Did you know that guinea pigs are the most commonly used animal for military laboratory testing? Their skin closely mimics that of a human, reacting the same way that a human would if exposed to sulfur mustard, or mustard gas. Many of these tests harm the animal, and they must be put down after the experiment. To combat the pain that these animals must go through, I helped to potentially create an In-Vitro Human Skin Model while at my internship at USAMRICD. If the model succeeded, it would be produced on a massive scale, limiting the need to even test mustard gas on animals!
There were three interns, each specializing in a different layer of the skin. I choose to work on the subcutaneous layer, which involves adipose cells (fat), hair follicles, and a vascular component. To start this process, I had to grow Human Mesenchymal Stem Cells (HMSC) from their infancy. As they grew older and multiplied, they had to be “passed” into a bigger flask. The term passed is defined as a passage of cells from one vessel to another. The purpose of this is to not allow the cells to become over confluent therefore allowing them to be more potent.
The passage of cells consists of moving cells that are stuck to the bottom of one container to an empty one and fixing them to that surface. This process involves two chemicals called Trypsin and Trypsin Inhibitor. First, the media (liquid that feeds the cells) is aspirated from the flask. This step is crucial. Then Trypsin is placed in proportion with the amount of cells on the flask surface. The cells and Trypsin mixture is then left alone for five minutes to unstick the cells from the container and then checked under the AmScope to verify that the cells are unstuck. The AmScope will then show white bubbles, signaling the bonds that hold the cells to the flask are broken. Trypsin inhibitor is then placed in the flask in equal proportion to the Trypsin. This stops the Trypsin from “eating” the cells up entirely. The mixture is then aspirated and placed in a new flask, until it is time for some more space to grow.
This routine is one that I regularly performed at my internship, leading one to believe that a process so simple, it could not be messed up. However, when I checked my new flask that should have contained my passed cells, they were not there! After having my mentor check the flask to make sure that I had not missed them, we dug the old flask out of the waste disposal and checked it.
The cells were still there.
In the process of trying to pass the cells, I had forgotten to aspirate the old media. This meant that when I added the Trypsin, and due to the present proteins within the media it inhibited the enzymatic reaction therefore making it useless in removing the cells from the original flask. My careless error cost a week’s time and killed fifty million human mesenchymal cells.
At the time, I was devastated. I thought that I had ruined any chance of further developing the In-Vitro Skin Model. Fortunately, we still had vials of HMSCs that were quickly cultivated. Despite the setback, we were still able to develop the basis of the skin simulation that will be used for future experiments at the army base.
From my experience, it is crucial to follow procedure strictly. As demonstrated, one tiny mishap can potentially kill an important aspect of your study. This can also be applied in your Biomed class. Follow procedure, and ask for help, if you truly need it! Had I checked in with my mentor to clarify what I was doing, the death of the HMSCs could be avoided. Despite my brush with cellular death, this experience definitely cultivated my interest in the microbial field, as I am pursuing to become a Microbiologist.