By: Chantal Dubé (MSc. Candidate in Bainbridge lab)
On May 16th, 2016 I boarded the train at the VIA rail station and embarked on an adventure to the big city, Toronto. The purpose for my visit to Toronto was to learn some placental research techniques from Dr. Caroline Dunk, a research associate in Dr. Stephen Lye's laboratory at the Lunenfeld-Tanenbaum Research Institute (LTRI) at Mount Sinai hospital. Dr. Dunk’s research focuses on understanding the mechanisms behind uterine spiral artery remodeling in early placentation. She is an expert in placental dissection techniques and has 15 years of experience working with placentas.
When I arrived to the LTRI, I was amazed. The large laboratory facility consisted of multiple lab benches all in parallel with an abundance of communal equipment, office space at the end of each set of benches and a long wall of windows at the back end of the facility. It was refreshing to see so much sunlight in the laboratory with a nice view of Toronto and the green-space below.
While I was in Toronto, Caroline taught me how to dissect extravillous trophoblast (EVT) explants from first trimester electively terminated placentas. This is a technique that I will be using in my thesis experiments to examine the impact of exercise-induced myokines on placental invasion.
EVT are trophoblastic cells that are outside of the villi. The EVT is what typically invades the maternal uterus in early pregnancy, a step that is vital in the establishment of pregnancy. Under a dissecting microscope, Caroline taught me how to dissect EVT explants from early placental tissue (<8 weeks’ gestation). We prepared a 24-well plate with 5 cell culture inserts coated with matrigel and 5 cell culture inserts coated with collagen. Upon receiving the 7-week placenta, Caroline put the placenta in a large culture dish and used tweezers to turn the placenta so that the membranes were facing upwards and the villi floated outwards. She then identified the villi that had “fluffy” ends and that were “sticky”. These are the EVT. They are fluffy because their ends consist of cell columns and they are sticky because these cells express adhesion molecules that allow them to invade into the decidualized maternal uterus. Once this portion of the villi was identified, she used fine tweezers and microscurgical scissors to cut small EVT explants from the stem. Once we isolated the explants and put them in a large culture dish with media, she had me use the tweezers to pin each explant down and use the scissors to clean the explants. This was done by separating the branches of each of the tiny villous trees and by removing the cap (cell columns) at the end of each of the branches. Removing the cap is important as it allows us to access the stem cell population below, which are the cells that will invade through the matrigel and collagen in the invasion assays. Once we cleaned the EVT explants, we carefully dropped one explant onto each of the 10 cell culture inserts in the 24-well plate
Using the scissors, we moved the explant gently in a circular motion to spread the branches outwards from the center. We then used a small gauge needle to very carefully ensure that the branches are well spread out in order to maximize the contact of the EVT branches with the gel. We added media to the outside of each cell culture insert and put the 24-well plate in the incubator overnight. The next day we put media onto each of the inserts (on top of each of the explants). This was done very carefully to ensure that the explants didn’t detached from the gel surface. Not putting media on top of the explants immediately allows them to adhere to the matrigel/collagen. Before returning the plate to the incubator, we took pictures of each explant (Day 1).
Twenty-four hours later we examined the health of the EVT explants and took more pictures of their growth (Day 2). We also removed the old media from each well and added fresh media to the outside and inside of each insert. After day 1, growth was already evident and the terminal ends of each explant were showing the beginnings of spiky outgrowth. By day 2, these spiky outgrowths were much larger and some of the terminal ends began to grow together. Using the pictures, the change in outgrowth area can be calculated in order to determine the total growth that has occurred. Caroline also took the time to show me how to isolate floating explants. These explants are isolated from later first trimester electively terminated placentas that are 10-12 weeks’ gestational age. We received an 11-week placenta that we used to isolate our floating explants. This time instead of looking for “fluffy” and “sticky” ends, we looked for smooth villi ends. We dissected the smooth-ended explants similarly to how the EVT explants were dissected using microsurgical scissors and tweezers, and cutting them from the stem. Once dissected, 2-3 of each of the explants were placed into a few wells of a 12-well plate filled with media. The plate was then put into the incubator overnight. These explants can then be used for further experiments but we didn’t perform any experiments with them because she was simply teaching me the dissection technique. I will likely be using floating explants for my thesis experiments in order to examine the impact of exercise-induced myokines on placenta viability, proliferation and hormone secretion.
On May 20th, 2016, I was already back at the VIA rail station and about to head home. Overall, the trip was a success and I learnt many valuable techniques. Upon my return to Ottawa, I presented what I had learnt in Toronto including many pictures and the protocols for the EVT and floating explant experiments at the Bainbridge lab meeting. I will likely be teaching this method to my lab colleagues so that they can perform similar experiments in the future. We have now ordered all the required supplies and I will be beginning my thesis experiments shortly. Here’s to hoping that all goes well when I go to perform EVT and floating explant dissections on my own!
Cheers,
Chantal