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Part II: Tissue Culture

By in2scienceuk Uncategorized

by Hafiza Irshad

During my two weeks in the lab I was given some cells to look after (A549), I developed a strong bond with these cells over the two weeks and loved them as my own. The cells divide constantly, if they outgrow their container they run the risk of dying, something I definitely didn’t want happening. To avoid this I ‘split’ the cells every two days. I was able to tell if the cell required splitting by tracking how confluent (closely packed) they were by looking at the cells through a microscope.

Blue coats are worn in tissue culture labs only, this prevents the cells from being exposed to dangerous chemicals we use in the lab. The hood provides a sterile environment.

Blue coats are worn in tissue culture labs only, this prevents the cells from being exposed to dangerous chemicals we use in the lab. The hood provides a sterile environment.

A pellet containing cells is visible at the bottom of the tube, and the pink medium above will be discarded

A pellet containing cells is visible at the bottom of the tube, and the pink medium above will be discarded

The splitting was done an aseptic hood in sterile conditions. I would extract the old media, perform a PBS wash and add an enzyme known as Trypsin (to ‘unstick’ the cells from the bottom of the flask). The cells would then sit in an incubator at 37° until they were visibly moving. I then add extra media and ‘spin’ them down in a mini centrifuge. This would create a pellet of my cells so I could tip out the media and then suspend in fresh media. I would then transfer a third of this liquid into a fresh flask and get rid of the rest. This means there is a third of the amount of cells previously in the same sized flask. The cells have more room and are able to live for another day or so before they require splitting again.

Tissue culture was challenging because of the strict sterile conditions involved, if your pipette touched anything other than the liquid you intended on measuring, you would have to dispose of it and get a new one. There are also different methods that people follow for splitting cells, for example, a PhD student in the lab told me I wouldn’t need to use the centrifuge if I added extra Trypsin. This method worked for him but my supervisor Laura had always used the centrifuge. It all depended on who taught you first and what you feel comfortable doing. I really enjoyed working in the tissue culture lab and looking after my cells (which I have been told are now frozen down for use later).