Mouse ES cell culture - ES cell culture for the purpose of gene targeting (Sep/09/2005 )

Greetings,

I have a problem with my ES cells in that they seem to differentiate in numbers after 3-4 passes. I don't grow them on feeders, so I was thinking the LIF in the media might be going off. Still, it's hard to imagine since LIF is frozen in individual aliquots and should keep forever.

Apart from clonfluence issues (certainly not the case) and LIF, I can't think of what else might be causing it since the serum is from the same batch they've been growing fine in, and so is pretty much everything else.

Any input welcome,

G

You don't need feeder cells when you add LIF. Theoretically, stem cells differentiate only when LIF is removed. So, the prime suspect is LIF. We have found that growth factors from several vendors are not stable (activity declined) over time, even you aliquote and store in -20 or -80. I will suggest:
(1) thaw a new vial of stem cell and start over; (2) purchase a new vial of LIF (we got ours from Epoch Biolabs, their LIF in our hands showed highest activity, stability over 1 month period in DMEM/F12 medium and stored at 4 oC).

may be you treat you stem cells too violently?

iriacal

As far as know, LIF(Chemicon) should be store in 2~8 Celsius!