10 plates stacked inside the culture flask). Is this something similar? I culture my MEF with DMEM + 10% HI-FBS, no antibiotics.
Here is the link to the one's we use= http://catalog2.corning.com/Lifesciences/e...s(Lifesciences)
They are roughly the equivalent growing surface of 22 T75 tissue culture flasks. However I suspect they will be of no use to you- as if you have never heard of them it is doubtful that you have the apparatus required to use them (you need a roller apparatus in your warm room or incubator). You could check with neighbouring labs, but I don't know that many ppl use them these days (we have a really old one which my professor wisely held on to for decades!!).....
I culture my MEF in MEM with 10% FCS.