Zero Order


Simple misunderstandings

Posted in Uncategorized by Amy Ross on July 30, 2010

In many ways,  lab techniques have a component of oral tradition. Sure, you can follow a protocol or an instruction manual, but it’s always better to have someone explain, or better yet demonstrate, a procedure. But as information gets passed from one person to another, like the children’s game Telephone, some of the details get lost.

I wanted to calculate the mesh size of my hydrogels, and in order to do that, I needed several measurements, including the volume when fully swollen. Our  hydrogel samples are fairly small (I typically do discs 2 mm thick and 6mm wide), so it’s hard to get an accurate measure by using calipers. A previous grad student had measured volume by employing Archimedes’ principle: the buoyant force on an immersed object (causing the apparent decrease in weight when compared to weighing in air) is equal to the weight of liquid it has displaced.  He weighed hydrogels in air and in butanol. This will give the weight of the displaced butanol, which can be divided by the density to obtain the volume. Why weigh in butanol?  The hydrogels need to be weighed in a liquid they can’t absorb. Water or anything aqueous could be absorbed and skew the results. So instead, the hydrogels were weighed in an organic liquid.

So I tried this, using the other grad student’s equations,  and I kept getting highly inconsistent and sometimes impossible  results.  I did six groups of polyacrylamide hydrogels, twice, and still couldn’t figure out the problem.  I put it aside to work on my experiments with cells. Meanwhile, one of our summer undergrads, Megan, needed to find mesh size for her PEGDA hydrogels.  I gave her the equations and showed her what I was doing, and she kept getting odd results as well. It turns out the way I was weighing the hydrogels was wrong.

To employ Archimedes’ principle, the object being weighed has to be suspended in the liquid. I weighed my hydrogels by dropping them in a (tared) vial of butanol. The density of the hydrogel was greater than the density of the butanol, so naturally, they sank to the bottom. If the hydrogel is sitting on the bottom, there is no buoyant force acting on it.

Oops.

Figuring out how to suspend the hydrogel was another challenge.  The gross physical structure of a hydrogel is like Jell-O: it can easily be mushed or torn.  Megan rigged up what looks like a miniature version of  an Easter egg coloring dipper, and she found it worked pretty well. I’m going to steal it from her when she goes back to school 🙂

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General Update

Posted in Uncategorized by Amy Ross on July 23, 2010

A few things I’ve been up to lately:
-Still working on cell attachment with my hydrogels. I’m trying single-layer PEGDA hydrogels, but the results aren’t much of an improvement over the bilayer hydrogels I had been using. One of our summer rotation students thought the environment might be too dilute of nutrients for the cells to survive, as I had been swelling them in phosphate buffered saline. She suggested I should try swelling them in cell  media.  I thought it was worth a try, so I did that experiment this week.
When I assessed cell viability using MTS, I didn’t see a significant difference in cell attachment between the hydrogels swollen in media and the ones that weren’t.  However, it seemed like there were fewer cells on the bottom of the well  for the media group when I looked at them under the microscope.

Oh well, I’m trying something new this weekend that I’m excited about. I’ll tell you all about it soon!

-I’ve been learning  gel permeation chromatography (GPC). In short, chromatography applies to a broad range of techniques that separate materials based on a specific property. In the case of GPC, it’s size. All chromatography consists of two phases, a stationary phase (typically something solid) and a mobile phase (a liquid or a gas). The components of a mixture that have a greater affinity for the mobile phase will elute first; as affinity for the stationary phase increases, the elution time lengthens.

GPC is primarily used for polymers. Allow me to use this crude drawing (that looks a lot like the 1980’s Atari game Defender) to demonstrate how separation occurs.

The polymer solution (the red and blue shapes) is dissolved in a solvent, and injected into a column (the grey thing).  The shorter polymers (i.e., the red circle) get caught in the gaps of the column, and leave the column last. The larger polymers (the blue ellipse) are too large to fit in the gaps. They leave the column first. After leaving the column, a detector (there are several varieties) determines the properties of the polymer solution.

I used GPC to find the molecular weight of the polyacrylamide hydrogels I had made.   I got decent results once I increased my solution concentration.

-We’ve had several undergrads in our lab this summer, and they’re wrapping up their projects and will be headed back to school in a couple weeks.  I enjoyed teaching them things, and learned a few things from them as well. The place is going to be awfully quiet next month.

Have a great weekend. I’ll have more updates as warranted.

My favorite things

Posted in Uncategorized by Amy Ross on July 16, 2010

Well, it’s Friday, and the extreme heat we’ve been having has made me lazy. In lieu of more serious content, I’d like to take a moment and recognize three things that make my workday a little easier:

1.  The Molarity Calculator page. My postdoc showed this to me one day, and it’s a lifesaver. You don’t expect me to do all those calculations by hand, do you?

