On Friday, I met with my mentor for the last time in 2013. We went over the data collected from my wound healing assay experiment last week. For the most part, my data was as expected. However, there were two wells that did not follow the general trend of my results. Possible error in these two wells could have been that I did not have the same amount of cells in these wells as the other six. Therefore, the graphs of these were significantly lower than the average. However, the cells in these wells caught up to the others after about 15 hours.

I also passaged my BSC-1 cells and MDCK cells. With practice, I am learning how to better passage these cells. The MDCK cells are the hardest to passage because they have tight junctions and are therefore difficult to separate from one another and the bottom of the flask. On Friday, I only used about one drop of cell culture in each of my new flasks because I will not be working with the cells for a few weeks. We do not want the cells to multiply and grow too quickly because they will use up all of the nutrients in the medium. Dr. Keese will be babysitting my cells while I am away, making sure they have enough medium and are healthy and pathogen-free!

Dr. Keese taught me how to manipulate the ECIS software to better understand my data. My favorite function saws the 3D graph option! He showed me how to put my graphs into three dimensions, and how to interpret these results. The 3D graphs plotted time (hours), frequency (hz) and impedance (Z). I would have loved to attach the graph of my results, but I had a bit of trouble getting the data onto my computer. I will try to get these images uploaded onto my blog for my next post. Sadly, I will have to wait until January 10th until my next visit to Applied Biophysics!

On Friday, I passaged my BSC-1 cells to make two new flasks of cells. I will be using these cells in a co-culture experient in which I will combine BSC-1 and MDCK cells together and examine their behavior with an ECIS run. I also passaged MDCK cells today. These cells behave very differently than BSC-1 cells becasue they form tight junctions and are tough to suspend. In order to get these cells off of the bottom of the flask, I needed to add three rinses of EDTA, which is a solution that chelates the cells (takes the magnesium and calcium ions off of the cells). I then added trypsin, which also helps suspend the cells. I then took my MDCK cells, added medium, and put 400 µL of this cell-medium mixture into seven wells of an 8W1E electrode. The eigth well is just filled with medium, no cells.
I then plugged my electrode array into the ECIS port, and started to run my experiment! In this experiment, I will be performing a wound healing assay. In a wound healing assay, the electrode sends out a current of electricity that will kill the cells in the vicinity of the electrode. In this experiment, I sent a 1400 uA current through four wells for 20 seconds.This process will cause the cell membranes to become porous, and medium will leak into the cell, causing it to die. Why would I want to kill of perfectly healthy cells? Because then we can see how the other cells will move in to heal the wound. We had to wait for the cells to settle to the bottom of the electrode and grow into a confluent layer before we wounded the cells. I set the wound time for 9 hours into the experiment, and I will see what my results look like this Friday!

I was able to connect some of my knowledge about genetic modification that I had learned in AP Biology to this wounding process. ECIS allows one to either wound their cells, completely killing the cells around the electrode, or electroporate them. Electroporating cells introduces a high frequency current that damages cell wall (not killing them), causing them to become more porous. The increased porosity of the cells allows substances (such as large or non polar molecules) that could not usually pass through the selective cell membrane to enter. Electroporation allows a scientist to introduce genetic information into the cells, often by a virus, changing the cells' DNA.

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!

Cell Culturing and Learning ECIS

Today I cultured BSC-1 cells on my own! I worked with the techniques taught to me last week, and split a single flask of confluent cells into two. I first practiced my mechanical pipetting skills, and then dove right into culturing my cells. Every 20 minutes I checked back on my cells to examine the rate at which they were growing. I found that as time went on, the cells began to assemble themselves into tiny clusters. When I arrive at my internship next week, I will take a look at my cells to see how they have multiplied and if they have grown into a confluent layer. (Fingers crossed I managed to avoid any type of contamination!)

Today, I was also introduced to the ECIS technology. Dr. Keese taught me how to run an RC test, which is essentially a computer chip that is engineered to mimic cell behaviors on an electrode. (How someone programs a computer chip to model cell behavior is something way over my head right now, but maybe with a few more internship visits I will begin to grasp this concept!) I then plugged this chip into the ECIS instrument, and was able to get a test graph. The graph was just eight horizontal lines of varying color and value. Next week, I will be putting the ECIS software onto my own laptop, and I will learn how to place my cultured cells in an eight-welled electrode. The graphs of living cells will obviously have a very different graph than the RC test chip. An ECIS cell graph will have multiple lines that originate in the same place, then steadily increase as they divide and multiply faster rate.

Our 8-welled RC test graph.

What our ECIS graph of cell behavior will look like.

I did not go to my internship today due to the Parent's Day schedule. Looking forward to next week!

