Hi Zhang Xiaojing . Your cells are way, way overconfluent. 100% confluency means that there is no space for adherent cells to grow into (without forming a compacted monolayer).

According to your pictures, the cells have formed a compacted monolayer with cells adhering more strongly to other cell than they do to the substratum. When cells adhere more to other cells than to the surface adhesion proteins, they come off as a sheet when agitated

Solution would be to treat the cells before they become confluent and form a tight monolayer.

Dear Steingrimur Stefansson .

Thank you for suggestion. I am troubled by the overconfluent.

I reprogram the cells based on yamanaka's protocol. My cells had been trypsinized on Day5 and reseed at a density of 100000 per 10 cm dish. According to articles, it will be cultured to 30 days for reprogramming and picking up colonies. But my cells seem to grow faster than others in ES medium.

I am afraid that reprogramming may be stopped if I treat cells againg at 10 Days.

seed 10000 cells/10-cm dish.

Thank you Krishan Gopal .I will try it.

And also start differentiation at 70-80-% confluence.

Hi Zhang Xiaojing, I completely agree with previous comments. When cells are over confluent, sometimes they do behave like your pictures. Try to split/differentiate them when they are not more than 80-90% confluent. I believe this strategy would be helpful. Thanks!

Agree with the previous comments reduce the seeding density and try out several cell concentrations and differentiate when their less confluent... good luck !

Dear Md Obaidur Rahman :

I appreciate your support. It is my first time to reprogramming iPSC, I will pay attention to it and split my cells in time.

Dear Dineshani Hettiarachchi :

Thanks for your suggestion and kindly wish . I will reduce the seeding density at different levels. Hope it works.