Hi,

Many factors can affect detachment: degraded matrix, infection, poor colony quality, colony differentiation, incubator failure, too strong pipetting, lack of ROCK inhibitor after thawing, long mixing of the plate after cells seeding, expired cell medium etc.

In my opinion, iPSC cells were very dense immediately before freezing and the medium was changed too rarely (when it was yellow). In addition, the colonies were very large at the time of freezing. Which affected the quality of the colonies after thawing.

The medium should be changed more often and the passages should be performed more often. Passage in small colonies with low density. Passage before the colonies merge. Use ROCK inhibitor for 24 hours after thawing.

Good luck!

Hi,

Sometimes too long passaging procedure also causes colony folding.

Good luck!

Hi,

Thank you for your answers ! I ended up trying ECMatrix-511 E8 Laminin Sigma and it worked really well. I did also change from home made media to a commercial one.

But ultimately I do think it was a mix of technique/passaging time/ reagents that made my cells act this way.

Thank you again !

I have good experiences with E8 Medium + rhVitronectin. Passaging with 0,5 mM EDTA in PBS without calcium and magnedium.

Good luck!