Cell Culture

Today at my internship I learned how to grow cells in culture! First, we created a liquid medium for the cells to grow in. Our DMEM medium consists of 90% nutrients including salts, glucose, amino acid, and vitamins. The remaining 10% of the medium is growth factors, specifically, serum. Serum is very important for cell growth as it provides cells with adhesion factors, hormones and lipids and other minerals that allow the cell to grow as a confluent layer across a surface. The serum used in our medium is Fetal Bovine Serum. It is a (somewhat creepy) amber liquid that is a remnant of the coagulated blood collected from a cow  fetus. The red blood cells are taken out of the serum by centrifuge, and growth factors and a small amount of antibodies are left. 0.55 mL of antibiotics was also added to our medium, which is a key factor in inhibiting any type of contamination in the cultured cells, including bacterial, fungal, yeast, mold, and viral growth. Avoiding contamination is extremely important in keeping healthy cells alive and multiplying. To avoid any type of contamination, we worked in a ventilated hood that filtered out any pathogens in the air, wore gloves, disposed of all pipettes after a single use, and cleaned the surfaces of anything that might come into contact with the cells with isopropanol.

Next, we took a flask of growing BSC-1 cells, which are African green monkey kidney cells. These cells are epithelial cells, which grow attached to a substrate. Our goal was to split these cells into two flasks, and in order to do so, we needed to get them off the surface of the flask which they were growing on. We did this by adding a solution known as Trypsin which is a protease that essentially eats away at the cell membranes, thus making them weak and unable to hold onto the surface. They were only exposed to the solution for a few seconds, as we did not want to damage the cells beyond repair. The suspended cells were round and more mobile compared to the initial cells that were growing on the surface of the flask. The initial cells were a mixture of round and long, football-shaped cells.

We then added the medium, and split the cells into two flasks. As they begin to grow and spread out as a layer on the bottom of the flask, they start to resemble tiny islands floating in the pink solution of the medium. My internship (sadly) only lasts three hours, so I will not be able to see the cells grow into a confluent layer until next time. However, as I checked on my cells every 20 minutes, I began to see them grow into these tiny clusters. We stored our BSC-1 cells in an incubator that is set at 37 degrees Celsius- about body temperature. The cells are kept in a humid environment, with 5% carbon-dioxide. The carbon-dioxide acts as a buffer zone that keeps the pH of the cell cultures at a healthy level- about pH 7.4.

I arrived at my internship (formally) for the first time on Friday. I was introduced to potential customers of Applied Biophysics' ECIS technology. I jumped in on their sales meeting and was instantly immersed in the complexity of the product. Through the next three hours, my brain churned as it tried to translate an incredible amount of technical terms into English. It wasn't easy to understand, but the brilliant people surrounding me seemed very enthused about new and altered features of the latest electrode arrays and ECIS graphing software. Both the clients and my mentors generously took a bit their time to catch me up to speed on the generalities of the company's history, the ECIS instrument, and software system. 

A major component of Applied Biophysics' product line are its different types of arrays and assays. An array is essentially a plastic slide with indentations in which cultured cells are placed. On the undersurface of the indentations are electrodes made of a thin layer of gold. Gold is a good conductor, and can therefore send an indirect, specified pulse of AC current to the cells. The AC current is able to be carried through the leads to the array, and back to the ECIS instrument. As the number of cells in each well multiplies, they cause less current to be transmitted to the ECIS instrument. This decrease in measure of current is called Impedance (Z). In the meeting, a new, more statistically accurate 96 well plate array was featured. 

I also learned the about different applications of the ECIS technology. Wound healing was one of the most interesting ability of ECIS. The cells in the array are sent a current of the highest voltage for a few seconds and are injured, but can recover. This leaves the cells permeable to otherwise impermeable substances, such as DNA or RNA. This method of cell wounding is preferred over manual wounding because it is precise, allows the researcher to deduce quantitative data, and does not remove necessary cell proteins. Along with this application is the idea of the "electric fence". The electric fence uses high voltage AC current to kill cells on the electrode. By killing these cells off, researchers are able to measure and observe the rate cell migration into that empty space.

Applied Biophysics is a company that works to create opportunities for research in drug discovery and cell biology through ECIS™, or Electric Cell-substrate Impedance Sensing. ECIS is method of studying cell growth and behavior in a real-time. Cells grown in tissue culture are placed in an electrode array. As the cells grow and divide, they cover the surface of the array. As the electrical current is sent indirectly through the layer of cells, a potential (V) across the electrodes results, and is then measured by the ECIS instrument. This impedance-based method of studying cell behavior has been applied in countless ways, such as in the study of the “invasive nature of cancer cells, the barrier function of endothelial cells, in vitro toxicity testing as an alternative to animal testing, and signal transduction involving GPCR’s for modern drug discovery.” (www.biophysics.com)

 The potential of ECIS was first realized when Drs. Ivar Giaever and Charles R. Keese were working at GE. Since then, the private company, Applied Biophysics, has been created around this product. The ECIS technology is now used in 6 continents and is utilized by major research foundations and universities.   

I am very excited to begin my STEM internship this year! I have heard so many incredible projects my fellow students have worked on in the past, and can’t wait to be a part of this program. From developing video games, to creating sustainable packaging with mushrooms, to learning about the evolution of molecular life, the stories I hear of my peers’ internships never cease to amaze me. One thing I love about the internships of my peers is that they were all able to explain how their work has real-world application. With my own internship, I hope to explore an area of science that will both challenge and inspire me. I am looking forward to working with people who are passionate about their area of study, and enthusiastic about sharing their love of science with others. I have confidence that my internship will be a guiding factor in what I want to pursue in college. Also, it will be a great experience for me to get a feel for working in a real lab setting before I go to college!