Hello Victoria Gillan

The important factor is keeping the Matrigel cold prior to polymerization at 37 degree C.

The 24 well plates should not be preincubated in a 37 degree C incubator. Instead, you can preincubate them in the refrigerator. When you work with these plates, place them on ice. When you prepare the matrigel-media mix the medium you use must be ice-cold.

It is crucial that any item that will come in contact with Matrigel should be chilled. Matrigel will solidify and adhere to any item that is above 10°C.

I hope this helps.

Good Luck.

Malcolm Nobre thank you for your reply. Incubating plates at 37 is standard practice when preparing organoids. The idea being that as soon as the cold Matrigel containing the crypts hits the plate the domes start to ploymerize immediately. If the plates were cold I imagine it would just spread across the well.

You are right. Why don't you first coat the wells with Matrigel? Would not that be helpful?

Get a new batch of matrigel to troubleshoot? maybe it's been freeze thawed too much

Agree with John, change your matrigel. Also try incubating at 37oC for longer time with current batch for polymerization, see if that works till you can get a new batch