Hello everyone. I have been experiencing problems with my endothelial cells. They are from the HBEC-5i line, and during the first 5-7 passages the cells had been proliferating rapidly for my permeability assays in transwell inserts, after the last winter vacation and when I returned to the lab, after thawing them to return to my experiments, they began to grow more slowly and now when they reach 70% confluence, the cells do not finish "filling" my bottles, in fact they begin to detach. I have also noticed that in my cultures there are at least 4 different phenotypes in the cells that I had not observed before. The cells are recent, from last year. Some extra information about my culture:

- I use DMEM:12 Glutamax + FBS 10% + antibiotic/antimycotic 1%.

- The bottles I use for the culture are 25 cm2 and before performing a passage, I add 4 mL of 0.2% porcine gelatin.

- I perform permeability tests with a 0.2% gelatin coating on the transwell insert before seeding them.

- I prepare the gelatin I use with double-distilled water and once prepared I sterilize it in an autoclave.

- I freeze the cells as recommended by the ATCC: using complete medium + 7.5% DMSO

- I attach images of the cells I observe.