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MEFs growth problem + dying when change to iPS medium


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#1 Maelle

Maelle

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Posted 03 October 2012 - 08:17 AM

I have problem with my Mouse Embryonic Fibroblasts (MEFs) which I use to support the growth of hIPS ...

I grow my MEFs relatively ok, I must say they have been less good than before but I thought maybe I made a few bad batches of primary MEFs or the trypsin was not that great....We have our mouse colony that we use to make MEFs either wild-type or DR4 (4 antibiotic resistance genes).
I gamma irradiated them, freeze them and thaw/ plate them on 0.1% gelatine coated cells the day before I need to split the hiPSC.

I put the hiPSC on them and before after a week I would start seeing MEFs cell death and just thought the hiPS medium was just not great for them...
Now it is getting earlier and earlier .
For example I plated them last Thursday , split the hiPSC Friday and today Tuesday more than ½ MEFs are dead.
However I had one well extra of MEFs g-irradiated that I did not use to put hiPSC on and left it in MEFs medium and it still looks fine ???
I tested them and they are mycoplasma free. I do not see any obvious bacteria or yeast infections ...

I am thinking I might have a problem with one component of the hiPS medium but I have been through a few bottles already... except the B-mercaptoethanol and the NEAA...
Last week I try filtering the medium before using it, no change... We changed our solution of gelatine used to coat the plates, no change..

Here are the recipes of my media:
‘hiPSC Medium’ : Dulbecco’s Modified Eagle’s medium (DMEM) F12 (Invitrogen)
20% KnockOut (KO) Serum Replacement (Invitrogen),
1 mM of L-glutamine (Invitrogen),
0.1 mM B-mercaptoethanol (Sigma-Aldrich)
1% non-essential amino acid solution NEAA (Sigma)
10 ng/mL of FGF2 (Peprotech).
1% Pen/Strep (Invitrogen)

DR4/MEF Medium Dulbecco’s Modified Eagle’s medium (DMEM) (Invitrogen)
10% FBS (Invitrogen),
1 mM of L-glutamine (Invitrogen),
0.1 mM B-mercaptoethanol (Sigma-Aldrich)
1% non-essential amino acid solution (Sigma)
1% Pen/Strep (Invitrogen)

Now I have heard end last week that Carl who grows MEFs for the same purpose than me (usually without gelatine coated plates) but for mouse iPS in our lab has the same problem.
His MEFs grow less good than they used too even though from the same batch of primary MEFs that I made a long time ago and were fine. So it is not a problem of bad or good batches of primary MEFs....
After irradiation they start to slowly die when they are put to grow onto mES or miPS medium, which I did not see when I used to grow the mES or miPS on them...
And today Wednesday, after splitting his MEFs on Monday, they attached on Tuesday and now half of the ones they were attached are dead floating...

I thought maybe it is the incubator but Carl has been using the other incubator than me as well and other cells are doing ok. We have only two incubators linked to the same Co2 source.

So again we are back to one component common in the media.

Here are the recipe of Carl’s media:
‘miPSC/mES Medium’ : Knockout DMEM (Invitrogen)
15% KnockOut (KO) Serum Replacement (Invitrogen),
1 mM of L-glutamine (Invitrogen),
0.1 mM B-mercaptoethanol (Sigma-Aldrich)
1% non-essential amino acid solution NEAA (Sigma)
LIF (Peprotech).
1% Pen/Strep (Invitrogen)

DR4/MEF Medium Dulbecco’s Modified Eagle’s medium (DMEM) (Invitrogen)
10% FBS (Invitrogen),
1 mM of L-glutamine (Invitrogen),
0.1 mM B-mercaptoethanol (Sigma-Aldrich)
1% Pen/Strep (Invitrogen)

Collectively Carl and I have been through a few bottles of those different components already except the B-mercaptoethanol, which I still can not imagine what could be wrong with it....
A few KO serum replacements... However it could be our University store had the same ‘bad’ batch for a while and we have been getting bottles from the same batch ? we unfortunately did not keep track of that...

I am going to try to eliminate the obvious components in common between the media starting with KO serum replacement and NEAA (even though they are not used in the MEFS media at least for Carl) but I am not sure it is going to be that easy...

If any of you on this forum as any idea what could be wrong either when we grow the MEFs or when we change the medium to stem cells medium... I would be glad to hear any suggestions

thanks a lot and sorry for the long post...

Maelle




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