Tissue Morphdynamics Lab: Week 5

So, its Danny, again, and this post will hopefully be a lot shorter than the last one. The fifth week was when the multiple hours in the afternoon spent dissecting chicken embryos would start to pay dividends in research. At the start of the fifth week I started to culture lungs in tissue culture, which was very challenging due to time constraints. In the previous post I mentioned that lungs would only be preserved in PBS solution for up to an hour, and then after an hours they would be considered "dead". Therefore I only had an hour to dissect four or more day 5 chicken embryos, extract the lungs and place them in culture dishes. Despite having had numerous hours of practice, I never felt pressured in dissecting the embryos as I had all afternoon to practice.

Needless to say, my first attempt at dissecting chicken embryos was mediocre at best, as I managed to get three chicken embryonic lungs in a little under an hour. Then Amira, the research specialist, showed me how to culture the lungs in wells. Additionally, all the work was done in a chemical fume hood and all surfaces, bottles of media|serum and gloves were sprayed down with 70% ethanol in order to prevent contamination.

To make the media that the lungs would grow in, we added 10mL of Hyclone DMEM/F-12 (1:1) glutatmine and 500µL of FBS serum into a 15mL sleeve tube. In order to mix the solutions, we simply turned the sleeve upside down multiple times, instead of vortexing it. We then got a 6 well plate and added 2mL of FBS|DMEM solution into a well using a 5mL pipette. Next we placed a single Whatman Nucleopore Track-Etch Membrane into the well with forceps, with the glossy side of the membrane facing down towards the bottom of the well. The Nucleopore Track-Etch membranes ensure no contamination, have a smooth flat surface that allows for high visibility of particles during a microscope, and have high chemical resistance. The membrane essentially sits suspended in the media. Lastly we used a transfer pipette to move the three lungs from the PBS solution on the membrane. The lungs are then grown in a carbon dioxide incubator that cultures the epithelial cells. We take pictures of all three lungs at 0 hrs, 24 hrs, and 48 hrs, which will later be used for analysis.

Day 5 Lungs (0 hrs)

Day 6 Lungs (24 hrs) 

Day 7 Lungs (45 hrs)

The purpose of growing the lungs in tissue culture is to analyze the results using ImageJ, a program that analyzes the area, perimeter, and other useful measurements of images. This data is then compiled onto an excel sheet, which is in turn used to create a graph that can be used to determine the tendencies of lung development. When the lungs are further treated with different concentrations of lungs, these graph also have varied results. The following are images of both the analysis I have done so far as well as the work that the lab has already done and published in an journal. In the excel sheet graph, the x axis represents the area of the epithelium, whereas the y axis represents the number buds located on the epithelium. The results also include all date from 0hrs to 48hrs.

Lung Development Graphs
Source: Jason P. Gleghorn et al (2012)

Lung Development from a few samples.

At the end of the fifth week, the microscopes were finally available and we were able to image the lungs I had stained using a phase contrast microscope. The confocal and phase contrast microscopes at the lab were to be used with extreme caution, as they had broken down multiple times over the past two months, according to Amira as well as other post-docs in the lab. Therefore I watched Amira image the lungs using HC-Image Live, and found it very interesting. Using a transfer pipette, we placed the lungs with PBS solution onto glass cover-slips and then using a beam of light, we found the optimal wavelength (around 460 nano-meters) that produced a good quality image. It was really interesting to see how the microscope was used, as there was a lever on the side of the microscope that changed the frequency of the light. The images are in black and white, but in the live feed from the computer they were a nice turquoise.

Day 5 Embryonic Chicken Lungs

Day 7 Embryonic Chicken Lungs