Hello,

I am keeping various prostate (cancer) cell lines in culture, e.g. BPH-1, LNCaP, DU145 and C4-2B, all of which grow perfectly in RPMI-1640 with 5% FBS.

Unfortunately, I keep having troubles with PC3 cells, which I got from a collaborator at passage 19. Every time I thaw a vial of them, they first grow very nicely (as the vials contain a lot of cells, I usually put them directly into a T75 flask) after thawing and get 90% confluent after 2 days. Then I passage them with 0.7 mL Trypsin (TrypLE Express Stable trypsin replacement Enzyme) for about 2-3 min and add 5.3 mL complete media as soon as the cells are disattached.

Then, the cells grow again in T75 flasks and the first 1-2 days they grow well and get to about 40-50% confluence. I usually change the media after 2 days but the cells stop growing and even start to die, as more and more cells disattach themself.

I grow them like the others in RPMI-1640 with 5% FBS and 1% PenStrep, as this were the conditions I got from the collaborator.

After I tried it now several times and even tried 10% FBS, they still stop growing after about 2 days after the passage. I don't tap them during the trypsinization or treat them anyhow different than the other cell lines.

Does anybody have an idea what I could do wrong with them?

Thank you very much in advance!