3.1.1. Derivation of MEFs From Pregnant Mice
1. Sacrifice one pregnant ICR mouse (see Note 9) on the 13th day of conception by brief exposure to CO2 (see Note 10).
2. Wash abdomen with 70% ethanol and dissect the abdominal cavity to expose the uterine horns.
3. Remove the uterine horns into a 10-cm Petri dish and wash three times with phosphate-buffered saline (PBS).
4. Using two pairs of watchmakers' forceps, open each uterine wall and release all embryos carefully without touching the fur.
5. Wash retrieved embryos three times with PBS.
6. Use the same tools to dissect each embryo from the placenta and membranes and discard soft tissues as much as possible.
7. Transfer clean embryos into a new Petri dish and mince thoroughly using sharp Iris scissors.
8. Add 6 mL trypsin/EDTA and incubate for at least 20 min.
9. Neutralize trypsin using at least 6 mL MEF culture medium and transfer the cells into conical tubes. Use MEF culture medium to wash the plate.
10. Centrifuge 5 min at 300g (see Note 11).
11. Divide evenly into T-75 culture flasks (see Note 12).
12. Add 20 mL MEF culture medium to each flask (see Subheading 2.1., item 4, with the addition of antibiotics). Grow the MEFs up to 3 d or until confluent, changing the medium at least once during culture (do not vacuum the lumps).
13. Freeze the resulting MEFs (see Subheading 3.1.3.).
3.1.2. Splitting MEFs
1. Add 2 mL trypsin/EDTA and cover the entire culture-flask surface.
2. Incubate for 6 min.
3. Tap the side of the flask to loosen the cells and add 4 mL culture medium to neutralize the trypsin.
4. Remove cell suspension into a conical tube and centrifuge for 5 min at 300g (see Note 11).
5. Remove suspension, resuspend in 2 mL culture medium, and pipet to fracture the pellet.
6. Distribute cell suspension to desired number of culture flasks.
7. Add MEF culture medium to a final volume of 10 mL.
3.1.3. Freezing MEFs
1. Remove all lumps possible.
2. Add 2 mL trypsin/EDTA and cover the entire culture-flask surface.
3. Incubate for 6 min.
4. Tap side of the flask to loosen the cells. Add 4 mL culture medium to neutralize the trypsin.
5. Remove cell suspension into conical tube. Let remaining lumps sink and remove cell suspension into a clean conical tube.
6. Centrifuge for 5 min at 300g (see Note 11).
7. Remove suspension, resuspend in 2 mL culture medium, and pipet to fracture the pellet.
8. Drop by drop, add an equivalent volume of freezing medium and mix gently (see Note 13).
9. Place 1 mL into 2-mL cryogenic vials (see Notes 14 and 15).
10. Freeze vials overnight at -80°C in Nalgene freezing box (see Note 16).
11. Transfer vials into a liquid nitrogen container.
3.1.4. Thawing MEFs
1. Remove vial from liquid nitrogen and thaw briefly in a 37°C water bath.
2. When a small pellet of frozen cell remains, clean the vial using 70% ethanol.
3. Pipet the contents of the vial once, and transfer the cells into a conical tube.
4. Drop by drop, add 2 mL of MEF culture medium (see Note 17).
5. Centrifuge for 5 min at 300g.
6. Resuspend the pellet in culture medium.
7. Remove cell suspension into culture flasks and add 10 mL of culture medium.
3.1.5. Preparation of MEF-Covered Plates
1. Add 8 |g/mL mitomycin C into culture flask and incubate for 2 h (see Note 18).
2. Wash four times with PBS.
3. Add 2 mL trypsin/EDTA and cover the entire culture-flask surface.
4. Incubate for 6 min.
5. Tap the side of the flask to loosen cells and add 4 mL MEF culture medium to neutralize the trypsin.
6. Remove cell suspension into a conical tube.
7. Centrifuge for 5 min at 300g.
8. Remove suspension, resuspend in 10 mL culture medium, and pipet to fracture the pellet.
9. Count cells and resuspend in desired medium volume.
10. Add cell suspension into culture dishes (see Note 19).
11. Let MEFs set for at least 2 h before plating hESCs.
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