Developing a Lung Mimic In Vitro

Today was an exciting day for Purdue iGEM; we received a pair of rat lungs. The lungs were ethically sourced and harvested from a rat that was sacrificed as part of a euthanasia training lab in Purdue’s College of Veterinary Medicine.

As we are creating our bacteria to be used in a medical therapy that involves our engineered bacteria being reintroduced into the lungs, we are studying how our bacteria will be distributed throughout the lung after our treatment. To do this, we will wash (also known as lavage) the lungs with a solution that contains E. coli that express a green fluorescent protein and then ventilate the lungs for 5 minutes. We will then slice the lungs with a machine known as a vibratome, which uses a vibrating razor to cut tissue into slices as thin as tenths of a millimeter. After getting slices two-fifths of a millimeter thick, we will culture them using a method known as organotypic culture, the growing up of tissue that was removed from an organ. You can learn more about organotypic culture and its methods here [https://link.springer.com/article/10.1007%2Fs00441-005-1111-y].

Our day began when a Purdue Biomedical Engineering graduate student, Brandon, taught us how to use the vibratome, a first for the team members of Purdue iGEM. Whereas many labs on Purdue’s campus use a vibratome to prepare preserved tissue for microscopy, Brandon’s lab also slices fresh tissue, rat brains, for neuroscience research.

 

The time then came to attend the euthanasia lab, in which the instructor, Carol Dowell, demonstrated euthanasia to the veterinary school students and then harvested the lungs for us. Carol first discussed the acceptable, humane methods of sacrificing rats that are used in research today. These included carbon dioxide suffocation, which was used to sacrifice the rat during this lab. She then showed videos demonstrating the carbon dioxide method and multiple methods of desanguination, which is used as a secondary euthanasia method to ensure that the rat cannot recover from the carbon dioxide suffocation. The class was then taken into the lab where Carol demonstrated the carbon dioxide suffocation method on a rat along with desanguination. She then performed a necropsy in order to remove the lungs for us and show the rest of the class. While the other students sacrificed their own mice, we returned to Bindley to work with the fresh tissue. We first swabbed the lungs with a pipette tip to determine the contents of the microbiome prior to introducing any new species of bacteria. We then lavaged the lungs with the GFP E. coli and ventilated the lungs for five minutes. Finally, we swabbed the lungs again to determine if this introduction of a new species of bacteria would have an effect on the microbiome.

We then brought our green fluorescent bacteria-infused lungs over to the vibratome, where we wanted to slice each lobe into about 10 slices and then transferred onto culture dishes. Through this process, we hoped to see both where our reintroduced bacteria will be distributed in the lungs.

Unfortunately, during this round of the protocol, we discovered that fresh lung tissue is difficult to slice on the vibratome. Luckily, we will have access to more ethically-sourced fresh lungs next week, and plan on trying again, this time using an agarose-gel solution injected into the trachea in order to help stabilize the tissue for cutting. Still, our experience today was quite valuable in helping prepare us for repeating this procedure again next week!

 

Example of a lung slice culture, courtesy of Bethan L. Barker and Christopher E. Brightling from their 2013 paper "Phenotyping the heterogeneity of chronic obstructive pulmonary disease" (http://www.clinsci.org/content/124/6/371)