2/21- Emma Talks

2/14 Snow Day

On Friday, Dr. Keese and I ran our first co-culture experiment with a 96 array! We used a 96w20idf, an array with interlocking pattern of electrodes. Because this is such a complicated experiment, Dr. Keese demonstrated the techniques needed for measuring out and pipetting our dilution series with the first 6 columns. He worked with the MDCK cells while I handled the BSC-I cells. It was helpful to get a refresher on how to passage cells, and how to handle 96 arrays. 

The first step was to get our cells to round up off the bottom of the flask using trypsin, and enzyme that temporarily damages cell walls (and also adding EDTA for the MDCK cells). Once the cells were  no longer a confluent layer, I added 20 mL of medium to the cells. I made sure the cells were evenly distributed throughout the solution by pipetting the mixture up and down, a technique known as titration. Then, I pipetted the cell-medium mixture into two tubes, and set those aside while I prepared the dilution series.

In this experiment, we were essentially running two separate dilution series: one with MDCK cells diluted with BSC-I cells, and the other with BSC-I diluted with MDCK. To recap the layout of this experiment, here are the tables I used in last week's post:

     

     Layout of cell ratios in the 96w20idf array: 

   
   Volumes of cells per column:

 

I labeled 5 tubes with each concentration ratio, and pipetted 4 mL of BSC-I cells into the first tube, 2mL into the second, then 1mL, .5mL, and finally, .25mL. Then, began to add in the MDCK cells that Dr. Keese had helped me passage. I did not put any into the first tube, as this will be pure BSC. The second tube I added 2ml, the third tube 3mL, then 3.5mL, and lastly 3.75mL. 

Now my dilution series is ready to be inserted into the wells of the array! After giving a quick flick to each tube to mix the cells around, I poured the first tube into a trough. Using the special, 8-tipped pipetter, I drew up enough liquid to fill each well (300 µL per well). I repeated this step 5 times, filling each column with a different BSC/MDCK cell mixture. Once Dr. Keese and I filled the entire array, it was off to the ECIS machine to plug in my array! Can't wait to see what this week's data looks like!

On Friday, I took a look at my data from my 8W1E co-culture experiment, dove deeper into the theory behind ECIS, and planned out my 96W20idf  experiment for next week.

ECIS Theory for Distribution:

Dr. Keese showed me a power point presentation that helped me understand the more technical side of ECIS. We spoke about the significance of different frequency levels and the physics behind the different types of 96-well arrays.

The first 96 array we spoke about is the 96W1E+, an array with two circular electrodes in each well. This array is used for measuring micromotion of cells, and for wound healing assays. Micromotion is the fluctuation of cell movement as measured by electrodes. Micromotion occurs even after a confluent layer of cells is formed, allowing us to take a close look at cell behavior throughout an entire experiment. 96W1E+ arrays are also used for wound healing assays. In a wound healing assay, electrodes send out electrical currents that will kill the cells in the vicinity of the electrode. This process will cause the cell membranes to become porous, and medium will leak into the cell, causing it to die. Wound healing assays allow us to examine the rate at which other cells will move in to heal the wound. 

96W1E+ with closeup on electrodes

The second array type we looked at was the 96W20idf array. This array has 96 wells, each with an interlocking pattern of electrodes which increases the total surface area significantly. More surface area allows us to take a look at more cells, therefore giving us data with greater statistical accuracy. However, for this accuracy we sacrifice the ability to examine micromotion and to perform wound-healing assays. Due to the large amount of electrode surface area, wounding would cause too many cells to die. I will be using this type of electrode in next week's experiment.

96W20idf with closeup on electrodes

Last week's data:

My data looked a little bit better than last week, yet I ended up with one outlier and my graphs seemed to suggest that my wells were a bit light on cells. Working with an 8 well electrode is much less statistically accurate because we are only looking at 2 wells of each cell mixture. If one (or both) of those wells differ from the true value, our data is more susceptible to error. Overall, I think my 96 experiments will be more  representative of what is really happening when MDCK and BSC-I cells are co-cultured. 

Next week's experiment:

I will be using a 96W20idf array to perform this co-culture experiment in a dilution series. It will be the most complicated experiment I've ever done, (and maybe even Applied Biophysics has ever done!) but here is the breakdown:

     Cells: BSC-I and MDCKII

     

     Layout: 

   
     Procedure: Using 10 tubes, we will dilute MDCK with BSC-I cells for the first five columns, and then dilute the BSC-I cells with MDCK in the last 5 columns. I will pipette 400 µl of cell mixture into each well. These will be the volumes of cells per column I will need for this experiment. 

Co-Culturing: Take 2!

Today at Applied Biophysics I worked with last week's data I from my co-culturing experiment. Sadly, the data did not turn out as expected. The experiment started out looking promising, however, the cells in every well died out around 15 hours. Our hypothesis as to why this happened? Using an array just after it has been etched is tricky. Arrays that have been recently etched tend to allow more liquid to wick up the sides of the wells. This means that the amount of medium in each well is thinly distributed, and the cells are unable to grow and divide in an environment with insufficient nutrients. Also, this causes the liquid to evaporate faster, thus leaving the remaining medium highly concentrated with saline. Cells cannot survive in such a hypertonic environment- they will shrivel and die due to osmosis.

