ES / MEF cell culture and electroporation of targeting construct

Day 0

One frozen vial of Murine Embryonic Fibroblasts (MEFs) is thawed quickly in a 37oC water bath. When the last bit of ice is melted, spray the vial with 70% ethanol and transfer the contents of the vial into one 75 cm2 flask (T-75) containing 20 ml of MEF media. Place the MEFs in a 37oC, 5% CO2, 86% humidity incubator. Every frozen MEF preparation thaws a little differently. If on Day 1, the MEFs are only 50% cconfluent, thaw another vial into your ongoing T-75 MEF flask. It is important for the MEFs to be maintained at a relatively high density, or they will not continue to expand.

Day 2

MEF passage 1: Expand the MEFs as follows: The MEFs should be 90% confluent (if they are not, feed them another day). Split as follows: Suction off old media, rinse flask x 1 with 10 ml phosphate buffered saline (PBS). Add 5 ml Trypsin/EDTA media (0.05%/0.02%) to the flask and incubate for 5 to 10 minutes at 37oC. After 10 minutes, the cells should be detached from the bottom. Now add 10 ml MEF media to this flask, pipette up and down to disaggregate the cells into a single cell suspension, and add the entire contents of the flask into one 150 cm2 flask (T-150). Add 20 ml MEF media to the flask, so that the final volume is 30 to 35 ml. Incubate at 37oC, 5% CO2, 86% humidity.

Day 4

You should now have 1 T-150 flask of confluent MEF cells.

MEF passage 2: Mitotically inactivate cells as follows: Rinse with PBS (10 ml) and harvest with 7 ml Trypsin/EDTA for 10 minutes at 37oC as above. Add 10 ml of MEF media and pipette cells up and down several times. Take 10 ml of cells from the flask and put in a 50 ml centrifuge tube. Refeed the T-150 ml flask with 25 ml of fresh media. To the 50 ml centrifuge tube containing the MEF cells, add 5 ml MEF media (bringing the final volume up to 15 ml). Take this tube to the blood bank and irradiate (3000 rads). These inactivated MEFs will be used to make two 10cm dishes of MEF feeder layers for your ES cell thaw on Sunday. Using a hemacytometer, count the MEFs before adding them to your dish. A good monolayer will be formed if you add approximately 1x106 MEF cells to each 10 cm dish. Plate inactivated MEFs in 2-10 cm dishes having a final concentration of 1x106 Mef's per dish and a final volume of 12.0 ml. We prepare 1 extra 10cm dish in case of contamination or poor monolayer.

Sunday, Day 5

Thaw ES cells as follows: Suction the existing MEF media off one of the inactivated MEF 10 cm dishes. Refeed the dish with 12 ml ES media. Thaw one frozen vial of RW4 cells (1x106 cells) in a 37oC waterbath. When the last bit of ice has melted, spray the tube with 70% ethanol and transfer the contents of the vial to the inactivated MEF dish. Rock the dish to evenly disperse the cells. Incubate overnight at 37oC, 5% CO2, and 86% humidity. The T-150 flask of ongoing MEFs is ready to be expanded today. Rinse with PBS, add 7ml Trypsin/EDTA, for 10 minutes, then add 10 ml of MEF media, and pipette up and down several times. Pipette 8 ml of these cells into a new T-150 flask containing 25 mls of MEF media. Refeed the existing T-150 with 25 ml MEF media to create 2 T-150's of expanding MEFs.

Day 6

Feed the ES cells with 12 ml ES media. Approximately 60% of the ES cells will form colonies in the dish. They are football shaped, shiny, and plump. You will need 4 x 10 cm dishes of irradiated MEFs for your electroporation on Day 7. You have 2 T-150's ongoing from which to make these 4 dishes. Follow the procedure for mitotically inactivating MEFs on Day 4. However, use both T-150s this time. Don't forget to refeed them, or you will not have MEFs for the end of the week. If the 2 T-150 flasks of MEF cells are not confluent today, feed them, and then inactivate them on the morning of Day 7. If you inactivate your MEFs on Day 7, you must either give them 2 to 3 hours to attach before changing the MEF media to ES media or initially inactivate and plate them in ES media.