I've had a few attempts in culturing 3T3 cells in my lab. We use DMEM supplemented with 10% FBS for them. From reading several articles, it's been said that 3T3 cells are relatively straightforward to culture, and it initials appears so when I thaw and plate them. Upon doing so, they grow as expected, with the doubling time of ~18hrs, and I split them every 2-3 days depending on the cell density. Everything should be OK from here onwards...
However, without any warning, the 3T3 cells suddenly stop attaching and duplicating after 3-4 splittings. There doesn't appear to be any infection (media colour doesn't change; no other contaminants present when viewed via the light microscope), but the cells don't adhere and thus don't replicate. I've ensured that I seed them at the optimal density (I count the cells after each splitting event because I've been paranoid about these cells...). Even after the seeding into the T75 flask, I visualise the cells under the light microscope to gauge the cell population and as expected, the cell population would appear healthy.
I've had this issue using 3T3 cells a few times now and it's bugging me since this is the only cell type that I've had issues culturing. I've also been culturing SV40 cells, which require the same media as the 3T3 cells (DMEM + 10% FBS), and I've had no issues with them at all. In fact, I've been maintaining both cell lines at the same time, but the 3T3 cells are the first to stop growing normally, while the SV40 cells have no issues whatsoever. I initially suspected cross-contaminations, but I've since prepared separate Falcons of media, PBS & trypsin for each cell line to avoid such risks. I also sterilise the BSCs when switching between cell lines, and I also have separate pipette tips specifically for the 3T3 cells since these are my problem cells. As a result, I've successfully maintained many different cell lines, including the 3T3 cells for the first few splittings. However, the 3T3 cells then stop adhering and replicating, and are the only cell lines I've struggled maintaining...
I'm wondering if there are others that have had this issue with the 3T3 cells? If so, were you able to rectify this issue, and how? I'm concerned because, as said already, 3T3 cells are reported to be easy to culture, but since they stop growing for me after a few passages, I've been trying to figure out where I've been going wrong...
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