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Extensive MEF death upon thawing - (Sep/17/2010 )

Hi All!

My lab is currently working with iPS and hES (in particular H1 and H9).

We had been using MEFs purchased through GlobalStem and MEFs prepared by other labs and stored longterm in liquid nitrogen. These MEFs were good but we wanted to start preparing our own as it seemed more cost effective.

As the lab technician, it was my job to isolate MEFs. I used a protocol given to us by another iPS lab at my school and with recommendations from others.

Our problem is odd. Having harvested multiple times, the cells grow well in P0 quickly filling the dish (within 1 day) and requiring expansion. Upon expansion cells continue to grow well into P2/P3 (depending on extent of splitting during passaging).

However the problem I've encountered is freezing down the cells. MEF that I prepared following the protocol I listed below do not thaw well (< 20% attachment). MEFs that do not undergo freezing grow well and thawing other MEFs are okay (>80% attachment) but it simply does not work with the MEF that I isolate.

The seems thus that the problem has to be with my freezing protocol/technique. I freeze other cells (MSC, dental pulp stem cells, etc) without a problem using the same/similar method. I've tried freezing media from other labs to compare with my own.

If anyone has any suggestions on what I could try or has experienced something like this before please do!

This is my first post here (though i'm a long time lurker) so thank you in advance for all your help and suggestions!

The protocol I use is as follows:

1. CO2 to knock out E13.5 mice (CD-1 or CF-1).
2. Cervical dislocation
3. Spray abdomen with 70% ethanol.
4. Remove uterine horn and place into 10cm dish.
5. Rinse 3x with 10mL PBS w/o CaMg
6. Cut open embryonic sac and release embryo into dish.
7. Using dissecting microscope, remove the head and visceral tissue (judged by color and morphology)
8. Cleaned embryo are placed in clean petri dish and rinsed 3x with PBS
9. tissue is minced with scissors/sterile blade
10. add 2mL Trypsin/EDTA per 3 embryo and continue mincing
11. Add 5mL trypsin and incubate @ 37oC for 5-10minutes
12. Pipet the embryos vigrously until few chunks remain
13. Neutralize with complete medium and transfer to 50mL tube
14. Allow larger chunks to settle and suck out supernatant (containing most of the free cells in suspension)
15. Dispense cells into p100 dishes or T75 flasks at ~1 embryo per dish.

16. Next day aspirate out media and unattached cells and refeed, passage or freeze based on confluence.
17. Passaging at P0 gone as high as 1:10 or as low as 1:5

Freezing (p100):
1. Aspirate out media
2. Wash with PBS
3. Add 1.5mL trypsin/edta and incubbate @ 37oC for 3 minutes
4. Lightly tap the edge of the dish and neutralize with 3.5mL complete medium
5. Collect media and cells in 15mL tube and spin @ 1000 rpm for 4 minutes
6. Aspirate out media.
7 Add 500uL freezing medium (90% heat inactivated FBS/10% DMSO or 90% FBS/10% DMSO) gently.
8. Pipette gently but break up the pellet in media and transfer to cryogenic vial.
9. transfer to freezing container (initially at room temperature) and put into -80 (no more than 2 minutes between freezing medium and -80)
10. Next day transfer cryovials to liquid nitrogen.

1. Take cryovial out of liquid nitrogen and warm in 37oC water bath.
2. When the inside is slushy, bring it to hood and spray down with ethanol
3. Gently add 500uL of complete media to the cryovial dropwise. Mix gently
4. Place cells into 15mL tube and spin @ 1000rpm for 3min
5. Aspirate supernatant and resuspend in complete media, plating 1 vial from p100 to 1x p100.



what passage are you freezing MEF???

if you freezing P1-2, the cell should attach and grow normally with regular DMEM+10% FBS medium.

But if the passage is P3 or later (especially after P4), cell will not grow. and should not be used for feeder layer