Another busy day for me at Applied Biophysics! On Friday, I learned how to freeze and thaw cells, how to take images of stained cells under the microscope, and re-created the 96w20idf experiment that I had previously done with the help of my mentor on February the 7th. 


Thawing & Freezing Cells:
It is common lab practice to freeze cells that are not being used in order to preserve them for long periods of time. This coming Friday, I will be using a new kind of cell: L cells! These cells are mouse fibroblast cells, which are responsible for laying down collagen- giving structure to living tissues. These cells have been stored in a container of liquid nitrogen for years at quite the chilly temperature, -346°F and -320.44°F. Before cells are put into this frigid environment, complete medium is added along with a cryoprotective agent such as dimethylsulfoxide (DMSO). This agent is extremely permeable, and slips right past the phospholipid bilayer of the cell membrane, and into the cell. DMSO will lower the freezing point of the medium, allowing the cells to freeze at a slower rate thereby reducing the chance of ice crystals forming which can rupture cell membranes. To prevent cells from dying, it is necessary to freeze the cells at a controlled rate decreasing the temperature by 1°C per minute. 

Thawing cells is much easier than the freezing process. To thaw our L cells,  we located the vial within the liquid nitrogen chamber, and placed them into a water bath at 37°C. Once thawed, we pipetted the L cells into a T-25 flask and added fresh medium. Then, we placed the cells in the incubator and waited for them to attach to the surface of the flask and begin to form a confluent layer. At the end of my internship on Friday, Dr. Keese and I took a look at these cells, and saw that after about 3 hours, they still had not begun to attach to the surface of the flask. These cells might have been frozen improperly, causing them to die.  Hopefully this is not the case, but I will post an update on the status of my L cells on my next blog post.

Results of Cell-Staining Experiment:
Last week, I created a series of pure and 50/50 MDCK and BSC-1 cell dilutions. Dr. Keese stained these cells over a period of time to demonstrate the growth and behavior of my co-cultured cells. We found that the MDCK cells and BSC-1 cells grew into islands of like-cell types. Here is an example of an island of MDCK cells surrounded by BSC-1 cells: 

I took this picture with a camera that can be attached to the eye piece of a microscope. (Getting the cells in focus is much harder than it seems due to the fact that the microscope itself has to be in focus as does the zoom function on the camera.)

**Side note: After spring break, Dr. Keese said that we will put together a time lapse video of the growth of our co-cultured cells!!)

96w20idf Experiment:
In any experiment, it is necessary to be able to reproduce your findings in order to draw accurate conclusions about your data. I re-did the very complicated 96w20idf experiment from two weeks ago so that we can be sure our data is reliable. This week, I reproduced this experiment 100% on my own, and realized how far I've come throughout my internship experience at Applied Biophysics. I remember on day one when I was lost in the jargon of brilliant scientists talking about frequency scans, dilution series, and plasma etching, and had not the slightest idea of what Electric Cell-substrate Impedance Sensing was. On completing this co-culturing dilution series experiment, I realized how much I've learned, and how this internship has allowed me to apply what I've learned about biology to the real world.