Cell culturing!

Hi y’all! Thanks for joining me again! This week was definitely a lot more interesting than the last two weeks, so I’m hoping this blog will be a lot more intriguing! 

This week was my first week actually doing lab work – the last two weeks were mostly remote and full of researching papers and annotating – and I’m super happy for the change. My mentor in the lab and I discussed cell culturing and passaging techniques in order to effectively maintain the cell culture. In a project as long as this, it is incredibly important to ensure that the cells are healthy over a long period of time and have enough nutrients and space to grow – which is essentially what is meant by cell culture maintenance. 

The most important parts of cell culture maintenance are feeding, hygiene, and timing. In cell culture growth, there are usually three phases – the lag phase, the log phase, and the stationary phase. Our goal is to keep the cells in the log phase, or growing phase. Since the cells need enough space to grow, we need to take into consideration the confluency, or the percentage of a culture dish or a flask occupied by any type of adherent mammalian cells, to ensure that we split the cells and move them to an empty flask so they have enough space to grow. My mentor and I also discussed aseptic work techniques, or hygienic techniques, in order to prevent any contamination of the cells that could potentially harm them and thus interfere with the experiment. 

This week, I, along with my mentor, set up the cell cultures, and started practicing passaging the cells, or moving them to a new flask. Essentially, I will be detailing the steps I followed to maintain the cell cultures. First, I created a medium (basically cell food) for the cells by mixing a 500 mL store bought medium with 50 mL FBS (fetal bovine serum) and 5 mL of penicillin (as an antibiotic to kill any bacteria). Next I learned how to correctly passage cells. First, I needed to suck out the medium that was already in the flask the cells are in by using a vacuum pump. Next, I needed to wash the cells with 2 mL of PBS (phosphate buffered saline) before dissociating the cells (as they are attached to the walls of the plastic – on a side note, since A549 cells, which are the non-small cell lung cancer cells I am using, are adherent, they attach themselves to the walls of the plastic flask). Next, I added 500 microliters of trypsin, which is an enzyme, directly on the cells – this enzyme causes the cells to break away from the plastic and essentially float around. However, trypsin can cause cell death – so it is important to move on quickly after trypsin is added. After incubating the cells for 3-5 minutes afterward, I added 4.5 mL of the medium (which counteracts the effects of trypsin) that I created and resuspended the cells. 

To transport some cells into the new flask, I added 4.5 mL of medium into the new flask. I then resuspended the cells from the old flask, and then added 0.5 mL of that into the new flask. To mix up the medium and the cells really well, I resuspended the cells again by repeatedly pipetting the solution in and out (essentially sucking in and spewing out using the pipette). This ratio is 1:10, meaning the cells will last about 3 to 5 days before I need to passage the cells again to ensure they have enough space to continue growing! 

My mentor and I also discussed and started seeding for the 96 well, but I’ll explain more on that in the next blog! Although all the pipetting and process for cell culture maintenance seems quite simple, I soon found it to actually be quite difficult. I have to remember to work with aseptic techniques, which required a lot of observation of where my hands were going throughout the whole process. The whole maintenance part took me about an hour and a half – so it definitely is not easy! Surprisingly, pipetting was actually kind of hard – but I think I got used to it by the end of the week! 

I had a lot of fun this week, and it was filled with a lot of hands-on activities and learning. I hope you will stay tuned for next week’s blog, where I will detail 96 well seeding as well as the proliferation assay that I will use for my results. Thank you for reading, and see you next time!

Adios! Au revoir! Sayonara!