Before using MEF for ES culture you must expand and inactivate (stop cell division) of MEFs to make feeders.
Expansion of MEFs
Day 1: Thaw 1 vial of MEFs per 10 cm dish. Use feeder media for culture. These cells do not have to be fed everyday, but can be fed every 2 days.
Day 2-3: When confluent passage each 10 cm plate 1:3. (3 x 10 cm plates)
Day 4-5: When confluent passage each 10 cm plate onto one 120 cm plate 1:1, or each 10 cm plate onto 3-10 cm plate (1:3).
Day 5-6: When confluent inactivate MEFs and freeze down.
Inactivation of MEFs
We use Mitomyocin C for inactivation. It is available from Sigma (M-0503) 2 mg/bottle in powder form
IMPORTANT NOTE: Mitomyocin C is toxic do not expose to skin
1. Make fresh mitomyocin C stock. Use a 5 ml syringe and inject 2 mls of PBS into bottle to yield 1 mg/ml. Mix well and draw back out into syringe. Pass through a 0.2 um syringe filter and place in a 15 ml tube. Wrap in aluminum foil to store at 4C.
2. Dilute 1:100 with feeder media when ready to inactivate MEFs. You will need 5 mls
of drug media for each 10 cm plate, and 15 mls of media for a 120 cm plate. For 9 120 cm plates you will need 135 mls of feeder media with 1.3 mls of mitomyocin C added to the media.
3. Add mitomyocin C media to plates and incubate at 37 C for 2-3 hours.
4. Aspirate mitomyocin C media into flask.
5. Wash with PBS. Trypsinize for 5 min at 37 C.
6. Inactivate trypsin with feeder media and collect into 50 ml tubes.
7. Wash plates again with PBS to increase cell yield.
8. Wash cells by spinning down at 1000 rpms for 5 min.
9. Resuspend in 25 mls of PBS. Do 3 washes of PBS while pooling cells into one 50 ml tube. During washes (or earlier) make freezing media.
a. 20% FBS, 20% DMSO, 60% DMEM and
b. 20% FBS, 80% DMEM
final freezing conc. will be 10%DMSO, 20% FBS, 70%DMEM
10. Count cells. Calculate vial concentration and number using worksheet.
i.e. 2.5 x 106 cells/10 cm dish
11. Have labeled appropriate 2ml freezing vials (Corning) with feeder conc., date, etc.
Put vials on ice with freezing media and loosen caps.
12. Resuspend cells at double the freezing conc. in 20% FBS, 80% DMEM
i.e. 12 vials at 1X conc. add 6 mls of 20%FBS, 80% DMEM
13. Add equal volume of freezing media 20% FBS, 20% DMSO, 60% DMEM to volume
of cells. Mix well.
14.
Aliquot 1 ml/vial quickly. Only aliquot 3-5 mls at a time to prevent streaming effect. Tighten caps.
15. Immediately transfer to -80 C freezer. Transfer to liquid N2 tank within one week.
Chart of plate size and corresponding feeder concentration
Size Feeder Concentration
10 cm dish 1X (two plates use 2X
6-well plate concentration)
24-well plate
12-well plate
96-well plate
60 cm plate .35X
2 wells of a 6-well plate
8 wells of a 24-well plate
2 nunc 4-well plates
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