Tuesday, October 25, 2011

Cancer Cells In Vitro

It's been a few years since my work as a biologist at the Cleveland Clinic, and sometimes I will read something in a scientific journal that will trigger memories of my work. This is an attempt to write a bit about my experience stemming from my many years working in the lab.

I was employed at the Cleveland Clinic first as a Research Technologist/Lab Manager in the Radiobiology Lab, and later as a Lab Manager in the MRI Research Center. In both departments, I designed and ran the tissue culture labs and performed experiments on cancer cells, ranging from human cancer cells to mammalian cancer cells.

The lab was essentially equipped with a CO2 incubator for the cancer cells, vented hoods, centrifuges, microscopes, refrigerator, freezer, sink, and UV lights overhead that turned on when closing the lab in order to sterilize the room. UV light destroys the DNA of the bacteria so that they cannot reproduce. Everything was positioned in such a way as to maximize sterility and efficiency.

The hardest part about growing cells in tissue culture was to keep them not only alive but clean from contamination by bacteria, viruses or fungi. The water used to prepare the media had to be pure, without contaminants so as not to interfere with the cell growth. All glassware was washed and rinsed several times with pure water to remove any soap residue. Even a little residue could affect the cells. Also, it was preferable not to use antibiotics, but if a cell line became infected, it could be costly, because they would have to be disposed of and a new batch prepared. So there were times when antibiotics had to be used.

Until our lab was up and running, I worked in another department of the Cleveland Clinic. In those days, they cultured the cells in an open lab, working over a lit burner (the heat was supposed to keep the bacteria away). I was initially trained in this method. Also, glass pipettes were used and had to be cleaned all the time. Any one of these methods were potential for contamination.

When our new lab was finally opened, we had disposable pipettes and range hoods that vented out. We also had pressurized rooms so that when we entered the room, the air from the outside would not enter the lab. I guess the difference between our lab and the older lab was the funding. We were the new kids on the block and had received funding that allowed for these upgrades, whereas they somehow didn't feel a need to do so.

Many times our work focused on radiobiological experiments, and we used radioisotopes often. We always worked with powdered latex gloves and often two pair at a time, one over the other, in case we needed to leave the room and return. When leaving, we would remove the outer layer and upon return, would put on another pair over the first layer.

Different cancer cell lines had different food requirements, but all cancer cells needed the basics, like fetal calf serum and L-glutamine, and glucose. The culture media varied, though, and the pH had to stay within a certain range. I could tell when cells were thriving or dying just by the color of their media. Yellow media meant they were overgrown, and needed to be recultured into another petrie dish, and purple media meant that the cells were dying.

Cancer cells were basically stored in a tiny capsule in a frozen state with DMSO,which helped them not crystallize and die. The freezing and thawing procedures were very delicate and came down to a fine art so as not to lose any cells in the process.

Cancer cell lines we used had finite lives before they began to mutate into something else, so many cultures are frozen at the very beginning of a cell line to use. After about 15 propogations, we would pull a new batch from the freezer and thaw it and grow into a new culture. If this was not done, and a culture was used continuously for a long time, the cells would start changing and the results would vary, making the cell line unstable.

Until next time....

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