March 14, 2014

I will be going on spring break for the next two weeks, as will Dr. Keese, therefore we did not start any new experiments on Friday. I prepared cells for freezing, cultured spare inoculations of BSC-1 cells, and analyzed my data from last week.

The L cells (mouse fibroblast cells) that Dr. Keese and I thawed last week are alive and healthy, and actually exhibiting some unexpected properties. Applied Biophysics acquired these cells from a lab in Korea because they were said to have unusual behavior when examined with ECIS. These cells supposedly caused the graph of impedance to ocilate. When I looked at these cells under the microscope, they looked very different than the BSC-1 and MDCK cells I am accustomed to working with. These L cells looked like they had grown in layers on top of each other. This behavior is abnormal, as cells are supposed to cease dividing and growing upon contact with other cells, forming a single confluent layer. This behavior is known as contact inhibition, and is characteristic of normal stoma tic cells grown in culture. Dr. Keese and I will look into the mystery of the L cells when we get back to break. I will be performing a dilution series experiment on these cells, coculturing them with BSC-1 cells. It will be interesting to see if the L cells and BSC-1 cells will form tiny "islands" like the BSC-1 and MDCK cells did.

Here are fluorescent stained images of the three cell types I have worked with in my internship thus far:
**(note the difference in cell junctions- these junctions are one of the main things we are looking at when analyzing these cells with ECIS.)

L-Cells (Mouse fibroblast cells)
Lay down collagen in the cell, giving the cell structure.
(Fluorescence Digital Image Gallery, FSU)

MDCK cells (Madin-Darby Canine Kidney cells)
Epithelial cells form tight junctions in confluent layer.
(MDCK Epithelial line, olympusmicro.com)

BSC-1 cells (African Green Monkey cells)
BSC-1 cells in the process of dividing
(Cell Division Mistakes, Laboratory News Network)

As for freezing the cells, I followed the same procedure outlined in last week's blog post. I suspended the cells, and placed them into a mixture of DMEM medium, 20% fetal bovine serum, and 10% DMSO (dimethyl sulfoxide). The 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. The cells are now ready to be put into small capsules to be cooled at 1°C per minute. Then, they are placed into a container of liquid nitrogen, which keeps them at a balmy -346°F and -320.44°F. I also resuspended a few flasks with a very sparse amount of cells, that way, they will reach confluence at a slower rate, and they will have enough medium to last them until my next visit in April.

My data from last week was in keeping with the results from our original co-culturing experiment. Because our discovery of these cell "islands" was replicated, we can now move on and study them under time-lapse, and test other cell types to see if they behave in a similar manner. Looking forward to putting together the time-lapse equipment for observation of these cell islands!

Figure 1: Micheal W. Davidson, FSU. (Oct 14, 2004).  Embryonic Swiss Mouse Fibroblast Cells (3T3). [Fluorescence digital image]. http://micro.magnet.fsu.edu/primer/techniques/fluorescence/gallery/cells/3t3/3t3cellslarge1.html

Figure 2: olympusmicro.com. (n.d.). Madin-Darby Canine Kidney Epithelial Cells (MDCK Line). [Fluorescence digital image]. http://www.olympusmicro.com/primer/techniques/fluorescence/gallery/cells/mdck/mdcksb10.html

Figure 3: Laboratory News Network. (Feb 2, 2011). Understanding how cell division mistakes lead to human disorders. [Fluorescence digital imaging]. http://labnewsnetwork.blogspot.com/2011/02/understanding-how-cell-division.html

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. 

Co-Culture results

I'm very excited to share my results from my co-culturing experiment! Dr. Keese and I came across some very interesting results, especially in the to 50% MDCK/50%BSC-I mixtures. This is the first time anyone has performed a co-culturing experiment using ECIS, so Dr. Keese was just as excited as I was to see the outcomes of this experiment. We expected to see these two different types of cells to integrate with one another, attaching to the surface of the electrode in an evenly mixed layer. However, we found that these cells joined together in like-groups, looking like islands under the microscope. With this conclusion, it would not be logical to perform a wound healing assay on our next co-culturing experiment as we had originally planned. A wound-healing assay would kill off any cells on the electrode, and therefore would kill one type of cell- whichever island had attached to the electrode. 

With this new insight into how different cell types interact with one another, we need to redesign the next steps in our ongoing co-culturing experiment. 

First, it is critical to take a deeper look into how these islands are formed, and why. To do this, I filled 16 wells of a cell plate with varying concentrations of MDCK and BSC-I cells. I filled two wells with pure MDCK, two with pure BSC-I, and 12 with a 50/50 mixture of both cell types (1ml of solution per well). This cell plate does not contain any electrodes, it is purely for the purposes of examining the growth of cells under the microscope. Dr. Keese will be staining the cells incrementally to see how they attach to the surface, and at what rate they will form like-groups. I will be working with different cell types in the future to see if all cells form like-groups when grown together in the same environment.  I'm looking forward to posting pictures of these stained cells in next Friday's blog post!

Finally, Dr. Keese and I analyzed our results in ECIS. Although the number of MDCK cells seemed to have been was lower than the BSC1 cells (a possible source of error), we still had nicely mixed cell population data. Our results are best demonstrated in a frequency scan. Here is the frequency scan displaying the results from every well: 

And here is the frequency scan that I have manipulated to display the average of all pure MDCK wells, pure BSC-I, .5/.5 mixture, and cell-free solution (medium):

As you can see from these two frequency scans, MDCK cells show up better at lower frequencies, and then switch places with BSC-I cells at around 20 hours. At about this time, the .5/.5 mixture is seen as having a higher resistance than both the MDCK and BSC-I cells. What frequency tells us about our data: At higher frequencies, impedance is more affected by extent of cell-coverage, whereas at lower frequencies, we  are better able to see the changes in the spaces between or underneath the cells.  

A few weeks ago, Dr. Keese gave me a presentation on how ECIS works, and spoke about cell migration. I remember that in one slide of his presentation, there was a video of cells, and how they grew in response to a wound healing assay. I asked him if using this type of time-lapse video would be beneficial to seeing how our two types of cells are interacting with one other. We will have to wait and see what his answer is next week! Here is an example of the time-lapse documentation of cell migration I am referencing:

2/21- Emma Talks

2/14 Snow Day