I'm new in handling CaCo-2, as I'm using it for my undergraduation conclusion project in Virology. I'm facing some issues with this cell, and I would like some advices.

I've already checked it's ATCC manual, but as many articles describe it, I'm using DMEM High, 20% FBS and 1X (1%) P/S. Still, basic handling tips aren't described in the articles I read.

I know its a very delicate cell, as in it will not adhere in at least 3 days when it's not in its peak of replication. I'm with this lineage for more than a month and I had a very hard time getting it to replicate, but after all this time and patience, I was able to expand and even freeze some cells, and I finally have a good amount of them now. However, while I thought they were already at a peak of confluence, forming "isles" and all, I'm still having trouble handling them. I know I have to trypsinize them gently, but they always form a big aggregation structure when I suspend them, becoming really hard to dissociate them. I've tried dissociating them with the pipette (up and down repeatedly), by tapping the flask, and the clot-like group of cells keep hanging on there. I'm afraid of trying to dissociate it by vortexing, as it could be very harsh to the cells and I really don't want to lose more of them.

Besides that, I'm having trouble subculturing it to make experiments. I calculate it to be confluent on the next day, and when I see my plate, the cells aren't even adhering on it.

I would like to know if this is normal, if people who work with CaCo-2 have to always wait at least 3 days before subculturing so they adhere and can be worked with, and if there's some advice on how to make them less "sticky" when trypsinizating.

Please, pardon my English and my poor academic language, I'm from Brazil and a bit frustrated with this lineage, but I really need to understand it for my graduation project.

I really appreciate any help!

I'm adding a picture of the "clot" of cells below.