On Friday I observed Dr. Keese while he performed an experiment using an array with filters. By growing cells on a porated filter, we area able to better simulate an in vivo environment, and to measure the barrier function between cell junctions. There are many different types of cell junctions,  each with a different strength. We used MDCK II (canine kidney cells) in our experiment which form tight junctions. These cells are very hard to separate, which makes it difficult for us to passage them. We added 3 rinses of a solution called EDTA which takes away the magnesium and calcium ions of the cells. Then, we added trypsin, which is an enzyme that essentially eats away at the cell walls, weakening their junctions. Once the cells became rounded and were no longer attached to one another or the surface they were grown on, we were ready to put them into our array!

The electrode array we used today has a very deep well which allows for our filters and a suspended electrode to sit in the well. Here is what our electrode array looks like:

Here is a diagram of a single well in our electrode:

As you can see, medium sits above and below the cells, therefore creating an environment that would be similar to that of the body. 

So what, exactly are we measuring in this type of experiment? Using the two electrodes, we can pass a current through the layer of cells growing on top of the filter, therefore seeing how tight the cells junctions are. This measurement is called barrier function, which is much higher for MDCK II cells than the BSC-1 cells I have been working with. The higher measure of barrier function, the tighter cell junctions are. For example, endothelial cells that comprise the blood-brain barrier would have an extremely high barrier function because they must have a low permeability in order to keep out microorganisms and other large or hydrophilic molecules. 

I also worked on passaging my BSC-1 cells. Here some candid shots of me working with my cells:
(Photo credits to my awesome mentor, Dr. Keese!)

Holding a flask of BSC-1 cells

Working in the hood

I learned a lot about the physics behind Applied Biophysics' ECIS instrument this week. (As more of a biology person, I need a lot of visuals to understand the physics side of things, so get ready for a lot of pictures and diagrams!) Dr. Keese and Dr. Renken gave me an in-depth presentation on how cells located in each well of the electrode array are subjected to varying frequencies to measure their behavior. There are two types of current- AC (alternating current) and DC (direct current). The ECIS instrument sends an AC current through the electrode, allowing us to get a constant reading. Here is a diagram I made to explain the main idea of using AC current:

For our purposes, the capacitor represents the cells that are sitting on the electrode. They act just like a capacitor because cells are mostly saltwater, and do not let current flow through.

In my experiment from last week, I used the most basic ECIS instrument, the Z. It sent out super-low frequencies of 1000 hz, all the way up to 96000 hz. At low frequencies, we were able to see how tightly joined our cells were. This is because current always wants to follow the path of least resistance, and will therefore travel around and underneath the cells. At higher frequencies, the current will pass right through the cell, and therefore allows us to measure how many cells are in the vicinity of the electrode. It is important to note that cells don't sit flat on a surface. Instead, they have little anchors that connect the cell to the surface it is growing and spreading out on. Here is a diagram that illustrates this concept:

With all this new information, I was able to understand the basics of my own ECIS graphs that were produced from the experiment I ran last week. My cells responded just as they were supposed to, with the exception of my control well which was filled with only medium. Apparently, I somehow got a few cells into this well and began to grow. (Opps!) 

Here is a graph of my data at a medium frequency of 16000 Hz. This frequency gives you a good overview of how my cells were growing, but it doesn't measure specific things like cell density or cell junctions. 

My Very Own Electrode: from Plasma Etching to Experimentation!

Today I began my internship by learning about the plasma etching process which is used to clean electrode arrays. The arrays are placed on a tray that is then put into a low-pressure chamber where the plasma etching will take place. Plasma is known as the fourth state of matter, because it is not a solid, liquid or gas. It is instead a mixture of atoms, ions, and free radicals. Plasma naturally occurs only in lighting and the aurora borealis. We created plasma by creating a vacuum, and then pumping oxygen into the chamber. After about a half-hour of this gas treatment, we sent an electrical charge through the chamber, thus creating plasma. The plasma ions become activated by this electrical field, and begin to vibrate and glow a bluish-purple color. (Seeing the glowing plasma was definitely the coolest part of this process!) The vibrating ions essentially “scrub” the arrays in the chamber, leaving them perfectly sterile. A vacuum pump then takes the contaminants that might have been on the arrays and removes them from the chamber. Plasma etching is incredibly important to the entire process of making an electrode array, because any type of contamination can interferer with the electrode's impedance or resistance, thus creating inaccurate data. 

I also passaged the BSC-1 cells I created last time (which, thankfully, turned out to be healthy!) to create new cells for next week, and also to use in my own ECIS experiment. I was on my own today to replicate the process of inserting cells into an electrode array. I then took my electrode that was filled with BSC-1 cells and plugged it into the ECIS-Z instrument, essentially the simplest version of the ECIS instruments to use. 

It was really interesting to see my electrode go through so many steps: from manufacturing, to etching, to its use in the lab, and finally into the ECIS instrument to conduct and experiment. 

Today Dr. Keese and I began an ECIS experiment with two different cell types, BSC-1 and D4BSC-1 cells. We first took two electrode arrays, an 8W1E (eight wells with one electrode in each well) and a 8W10E with ten electrodes per well, and flooded the inside of the wells with 10 mM of cysteine. Cysteine is an amino acid that reacts with the gold that plates each of the electrodes, allowing proteins to be adsorbed more quickly and inoculated cells to attach and spread easily.  Each electrode is 250 µm and looks like an tiny white in the center of a gold circle. These electrodes connect to a larger electrode running down the center of the array, completing the circuit. Here is an image of an electrode array:

To graph our cells, the ECIS technology measures cell growth by sending currents of varying voltage through the electrode. The cells'  rate of growth is measured through impedance, the amount of opposition to the electrical current. Sadly, one needs more than just a three-hour block of time, so next week at my internship I will see how the ECIS graph of our cells turned out. After 38 minutes, this is what the graph of our cells looked like:

I am also looking forward to next week's internship because I will be applying this process to my own BSC-1 cells that I cultured!