Composition Feeder medium (for 1L)
• 2x 500 ml H2O (Sigma or Gibco; embryo, cell or tissue culture tested)
• 13,5 g DMEM powder (Gibco; cat. 12800-116)
• 120 ml FBS
• 26 ml non-essential amino acids (Gibco)
• 24 ml glutamine (Gibco)
• 8,6 μl mercapto-ethanol (Sigma)
• 3,79 g NaHCO3(Gibco)
• 24 ml Penicillin/Streptomycin (optional)
• Filter sterilize with a 0,22 μm filter
1.Plating of embryonic feeders
Method A:
1. Take a cryovial from the liquid nitrogen tank and place it immediately in a hot water bath of 37°C(for 2 to 3 minutes).
2. As soon as the cryovial is thawed, rinse the outside of the cryovial with 70% ethanol before placing it into the flow cabinet.
3. Pipette the MEF cells up and down in the cryovial with a 1 ml pipette.
4. Transfer the contents of the cryovial (all MEF cells) to a 15 cm dish, which already contains 25 ml of feeder medium.
5. Shake the dish smoothly in a circular and 8-shape movement for an optimal distribution of MEF cells.
6. Place the dish in the incubator.
7. After 30 minutes, shake the dish again.
8. After 1 hr, shake the dish again.
9. After 2 to 3 hrs, tap the dish and if the cells are well attached to the dish, refresh the feeder medium (not essential).
OR
Method B:
1. Take a cryovial from the liquid nitrogen tank and place it immediately in a hot water bath of 37°C(Take it back out of the water bath before the last clump of ice are melted in the cryovial).
2. As soon as the cryovial is thawed, rinse the outside of the cryovial with 70% ethanol before placing it into the flow cabinet.
3. Aspirate first 10 ml of feeder medium in a 10 ml pipette, followed by the thawed cell suspension. By sucking up very slowly a few air bubbels, one by one, the air bubbles that rise in your pipette will mix the thawed cell suspension (which still contains freezing medium) with the feeder medium.
4. To get rid of the freezing medium, transfer the cell suspension into a Falcon tube and centrifuge for 5 minutes at 1000 rpm.
5. Aspirate the supernatant and resuspend the cell pellet gently in feeder medium.
6. Transfer the contents to a 15 cm dish and add feeder medium till your reach a total volume of 25ml.
7. Shake the dish smoothly in a circular and 8-shape movement for an optimal distribution of MEF cells.
8. Place the dish in the incubator.
9. After 30 minutes, shake the dish again.
10. After 1 hr, shake the dish again.
2.Passaging/splitting the MEF cells
1. Refeed the dishes each day with fresh feeder medium.
2. When the cells form a confluent monolayer (after about 2 to 3 days), they could be trypsinized and splitted to expand.
3. Aspirate the feeder medium.
4. Rinse the cells with PBS and trypsinize them to single cells using about 3 ml trypsin for a 15 cm dish.
5. After 5 minutes in the incubator, tap to dislodge the cells from the dish.
6. Add feeder medium to block the trypsin.
7. Divide your cell suspension over 2 x to 3 x 15-cm dishes (add feeder medium to the dishes so that they contain about 25 ml per dish)
or
Perform first a centrifugal step before counting and dividing the cells over other dishes (for a centrifugal step, see method B of the paragraph 'plating of embryonic feeder cells', points 4 and 5).
3. Inactivation of MEF cells before adding ES cells to the dishes
1. Take a confluently grown 15 cm dish out of the incubator.
2. Aspirate the feeder medium.
3. Add 25 ml prepared mitomycin medium onto the dish with a 25 ml pipette (in the corner, otherwise you will destroy the confluency of the feeder layer).
4. Incubate the dish for exactly 3 hours.
5. After 3 hours, aspirate and discard the mitomycin medium.
6. Wash the monolayer of cells with PBS, add fresh medium and incubate again for at least one hour.
7. The dish can now be used directly to plate out ES cells on it
or
The dish can be used for a passaging/splitting step to plate out the inactivated feeders on other dishes/well-plates (note that inactivated feeders do not grow anymore and so that you have to plate out enough cells on your new dishes/well-plates to get a nice confluent feeder layer.
Remark: Ideally, inactivate the feeders by a mitomycin treatment one day before plating the ES cells on the feeder. Replace the medium with ES cell culture medium before adding ES cells to the dish.
