2/24/2017: Week 3 – Time to Get Technical

Normally, my Week 3 wouldn't be over until ranch duty ends tomorrow, but unfortunately, I won't be able to make it to the ranch again this week (due to a piano competition). However, this does mean an earlier post for all my fans who I know are dying to read more. 😛

One quick update on the ranch before I begin: Cheyenne is not yet on yellow alert, but she is close to giving birth. Also, there's a new horse at the ranch now. She's a beautiful white mare named Lansdowne. 

Lansdowne Checking Out Her Temporary Home

LET THE LAB WORK BEGIN

Tuesday:

As I mentioned in my previous post, Lil gave birth last week, so we finally had collection material to work with. Although we currently have both amniotic fluid and amnion, we spent this week working only with the amniotic fluid.

You might remember this from one of my earlier posts, but amnion/amniotic fluid contains three substances that make it ideal to be processed into a treatment: extracellular matrix, growth factors, and of course, stem cells. However, the amniotic fluid can't just be used as it is. Everything must be thoroughly cleaned (collection is messy, after all), and the cells must be isolated from the rest of the solution. This is important because the cells and the rest of the fluid are stored under different conditions, and one type of product AniCell produces actually contains no cells. 

On Tuesday, we worked on separating the stem cells from the rest of the fluid. To do this, we transferred all the fluid into sterile 50 ml tubes using serological pipettes and spun the tubes using a centrifuge. Spinning these tubes at high speeds using the centrifuge basically made the relatively heavy cells accumulate in a pellet at the bottom of the tube, effectively separating them from everything else. The simple diagram below shows this. 

Simple Pellet Supernatant Illustration

Our Lab's Small but Fast Centrifuge

After we had the pellet of cells at the bottom of the tube, we then transferred the supernatant (which we need) to a new sterile container, making sure not to disturb the pellet in the process. Because we had about 20 tubes of the material, we also transferred all the pellets to one tube. To do this, we resuspended each one in PBS (phosphate buffer solution, not the Public Broadcasting Service), a sort of resting solution for the cells, and transferred all the liquid to one tube. Something important I haven't mentioned yet is that all this work must take place in an extremely sterile environment. We all wore gloves, hairnets, and shoe covers during the entire procedure and during any time we spent in the lab's sterile area (mopped using isopropanol). All the procedures were also performed in a biosafety hood. 

Here's a video of me using a chair to exit the sterile area to save shoe protectors:

To check for bacterial contamination (which can ruin the whole product, as you obviously can't treat an animal with infectious material), we left an agar plate open in the hood where we worked. Later, we stuck this plate in an incubator, as well as a plate with some of the collected fluid rubbed on it. After letting these plates sit for a day, we can then see how sterile our working environment, and the collection material, were based on bacterial growth on the plates. 

Irene Pipetting Some Amniotic Fluid

Two Plates Mentioned Sitting in the Incubator

Equipment Close-Up

Anyway, by the end of Tuesday, we had one container with all the amniotic fluid minus cells, and one tube with all the cells suspended in PBS (pictured below). We ended the day by adding a small vial of antibiotic to the cells, hoping to thoroughly clean the solution containing the cells.

SC = Stem Cells

Thursday: 

Before starting on anything new, we checked out the plates we left in the hood on Tuesday. Fortunately, the control plate (the one we left open in the hood) was bacteria free, indicating we did indeed have a clean working environment. However, as expected, the plate which we rubbed some of the initial amniotic fluid on was caked with bacteria (it was pretty gross). Hopefully, the solution containing the cells was thoroughly cleaned by the antibiotics, since it only contained small remnants of the initial fluid. However, the initial fluid (which we also need) would need a lot more cleaning than simple antibiotics.

So, Thursday consisted of cleaning the amniotic fluid. To do this, we first spun all the fluid, hoping to let larger bacterial cells and dirt particles clump together at the bottom of the tube, and put all the liquid through a filter. The filter we used was made of a special material which repels proteins, effectively letting all the good growth factors through. The whole apparatus is also attached to a vacuum, which helps pull the solution through the filter.

Lemonade to White Wine

As you can see, the filtrate (on the right) is much clearer (and cleaner) than the initial solution (on the left). After this initial filtration, we spun the material once again and then put it through a more powerful filter. 

Even Better White Wine

Now that the material had been spun and filtered twice, it was finally ready to go into the -80° F freezer for storage until it's needed again. 

Clean and Clear Amniotic Fluid

For us to be able to check the efficacy of the filtration, we took another agar plate and split it up into four quarters, which were treated as follows:

1) With the antibiotic-treated cell suspension solution (from Tuesday)
2) With unfiltered amniotic fluid
3) With once filtered amniotic fluid
4) With twice filtered amniotic fluid

*Leftovers were used for treatments 2 and 3

When I go back to the lab on Monday, I can see the plate and observe how the amount of bacterial growth changes between the samples. Hopefully, it decreases! I'll include a picture of the plate in my Week 4 post. 

So, to quickly summarize, my work in the lab this week was essentially the first steps of the production process: separating the stem cells from the amniotic fluid and cleaning it all. I can't wait to go back and see what's next!


ADDITIONAL NOTES

I will probably have a smaller post before my Week 4 post where I will talk a little about the SOP (Standard Operating Procedure) I mentioned in my previous post, as well as the papers on stem cells and cancer I'm currently reading. Stay tuned, it's really getting exciting now!