I encountered a similar issue in the past, and I believe a couple of factors could contribute to this problem. One possible cause is that the Matrigel might have been thawed too quickly or kept for too long before use in 4 degree, which can affect its performance. Another factor that could be relevant is the concentration of Matrigel. Initially, I was using a 1% concentration, but after increasing it to 5%, I no longer experienced this issue. hopefully this could help

Said M Afify Thanks for your answer!

Hello Martina, which brand do you use for plates/dishes? We started to have issues like sudden balling up of colonies and dying, and spontaneous differentiation since January in multiple labs, and everyone use different media and coating reagent. So now we suspect the plates, as it is common among the labs. I would suggest you to simultaneously test an old batch and new batch of your plates, along with another brand maybe. It would be nice to know if and how you could solve the problem.

Izzet Mehmet Akcay Thanks a lot for your answer. We use the same 6-well plates from Sarstedt now for years and hESC-qualified Matrigel. The thing is, that it worked with exactly the same batch from both for months and now we had troubles. Meanwhile it is working again - we assume that it was some error in preparing the Matrigel-plates (maybe some of the aliquots were not working?) - but this is just a guess.

Second thing what came to my mind was, that I could have been a contamination with Mycoplasma but we tested and cultures were negative.

Hope you can also fix your probem!