This week was my first full week in the lab and I have learned an immense amount about cell culture and the vascular biology of our arteries. I also followed some really awesome experiments conducted by researchers in the lab that I will share more about next week!
In order to determine which drug (paclitaxel, sirolimus, or drug x (still TBD) ) in drug-eluting stents produces the best anti-inflamatory response I am going to conduct a proliferation assay experiment. This experiment will model cell growth of both endothelial and smooth muscle cells in a vessel wall after vascular injury caused by the insertion of a stent.
A proliferation assay is a quantitative way to measure cell division or proliferation.
In order to conduct this assay I need to culture cells. I cultured both cell types this week. The cells were isolated from a saphenous vein of a patient that suffered from developed cardiovascular disease and then grown in the cell culture lab.
If you are curious about how I did this, here are the steps I followed: Cell Culture Protocol. My smooth muscle cells grew evenly and were almost completely confluent by the end of the week.
Confluency is a way to describe the number of cells adherent on the surface of a plate. 100% confluency means that the surface is completely covered by cells.
Unfortunately, my endothelial cells lived a very short life 😦 . The death of my cells was most likely caused by contamination or the use of expired media (a nutrient solution added to the cells to aid in growth). Under the cell culture hood it is extremely important to sterilize everything by spraying 70% alcohol solution. Moving forward I need to be more careful to make sure I am not touching the inside of the plates and pipet tips, not using expired media, and being as sterile as possible.
I am going to culture both cell types again next week and hopefully they will grow as expected. Using these cells I will conduct the proliferation assay, first without the drugs and a second time with the drugs. I am attempting the experiment without drugs first as a “test run” to learn the basic techniques needed. For this assay I am going to first culture cells and closely watch them under a microscope until they are confluent (Day 0). Next, I am going to add alamarBlue to read the number of cells initially present (Day 1).
AlamarBlue (AB) – a compound that turns fluorescent and can be read by a fluorescence spectrophotometer. The fluorescence or absorbance of AB is proportional to the number of living cells. Living cells will reduce AB from its reagent form, resazurin (blue), to resourufin (red and highly fluorescent). A fluorescence spectrophotometer will count the number of bright red fluorescent tags or living cells. To learn more: AB.
On this same day I will add the drug to the cells (not the first time I am doing the assay) and change the media (Day 1). I would let the cells grow for 2 days (the typical cell cycle duration for vascular cells). I would then add more alamarBlue to read the new number of cells, change the media, and add more drug resolvent (Day 3). Wait another 2 days and repeat these steps for about 10-12 days. I would also have cells without any drug treatment to serve as a control group. I would repeat this assay for all three drugs and calculate which drug slowed the growth of the cells (or proliferation) most compared to its counterparts.
I will share more details about the experiment and the protocol when I start, but I hope this brief outline helps clarify what I am trying to achieve. Please comment below if you have any questions!