2. MTS assay. I’ve talked a little about MTS before. It’s a measure of how many live cells are present in a given sample.  The MTS dye is reduced to formazan in the presence of mitochondrial reductase, changing the color of the solution. The color change can be measured by spectrophotometry. It’s an easy test to perform and there’s no solution to mix.

3. Last but not least, the automated cell counter. Cells can be counted by hand using a device called a hemocytometer (named because it was initially used to count blood cells). The hemocytometer consists of two blocks divided into nine squares each. A  couple of drops of cell-media suspension are placed on the hemocytometer, and s cover slip is placed on top. Using a microscope, cells in the squares are counted, averaged per square, and multiplied by 1000 to get the cell count per milliliter.  I’ve done this once. It wasn’t too bad, but I only had 12-20 cells per square. If you get into the hundreds, it’s much worse.

The cell counter is faster,  more accurate, and fairly easy to use. The one in our lab measures particle size by measuring change in electrical impedance. The only downside is it can’t process large cell counts.

An honorable mention goes to Wikipedia chemistry articles. How did anyone get through Orgo without them? I guess they had to look up all those densities and boiling points in a book! 🙂

Other Uses for Hydrogels

Posted in Uncategorized by Amy Ross on July 12, 2010
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A few weeks ago, I talked a lot about using hydrogels for drug delivery. Today I’m going to talk about two other uses for hydrogels: one commercial, one experimental.

Use in Electrodes

Biological signals tend to be of low voltage, (e.g, 0.5-4 millivolts for cardiac potential), which I suppose is a good thing for us. The downside is that potentials measured from other sources-often referred to as noise-interfere with  the signal measurement. One of the problems with electrodes is that noise can occur if the electrodes slip out of place, or motion artifact.

Electrodes function by forming a series of opposite charges at the interface between the electrode and what it’s placed on. If the electrode moves, the charges on the other side of the interface are disrupted, and will have to redistribute to re-establish equilibrium.  During that redistribution, a potential difference forms, which will be measured and considered part of the signal.

Hydrogels that contain electrolytes (such as chlorine ions) are used as an interface between electrodes and the skin.  They keep the electrodes in place and reduce motion artifact.

Tissue Engineering

Tissue engineering is exciting stuff.  Can we grow skin, bone, or more complex body parts  from cells?  If we can, it would be a benefit to people with chronic wounds, burn patients, and (hopefully) people waiting for transplants. No doubt you’ve heard a lot about stem cells and the research done to get them to differentiate into a specific tissue type. One approach is to use a hydrogel as a scaffold, or support structure, for stem cells.

The properties of a hydrogel are similar to human tissues-they’re relatively soft and elastic. The premise is that the stem cells will take cues from the environment that will direct their differentiation. Additionally, hydrogels provide a 3-D environment more similar to the human body than the typical 2-D environment  in which cell studies are performed.

Our lab has approached this by using superporous hydrogels as a scaffolding material.  When the hydrogel is polymerized, the pores are created using sodium bicarbonate.  The pores that are created interconnect and allow stem cells to infiltrate the structure and proliferate.  One of the PhD students, Melanie, is currently working on this project. She’s using superporous hydrogels to differentiate human mesenchymal stem cells into bone tissue.

There’s way more than I could possibly say about the tissue engineering angle, but I’ll leave it at that for now.

A research update

Posted in Uncategorized by Amy Ross on July 9, 2010

In a previous post, I talked about the difficulties I had getting cells to stick on the surface of my hydrogels.   Here’s an update:

I’ve been trying to find the right concentration of RGD such that the cells will not only attach to the hydrogels, they will also proliferate at a rate comparable to what they’d do on a plain tissue culture plate.  If the RGD concentration is too high, the cells will attach, but not proliferate. I tried an experiment using different concentrations from 100 μM (micromolar) to 1 mM (millimolar, ten times the 100 μM), and found I was getting the best results at about 500 μM.

However, I was still getting more cells falling off the hydrogel surface than staying on. I thought, if cells landed on the surface and attached, would they stay attached after movement? That way, I could move the hydrogel to a new well, anything that was on the surface would stay on the surface. Any cells on the bottom of the tissue culture plate would stay there. The well I would move it to would only have cells on the surface.

I tried this by moving hydrogels at specified time points after adding the cells-2, 4, 6, 8, and 24 hours. Unfortunately, cells still fell off the hydrogels after movement, no matter what time point I moved them.

After sharing these results with my PI,  he recommended trying experiments with single-layer hydrogels instead of bilayers.   I would be lying if I said I wasn’t disappointed. But as we know, projects not working as expected goes along with the territory.

I’m starting a new round of cell experiments next week. We’ll see how they work!

I’m not dead yet!

Posted in Uncategorized by Amy Ross on July 3, 2010

Hey everyone. This week has been crazy busy, so I haven’t had a chance to write a post.  Next week I’ll have an update on my research and some new things I’ve been up to.

In lieu of actual content, here’s a lab-themed parody of Lady Gaga’s “Bad Romance”.

Have a great Independence Day if you’re in the U.S., and if you’re not, have a good weekend.