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