how to maintain iPS cells - (Aug/26/2008 )

Hi. I have started working on iPS cells without any experience on stem cell culture and I am in trouble now.

I could get iPS colonies from human fibroblasts with written protocol and picked up them into 24-wells with MEF feeder cells. They grew but went to differentiate. I passaged them with mechanical disruption. Usually, a half of colonies went to differentiate after each passage. Sometimes, center of colonies started differentiating. So, I lose too many cells and cannot expand them.

I use primate ES-cell medium from reprocell, supplemented with 4ng/ml bFGF and penicillin streptmycin. Feeder cell is commercial SNL 76/7, passage 7-9. I change media every 48hrs.

Does anyone know tips to prevent differentiation of iPS or hES cells?

Thanks in advance.

-Fum-

QUOTE (Fum @ Aug 26 2008, 04:36 AM)

Hi, Fum. I also had some problems working with iPS cells at the beginning, but here is how I am culturing them right now (though in mice-derived iPS):
(1) ES Medium: D-MEM supplemented with 15% FBS, ESGRO (LIF), penycilin and streptomycin;
(2) MEF feeder cells, passsage no further than 6 (I've tried more than P6 feeder cells and I got the same result when I put them 24h prior to the iPS cells);
(3) When trypsinizing, try to disrupt as many colonies as possible (pipetting with 1mL pipette does) when passaging;
(4) Passage them every 2 days (3, maximum); and
(5) You have to change media everyday.

Well, I hope it helps you.
Good luck.

-Doda-

Hi, Doda. Thanks for your suggestions.
I will try to use early passage feeder cells and also change media everyday.

Good luck on your work, too!

-Fum-