Hello,

I am a PhD sudent that has been working with iPSC for more than 2 years. They are being seeded in Matrigel and cultured with mTeSR media.

I am dealing with partial detach of the cells in the well. Only in certain parts, usually the bottom or the margins. The rest of the well looks normal.

These lines are reprogrammed from a control individual and have undergone 2 rounds of crispr. However the problem occurs with any type of iPSC cell, the ones that have undergone crispr and the ones that have not.

I have cheked everything: incubator, hood, room, temperature of the media, not using to much strenght to change the media. The matrigel etc etc.

I think the problem might be that I passage them with ReLeSR and the cells attach very well after using it. So I think there might be too many colonies in each well, causing them to detatch in the most confluent parts of the well when they cannot grow without growing all over each other.

It usually never happens with low-conlfuence wells and also it only happens when I change the media, I think it might have something to do with some kind of composition of the media shock. The cells that remain in the well look great.

I'm the only one in my lab dealing with this, but I am also the only one that passages the cells EXCLUSIVELY with ReLeSR and at this confluence.

Here you can see:

1. How the floating colonies look and how the remaining ones look

2. How the remaining parts of the well look

3. How the holes in the plate look

Please if someone has dealt with something like this before I would greatly appreciate the help because I have already changed all the conditions I can think of 1 by 1.

Thanks!