Is it possible that they are acquiring anchorage-independence? perhaps an EMT transition? you can check for protein expression of EMT drivers such as Snail, Vimentin, N-cadherin etc...

Hope this helps

Tam

Firstly, Do you have a lot of cells? What is the confluence ?. I belive you are watching clones. It's very common when you start cell culture, you probably did not put enough of the inocul then the cells that were divided are clones. Other posibility is if you started the culture from a cryovial or a flask, maybe you did'n homogenize the cells in right way.

If the cells that you saw look like stars, it's very likely that they are like clones. Then the solution is easy, in the next pass you have to use trypsine for more time and when you get the pelet, you mix well the pelet whit the medium before put the cells in a new flask.

I hope that you can undertand me. I am not a english native speaker.

From a confluent flask one third was transferred to a new flask while passaging. cell clumps are looking like stars.

Here's a nice article:

https://www.sigmaaldrich.com/technical-documents/articles/biology/cell-culture-troubleshooting-cell-clumping.html

I faced this problem quite frequently. I hope you don't vortex the cell pellet vigorously after centrifugation as excess mechanical shear tends to do that. It is always best to use pipette aspiration to homogenize cells. However, in my case, I noticed that I was getting low level bacterial contamination. By morphology, they appeared to be coccus. I found them when looked at the cells very carefully in 40x magnification Phase Contrast. The cells appeared very elongated after 3-4 passages