Cell culture woes.

From ATCC, T47D breast cancer cells grown in culture.

For my non-scientist readers some background:

Many of us use cells that we grow in special little dishes for our research. These can be different types of cells that are isolated from different organisms. They can be healthy 'normal' cells (although they aren't exactly normal, since they are growing in a dish in a lab somewhere and not in their natural environment- the body). I use cancer cell lines, which means someone at some point took a tumor out of a patient, isolated the cancerous cells, and those tumor cells have been happily growing in our labs ever since. A primary cell line is one that has been very recently isolated- days old. Established cell lines have been growing for years or even decades. Some of these cancer cell lines have survived long past the patient. The T47D breast cancer cell line shown in the picture above was isolated sometime before 1980, and perhaps even as early as 1974. This technique of cell culture is a well established practice, but that doesn't mean it is foolproof. Cells in culture don't always behave the way you want or expect them to, and that can be very frustrating. Case in point:

I work with a lot of different 'primary' –oma cell lines. I use the word 'primary' with some caution, because although our manager of the tissue culture facility goes down to the OR once or twice a week to get new tumors from which to establish these cells, by the time I get them they are really at a passage past the point that qualifies them for still being primary, yet they aren't immortalized either. This is significant because although they do continue to grow for some time, at any unpredictable and random point in time they will reach a crisis and just up and die. It has nothing to do with passage number; and the frustrating thing about it is that they all seem to die at the same time. I will come in one day having plated mass quantities of cells in preparation for big experiment, and lo and behold all my cells are dead.

This has happened to me several times, and I am quite frustrated about it because it makes my progress on this project sporadic and sloooooooooooooooooooow. Several of us are working with these cell lines, each investigating our own angle, and this happens to each of us, but each at different points. I can rule out equipment failures, I can rule out media problems. It isn't contamination. It isn't that they are overgrowing, because that will actually cause this same problem so I am meticulous to the point of being anal about preventing that particular phenomenon. I have been doing tissue culture for 16 years now, and this is the first time I've had this problem, and it is driving me batty. My only other option is mycoplasma, so I am testing for that now, but even treatment with Ciprofloxacin doesn't help.

It makes me want to give up. I am incredibly discouraged. My PI keeps telling me not to wait for the cells, but since all of my experiments hinge on having these cells to work with, I don't really think there is any other option. Grrr. Meanwhile, I'm a cell miser. I don't throw anything away; I'll freeze the cell pellets, or freeze lysates at -80 and stock them away for future use. It isn't my favorite way to work, but at this point, it is the only way. Sigh. Maybe it is time to find a new model system. I'm thinking zebrafish.


Anonymous said...

I know this blog is old... but I am also working with breast tumor cells, T47D to be exact... and they will stay healthy for quite some time, but then out of nowhere BAM, they die... it would seem that no matter who touches them, they are bound to die, just some of us have more luck then others haha