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Cell Culturing at Dana-Farber Cancer Institute, Boston
Hi guys, my name is Yesh and I am a rising senior. I'll be writing about my research internship at the Dana-Farber Cancer Institute (DFCI) in Boston during late July. At DFCI, I worked with brain tumor cell lines, learning how to culture and to run assays on them.
While interning, I was soooo intensely focused that I didn't have time for (read - I actually didn't even think of) taking pictures of my lab work -.- . So, in the moments of panic before making this blog, I sent a quick plea to Dr. Gokhale, the researcher I was interning under, to send me a few pictures of the work I helped on. Thankfully he responded and with the help of him, a bit of google images, and a little writing I can give you an extensive look at my week and a half in Boston.
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Part 1: Cell culturing
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So what is cell culturing?
Cell culturing is the process of removing cells from a living organism and enabling them to grow in an artificial environment. During my internship, I focused on the latter part, making sure that the brain tumor cell-line (GL261) I was working with continued to proliferate. The cell culturing process is very lengthy and I will try to outline it below with pictures of my own work.
I cultured the GL261 cell-line in a glass environment, also known as in vitro (Latin: within the glass) culturing. The glass environment is a T-75 flask which has a 75 cm^2 surface area for cell growth. The pink liquid is called media, providing nutrients for the cell culture.
The problem with in vitro cell culturing, however, is the tedious amount of work required to maintain the cell-line. For my GL261 cell-line, after 2-3 days of growing inside this flask, the cells proliferated to an extent where their population size was no longer viable in the small confines of the flask. When a cell culture becomes very packed along a surface, they are said to be confluent as shown with the purple dyed cells below. The cells can be seen with an inverted microscope of 100x power.
During the course of my cell culturing work, I had to regularly transfer cells from one flask to another to make sure they stayed alive, using a small protocol and chemicals like Trypsin and PBS.
This is the protocol that I wrote after first observing Dr. Gokhale transfer the cell line culture between flasks. I will explain most of the steps written here below in the other pictures.
Trypsin is used to dissociate the adhered cell culture from the flask, like the one seen 5 pictures above. It does so by acting as a proteinase that breaks the protein bonds of the confluent and adherent cells. The application of trypsin is appropriately called trypsinization (when scientists have the chance to be creative with names, ofc they don't take it, why would they?) ((I promise my jokes get better as you read on)).
PBS (Phosphate-Buffered Saline, for long, lol) ((maybe not this one)) is also known as a cell wash to get rid of the excess media that still remains after the majority of the media has been aspirated out of the flask. PBS is crucial because excess media can neutralize the affects of the trypsin and the cells may stay adherent to the flask and not be transferable. The buffer and saline concentration in the PBS wash are meant to maintain the cell culture's pH and salinity levels.
After the cells were washed and suspended in the trypsin, the solution had to be neutralized with media and then centrifuged to create a pellet of pure cell culture. The pellet is the white clump at the bottom of the test tube surrounded by the pink trypsin and media solution. (picture courtesy of google and my absentmindedness for not taking pictures :) )
Next the pink supernatant trypsin and media solution had to be aspirated from the tube and the now isolated cell pellet had to be diluted with media so that not all of the cells from the original flask were transferred into the new flask. But first, I had to find out how many cells were existent in the original flasks' culture, so I could tell how much diluted solution of cells to put in the new flask. I took a small sample of the diluted solution and found the cell concentration using a hemocytometer shown below in a process called... wait for it.... CELL COUNTING! (eh.)
The left image shows micropipetting of the diluted solution, dyed with a chemical called trypan blue to identify dead cells, into the hemocytometer. The right image is the magnified image under the hemocytometer with 1 millimeter sized squares to help manually count the cells.
Once the cells were counted, the cell count in the diluted media solution was calculating using the formula:
Avg. cell count per quarter square in hemocytometer X Dilution Factor of solution (usually 2.0) X 10^4 cells/ mL
= Total cell count in the diluted media solution
The appropriately calculated amount of the diluted media solution was then added to the new flask and the culture was allowed to grow for the next 2-3 days in an incubator before the cells became confluent and nonviable again.
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Part 2: Special Lab Equipment
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This is a standard fume hood which uses air currents to maintain sterility within the hood. In cell culturing, sterility is an absolute imperative because any contamination can ruin the integrity of the cell-line and waste a lot of research grant money.
This is an incubator used to provide a temperate environment for the cell lines when they are not being used or are growing. The temperature is kept at a constant 37 degrees Celsius. And more importantly, the CO2 level is kept at at 5%, ensuring that a stable pH level is maintained despite the ongoing release of CO2 from normal cellular respiratory activity.
[Insert picture of the really cool laboratory fridge here]
LOL just kidding!! I don't have one because I didn't think of taking a picture of the coolest thing in the entire lab. *face palm*. Anyway, this fridge was NEGATIVE 80 degrees Fahrenheit and the inside of it looked colder than a mid-winter blizzard in the antarctic. When I opened it for the first time to store a cell pellet a huge chunk of ice that was hanging over one of its racks crashed to the floor. Too lazy to pick it up, I kinda just kicked it under the fridge hoping it would melt lol. (My lab etiquette is probably beyond salvation ngl)
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Part 3: Running an assay
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Well, what's an assay?
An assay is an investigative laboratory procedure to measure functional activity of a desired item. In my internship, I helped run a cell proliferation assay to determine the effect of a cancer drug on the GL261 tumor cell-line. The assay protocol and materials were provided in a kit, so all that was left to do was run the assay. First there needed to be some preparation as seen in the excel template created below (sent from Dr. Gokhale after I kindly asked :) )
The excel chart provides a map for the concentration for cells, cancer drug, and assay chemicals to be administered in each of the 96 wells of the well plate. The well plate schematic includes experimental controls and various dilutions of the solutions to make sure the assay collects all data accurately, factually, and with little error. The right side of the excel chart has the calculations for the dilutions of the cancer drug LEE011 which inhibits CDK development, effectively preventing G1 stage of tumor cell mitosis.
The indication that the LEE011 cancer drug worked was based on the affects of the assay chemical on each of the well plates. The specific cell proliferation assay kit we used used luminescence as an indicator for cell proliferation. The less light given off by the well plate, the less mitotic activity, and the more successful the cancer drug was on reducing the tumor cell proliferation.
This machine tests the absorbance of each of the 96 wells in the well plate. Based on the reported absorbance of each well, we saw which dilutions of LEE011 were most effective on the various amounts of GL261 tumor cells.
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Part 4: Closing Remarks
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Interning and learning new things in a lab environment, especially in one of the most distinguished facilities in the country, was a fulfilling experience and provided an invaluable education. I got to get hands on experience in things that I have never done before and now feel more capable in and confident than ever with a lab setting. In the same time, I also got to meet very talented researchers and make friends and connections in a field that I one day want to pursue.
I now have a new perspective on and appreciation for the triumphs over cancer that are being made every day by scientists all around the world.
If any of you guys have questions feel free to hmu :)
Thanks for reading,
Yesh
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