Preparation of NeoR Murine Embryonic Fibroblasts

<center> <h1>Preparation of Neo^R Murine Embryonic Fibroblasts</h1> </center>
Three weeks prior to embryonic fibroblast isolation, a PGK-neo male mouse is mated to a heterozygote female. 13.5 days to 14.5 days after the plug is observed, sacrifice the pregnant female either by cervical dislocation (after appropriate anesthesia) or CO₂ asphyxiation.

Prepare three 10cm dishes containing 15ml PBS each. With the mouse on its back, wipe down the abdominal skin and fur with 70% alcohol. Make an incision down the midsection using scissors, thereby exposing the uterine horns. Observe how the embryos are located on each side of the uterus and in the amniotic sacs. Pull the embryos and uterine horns away from the abdomen and carefully detach them from the animal, placing them in a dish of PBS.

Using fine tipped scissors, cut open the uterine horns and release the embryos into the PBS. Next, carefully detach embryos from the amniotic sac and place in a fresh dish of PBS. While holding the embryo with a forcep, carefully pull out the liver, decapitate and rinse in another dish of fresh PBS, trying to get rid of as many red blood cells as possible.

Prepare 1 dish of Trypsin/EDTA, 15ml/dish. Place the decapitated embryos in the dish containing Trypsin/EDTA. Using curved Iris scissors finely mince the tissue quickly until it can be taken up in a 10ml pipette. Pipette up and down 6-7 times and place the dish of minced embryos in the incubator for 15 minutes. Add more Trypsin/EDTA to the dish and again pipette up and down with a 5ml pipette. Then place the dish back in the incubator for 10 more minutes.

Put all the contents of the dish in a 50ml conical tube. Allow the pieces of cellular debris to settle out over a period of 2 minutes. Remove the supernatant into a fresh tube, bring the volume up to 50ml with MEF media and spin at 1000 RPM for 5 minutes at 10^o C. Resuspend the pellet in MEF media and place the cells in a T-150 flask. Let the cells grow overnight in the incubator. Leave the flask undisturbed for 24 hours. After this time, change the medium to remove any cellular debris and place your flask back in the incubator for another 24 hours.

The next day, if you want to freeze your MEFs at passage 1, take up the cells with Trypsin/EDTA and place all the cells in a 50ml centrifuge tube, bringing the volume up to 25 ml with MEF media. Spin cells in a centrifuge at 1000 RPM for 5 minutes at 10^o C. Resuspend the pellet in 5.0ml MEF media, pipette up and down to break the pellet into single cells, add 0.5ml MEF cells in each of 10 Nunc cryotubes, add 0.5ml 2X MEF Freezing Media to each tube. Freeze vials in -70^o C freezer for a week then store in liquid Nitrogen.

If you want to freeze your isolated MEFs at passage 2, then split your original T-150 into 3 T-150s and incubate for 24 hours. Next day feed these 3 flasks with fresh MEF media. The following day take your cells up with Trypsin/EDTA, pool all your cells, spin at 1000 RPM for 5 minutes at 10^o C, and resuspend them into 10mls MEF media. Add 0.5ml cells in each of 20 Nunc cryotubes, add 0.5ml 2X MEF Freezing Media to each tube. Freeze vials in -70^o C freezer for a week then store in liquid Nitrogen. At passage 1, you can get 10 frozen vials from 1 T-150 flask, but at passage 2, you can get only 6 to 7 frozen vials from each T-150 flask.

Usually one frozen vial of MEFs (thawed and expanded) will be enough cells for feeder layers for 1 entire electroporation procedure. If thawed at Day 0 into a T-75 you should have been able to expand it into 2 T-150's 7 days later. If you thaw your vial at Day 0 and on Day 1 it is only 50% confluent, you may want to thaw a second vial into that ongoing T-75. MEFs like to have some contact to enable them to stay in their growth phase. If they are too sparse they will begin to senesce. After passage 9, you will see these cells begin to grow more slowly and senesce. At this time, it is time to get a new thawed vial of MEFs expanded if you are preparing to do another electroporation.

After your MEFs have been frozen down, thaw a vial into a T-75 flask to see how well they have survived the freezing procedure. You no longer need Pen/Strep in your MEF media. When you expand your MEFs to a T-150 flask, divert some of your cells to a T-25 with MEF media without Pen/Strep. Leave this flask in the incubator for 5 days without changing the media. On the fifth day take at least 4 ml of the media off the cells and put in a 15 ml centrifuge tube, label the tube with the type of cell supernatant, your name, date and phone number. You should use this to evaluate if there is any mycoplasma present, either using your own institution's facilities or by the use of one of the commercially available kits on the market. You should test every new MEF prep. that you prepare.