Hi Nasibe,

At what density did you plate the MEFS? If the MEFs are plated too dense, the feeder layer may detach from the plate, and the culture will be lost.

Hi Nasibe,

as you have feeders, you are not expecting them to growth :-)

However, i do not understant why they died....you medium composition seems correct. Do you plate them on gelatined plates at the manufacturer recommended concentration?

Hi Nasibe,

As Daniel said- check your plating density. Your media sounds like it is right- we do not use high glucose DMEM, however. My best suggestion is to contact GlobalStem and get a lot of their FBS to test with your MEFs. I have found this to be the biggest factor in growing MEFs. Also, how long have your cells been in the LN2? This also can have an effect. Lastly, you can try coating your flasks with gelatin.

Best of luck

irradiated cells are not supposed to grow (that is why you irradiate them!) You should grow them first, THEN irradiate them and aliquot them.

Which Gluc concentration did you use?

As others already stated: Your irradiated MEFs will not grow at all. I use them at a density of 1.6Mio/10cm dish for iPS cell culture, pre-gelatinized dishes; growth medium: DMEM:F12 + 1%PS + 1%NEAA + 1%Na-Pyruvate + 0.5% L-Glutamine.

Good luck

as already written the irradiated MEF do not grow, the medium used is correct and if you followed the protocol for the density there may be a problem of freezing or maintenance of cells at low temperatures. Being irradiated MEF are very sensitive, then keep them up in nitrogen and only for a very short time at -80 ° C, when you thaw them used gelatin 0.1% coated dish and don't centrifuge them, rather changes the medium on the following day.

Hi,

Irradiated cells can't graw. For ESCs maintenance you should cover plates (dishes) with collagen (0.1%) or other, and culture MEF under high density (4/5-3/5). As rule (in our lab), MEF (irradiated or mitomycin-treated) live in a culture about 10-12 days, then go to death.

Hello Nasibe,

We always coat our flasks with 0.1 % (w/v) gelatin in PBS for 1 hour at 37 degrees celcius before seeding inactivated MEFs.

We use high-glucose DMEM suppliemented with 10 % (v/v) FBS (South American Origin, EU approved), 1 X Non-Essential Amino Acids and 2 mM L-glutamine). All components obtained from Life Technologies. We do not routinely use Pen/Strep as it can affect the performance of cells.

Q: Did you suppliment your medium with glutamine?

We seed 3.6 x 10^6 cells per T75 flask for the production of conditioned medium. Seeding densities for other applications may vary, and I would recommend performing a titration experiment to determine the best performing seeding density for your process.

When obtaining fresh MEFs, we expand the cells (obtained at Passage 0) on geltain-coated plates for 2 passages. On passage 3, we inactivate the cells at half confluency using mitomycin-C (our experience with irradiated MEFs has not been especially good). Then we cryopreserve the cells using 10 % (v/v) DMSO in FBS. Doing a test thaw with one vial is encouraged at this stage, just to make sure the cells are not contaminated, and that they retain the ability to adhere to gelatin.

If all is well, cells should survive as a monolayer for at least 2 weeks, given media changes every 3 or 4 days.

Good luck

You have to coat the flasks with gelatin and you need L glutamine in the medium (1%)!

good luck!

Our University, especially our Graduate School of Agricultural Science is very poor, and very limited research budget. Furthermore, very limited number of people in cell biology area. Based on such kind of poor department, we are currently using Mytomycin (MMC) for the feeder prep. Since our protocol is based on the chemical treatment, it would be just the suggestion.

1) Radiation dose

If the radiation dose is too much, cells will be killed very quickly. You need to optimize the best dose, which is enough to stop the cell proliferation, which is not enough to kill the cells immediately.

2) Recovery period

After the treatment, we need to wait for the recovery. Please imagine. After the big damage, if the additional frozen damage comes, they can not survive. In our laboratory, we are washing out the MMC with PBS twice, and replace to the medium. After the overnight recovery of the cells, we will go into the frozen process.

3) Wake up process

For the wake up, we need to help them little bit to attach to the bottom. For that help, we will coat the bottom with gelatin, which suggested in the previous replying.

Anyway, in case of Radiation, non necessary to wash out, non necessary to replace. That is good. I envy you so much, you have a good condition for the research.

Good luck.

Check the cell viability right after thawing your cells. Then you'll know where you're standing and what to expect.

The media that has perfectly worked for me is DMEM (ATCC 30-2002), 10% FBS (Hyclone ES screened FBS), 1/100 PEN/STREPT on flasks coated with 0.1% gelatin (in PBS and autoclaved for 15 min) and placed in the incubator x at least 30 min before adding the MEF's. If healthy, they will grow like grass!

Dear Nasibe

Based on instructions of a workshop on human embryonic and iPS stem cell culture held by Royan Institute, Iran:

I offer you use the Culture medium and components as follow:

Deionized water: 837 ml

DMEM(high glucose & +pyrovate) 13.5 g

NaHCO3 3.7 g

Pen/Strep 10 ml

2-ME 7 µl

FBS 150 ml

pH adjustment 7.2-7.4

and remember that if you use irradiated cells and your culture conditions be appropriate, they will survive but they'll not grow and expand. Irradiation does not kill the cells but prevents their growth and proliferation. this characteristic is crucial for preventing over-growth of feeder cells, and their blending with stem cells co-cultured on them.

hope you succeed.

bests.

Hi Dear Marie Wattenhofer-Donzé ;

How the concentration of gelatin could be important in the cell attachment?

Hello dear Min-Joon Han

the problem is that they are not attached to be used as the feeder layer cells.

Hello Dear Lisa Trump;

The cells were kept in LN2 about 2 days after shipping,by the way,I thawed one vial of the cells in two T25 Flasks plus 4 ml media and as I checked by the supplier of the company my serum has no problem to be used.

Hello Dear Daniel Medina;

as i mentioned earlier I tried to culture the cells of one vial in two T25 flasks which seems not to be that much dense to prevent cells to be attached.

Hello dear Ali Rezaei-Golmisheh;

Thank you very much for you guidance,I have one more vial now,so based on the data that I got from my friends I will try to thaw it ,hope to be successful this time.

Hi Nasibe

Sorry, i was not clear enough, you misunderstand me. I was thinking about the cell concentration and not the gelatin concentration. (By the way the gelatin concentration is generally 0.1 %. The gelatin helps a lot the feeders cells to attach to the vial/plate) and thus avoid a huge mortality after thawing. The cell concentration is important. As the cells have been irradiated, you can not expand them, so you need to plate them directly at the right density (recommended by the manufaturer) and in the right support. The goal is to have a nice tapis of cells into which you will plate your ES cells.

How much cells do you have in your vial? It will determine the surface in cm2 you can cover with your feeders cells.

I have another question for you. Remember that the flask/plate you use to thaw you feeders cells will be the suuport of your ES culture. If you thaw your feeders cells into T25 flasks, it means that you will then cultivate your cells into these flasks. Are sure it is really what you want to do? Don't you want to cultivate your ES cells into smaller surface (eg 6 well plates?).

Dear Nasibe...I would get regular MEF's (not irradiated) and grow them to expand so that you can have a back up stock (about 20 vials of 10E6 cells). Once they start growing healthy they grow like grass. Then when you want to use them as feeders you treat them with mitomycin before adding ES cells. I think that irradiation + freezing and thawing is little harsh on cells and won't help them to be good feeders. That's what I have done and started with 1 vial of frozen MEF's.