Help: How can I make cell line from MEF cells? - (Jun/02/2003 )
Hi, I want to set up cell line from mouse embryo fibroblast, who can give me some good advice? Thanks!
Making a primary cell line from mice is quite easy to do, but doing so with no contamination is what makes it a little tricky.
Firstly you need to visualize the vaginal plug...that will tell you approximatly what day conception took place...if you dont know what that is then i suggest you read up on mouse breeding from there i usually count 13-14 days. On day 14 this is whre the magic happens:
Things you need....sterile container with sterile filtered PBS. You need to make MEF media too....the recipe can be found online, i cant remember the molar concentrations but all i remember is (from doing it so many times) is that you need 440ml DMEM, 50ml FBS, 5ml Essential amino acids, 5ml L-Glutamine and 0.5ml Pen/Strep 10,000. I use all Gibco products.
This should be sterile filtered.
You need to sacrifice your female mouse and using all sterile instruments, wipe her belly down with alcohol and carefully open her abdomen with scissors...you should be able to see the embryos. With forceps gently pickup the sac of embryos...should look like a string of pearls and place into a container containing sterile PBS.
Now you have to move to the hood. Not compton...hehe
so now you have the embryos:
this is kinda brutal and icky, and i didnt like it when i first did it...but open the sacs and release the embryos in the sterile PBS. transfer them to another dish with PBS...there with sterile scissors you need to cut off their heads and open their chest cavities and remove all their insides...
i know ick!
next transfer the carcass remains to another dish and wash it with more PBS...then with fine tipped scissors you need to mince the tissue. Mince the tissue very finely and add some trypsin...i never measure, just add enough to cover the tissue. Leave for about 10min....sometimes while it is in the trypsin/edta i mince it a little more. You then need to transfer all this material to a 50ml tube and add MEF media to the top and let all the pieces settle. Once it has all settled, take the supernatent and spin that down at 1500 for 5min @4 degrees, and you should end up with a pellet. YOu should have fibroblasts in here now....if not repeat the process by adding more trypsin to the embryonic parts and mice and spin and remove supernatent and spin that down...etc... then once you have a pellet you need to culture it in a flask. I usually use a 50mm2 flask with 20ml of MEF media, resuspend your pellet in 1ml and add to the flask...the next morning you should see some cells have adhereed and they are fibroblasts.
i hope i helped...email me @ firstname.lastname@example.org if you have any more questions...
I just did that a few months ago and found the following references really good:
Freshney R. I. 1994. Culture of Animal Cells: a manual of basic techniques.
Check your local University library for that one, it has pictures and fairly detailed instructions as well.
I have been using DMEM and Antibiotic/Antimycotic from Sigma with 10% FBS for maintenance of my lines, they seem to be doing fine.