I treated each of the wells with cysteine, which usually helps with the problem of liquid wicking up the sides of wells, but my array might have been too fresh (last week, I took my array straight from the plasma etcher to the hood). So, this week I repeated our experiment. All conditions remained the same, except I used an array that had been etched a few weeks ago. That should have fixed our problem! Hopefully next week's data goes smoothly!

Friday was a busy day for me at Applied Biophysics. I learned how to plasma etch an array, interpreted the data from my 96 array experiment last week, and began my newest project: co-culturing. 

Plasma etching:

At Applied Biophysics, we use plasma etching to sterilize electrode arrays. On Friday, Dr. Keese put me in charge of the etching machine! First, I placed my array (an 8W10E+) into the chamber that will be pumped down to create a vacuum. We then pumped oxygen into this low-pressure chamber. After a few repetitions of pumping down the chamber and adding gas, we were able to start the plasma. 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. We sent an electrical charge through the chamber to create the plasma. The plasma ions become activated by this electrical field, and begin to vibrate and glow a white to purple color.  The vibrating ions essentially “scrub” the arrays in the chamber, leaving them sterile. Here is a picture of the plasma we created inside the chamber (and a super nerdy picture of me in front of the etcher):

Data from last week:

Last week Dr. Keese and I created a 96 array dilution series, with half MDCK-II cells and half BSC-I cells. Here is our graph using the ECIS software: 

Kind of crazy looking! Dr. Keese added a wound to the cells at 48 hours, which can be seen in the dip and recovery of the green and dark blue lines. The rainbow series of dots in the left hand column is the layout of the array wells. Columns 1-6 are MDCK-II cells, and 7-12 are BSC-I cells. Each column has same type and about the same concentration of cells, therefore we can group each column (take the average of all wells per column), making a much more manageable graph:

As you can see, the lines that are a shade of blue (representing the MDCK-II cells) take much more time to attach to the surface of the wells and form a confluent layer compared to the BSC-I cells. 

Co-culturing:

On Friday, I began my own study on the subject of co-culturing. Co-culturing cells means growing two different cell types together in one culture (or in my case, well). By studying cells in this way, we are able to see how cells interact with one another, in this case, how BSC-I cells will affect MDCK. Will one cell type overtake the other? Will the cells grow in isolated groups? Will they together form an evenly distributed confluent layer? To answer these questions, I created a dilution series, (explained in last week's post), diluting MDCK cells with BSC-1. I filled four tubes with 1ml of MDCK cells, and then added 1ml of BSC-I to the second tube. I then mixed the contents of that tube, took 1ml of the mixture, and dispensed it into the next tube. The overall array had 2 wells of pure MDCK cells, 2 wells of a 50/50 mixture, 2 of .25/.75, and 2 of .125/.875. I am looking forward to seeing these results next week! 

96W1E

It's been so long since I've been at my internship! It was a good thing I didn't forget what I had learned over winter break, because we started a much more complex experiment today! Today I got to work with a 96 well electrode known as a 96W1E. This wellpad works the same way an 8 well electrode would, but looks quite a bit different! Here is an image of my 96W1E, all filled up and ready to be placed into the ECIS station:

In today's experiment, we used two cell types: MDCK and BSC-1 cells in a dilution series. Dr. Keese demonstrated how to work with a 96 electrode, and proper pipetting techniques using an 8 tip pipette. He first pipetted 150 µl of cysteine into the first six columns of the wells. Cysteine is an amino acid that reacts with the gold of the electrodes, allowing proteins cells to attach and spread easily. While we waited for the cystine to react with the gold, a process that takes at least 5 minutes, he began to treat the MDCK cells with EDTA and trypsin to get the confluent layer of cells to be free-floating. Setting those cells aside, we began to prepare our dilution series. The main idea behind a dilution series is to examine how cells grow in environments of varying sparsity. We created cell mixtures in six test tubes with the following cell compositions: 1x, 1/2x, 1/4x, 1/8x, 1/16x and medium (0x). We got these mixtures by filling each test tube with 3mL of medium (except the 1x tube). The 1x tube was 6mL of a suspended MDCK cell mixture. We took 3mL from this tube and dispensed it into the second tube- 1/2x. We mixed the cells and the medium using a titration technique (aspirating and dispensing the mixture over and over again to make sure the cells are uniformly distributed). This step was repeated for each of the subsequent tubes, therefore making each tube half as concentrated as the preceding tube. Dr. Keese then pipetted the mixtures of varying concentration into 6 of the 12 columns. I repeated these steps on my own with the last 6 columns, using BSC-1 cells. Here is an image that shows the layout of the 96 wells:

Finally, we were ready to place our prepared 96W1E into its station! This station looked different than those that I have used with 8 well electrodes. Instead of teeth that attached to one end of the electrode, this station uses what Dr. Keese called, a "bed of nails" to transfer signals from the electrodes to the ECIS machine. Each of the gold "nails" would connect to a single electrode, relaying the electrical signal to the more advanced ECIS machine, the Zθ. This machine is different than the Z (which I am used to working with) because it uses a "complex impedance spectrum", meaning it can read cell behavior in Z, R, and C (impedance, resistance and capacitance). 

Here is a sneak peak of the first 15 minutes of my graph. Looking forward to seeing my results next week!