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!