Video Description for the Visually Impaired
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SCENE ONE
Visual: Dr. Carl Simon on camera.
Text: Dr. Carl Simon, PhD
Project Leader, Biomaterials Group
National Institute of Standards and Technology
“The goal of my research is to understand how cells interact with polymer scaffolds. Polymer scaffolds are 3D templates that we use to grow cells on to generate tissue for tissue engineering applications.”
Text:
Fabrication of 3D Combinatorial Polymer Scaffold Libraries for Tissue Engineering
Text:
Salt Leached Polymer Scaffold Librares
Narration:
Billions of dollars have been invested in developing tissue engineered products. But, the return on that investment has yet to be fully realized.
Between 2000 and 2003 tissue engineering, for a number of reasons, was in dire straits. Since then, a remarkable turnaround has taken place.
Still, few profitable tissue engineering products have made their way to the market place. There is, therefore, a need to accelerate the research in this field. To this end, the Polymers Division of the National Institute of Standards and Technology has
developed several approaches for fabricatingcombinatorial scaffold libraries for screening cell material interactions in 3D.
Narration: (con’t.)
One such approach is to assemble simple, low cost Salt-Leached Polymer Scaffold Libraries.
Visual: Dr. Carl Simon on camera.
“We’re working on combinatorial methods of seeing how cells interact with materials. A lot of previous work has been focused on how cells interact with cells that are presented in a flat form, for instance, a surface of a film because this is easy to study. However, for tissue engineering, we want to create a 3D tissue because we live in 3D. So, the methods that are currently available are okay, but they’re not great so we’re trying to develop new methods that are better for 3D structures. So, the 3D porous structure and the cells that infiltrate it and grow in 3D to generate a tissue in 3D so you have a tissue mass instead of just a film. So, the way cells respond to materials in 3D and 2D is different.
What we’ve done is we’ve instead of researchers having to make one scaffold at a time to test it, we’ve used combinatorial methods to create a lot of scaffolds at once and many different properties in a small miniaturized platform so you can rapidly and efficiently see how cells interact with it and see how efficiently tissue can be generated in this context.”
Visual: footage illustrating the procedure Dr. Simon is describing
“Okay. This is what we’re gonna make here. This is a combinatorial polymer scaffold library in an array format. This is in a gradient format. This one, you can see, has a linear change from clear polymer solution to red polymer solution. This one is discrete in composition. Individual scaffolds, discrete compositions and it runs from here, from white and here the red.
And, these are some control all white and the control all red scaffolds.
So, the first thing is to pull some solution up into the syringes. We’re gonna do. This is the poly (caprolactone), the red dye is Sudan IV. You can use any dye that’s soluble in the solvent, The solvent is dioxane. This is the tubing. These are standard tube connectors and some tubing and a T-junction, more tubing. This is a static mixer that’s going to mix the two solutions and break up the laminar flow so what it comes out here on this end is well mixed and homogenous.
So, you use one milliliter of solution on the left and two and a half milliliters of solution on the right and you pump the solution through. And you put these onto the mixer and you mount this onto the syringe pump.
This is a lab stand and a clamp there to hold it. You want this to be at a thirty degree angle. this reduces your bubbles. And, if this is pumping up or flat, I’ve noticed, sometimes you can a little
runback and more bubbles. If you have a slight decline and you don’t get that.
We also want the pipet tip here to be down at a thirty degree angle also. If it’s flat you can get drips coming back along the tip.
We’re gonna start with the syringe pump that as a clear polymer solution in the ‘on’ position. This one’s gonna start in the ‘off’ position. This one’s gonna ramp down, this one’s gonna ramp up. So we’re ready to start. First thing you’re gonna do is prime. I’m starting these. And, they’re priming the lines right now. If you pump and prime with a little extra of the clear on the left 300 microliters you get much more linear stable reproducable gradient.
You see the clear one coming in right here. I’m gonna prime it up to right there. This one is primed up to the ‘T’. I’m gonna tunr it off till the red gets up to the ‘T’.
All right. Now, I’m priming 200 microliters down the line. And the pump gives you a readout of how many microliters you’re pumping. This is the microliters that are getting primed down the pump--215, 220.
All right. We’re now ready and we’re turning on the pumps. I’m gonna start them now. You’re gonna hear this one ramp down and this one’s gonna ramp up. We don’t start this stage until
the solution starts to elute from the tip here.
Right now, you can see, you’re getting laminar flow as the red and the clear solutions mix. And when they get into the static mixer, the corkscrews will cut and fold that and make an homogenous solution so that what we get out here is well mixed.
Okay, so now I’m starting it. There we go. We’re gonna have the drops falling in the center. You can see as they hit they get soaked into the salt and kind of spread out. Right here is the end. I let a couple of extra drops fall just to fill up the edge and get a nice square edge.
So, the next step would then be to submerge it in liquid nitrogen to freeze it. I happen to have some liquid nitrogen right here. It’s still liquid right now, but now it’s gonna freeze slowly as you fill it. This is what you want. You don’t want to dump it right onto the scaffold or you’ll make a mess of it. Want to pour it in gently from the side and let it come up and over the scaffold and it’ll freeze it.
That’s it.
You let it sit in the liquid nitrogen and thoroughly freeze for about five minutes and then you put it on the freeze dry overnight and that will remove all solvents and the next day you’ll be left with a polymer matrix around the salt.
Now, we’re gonna make a discreet 96 well scaffold library. We’re gonna do it on a 96 well plate using 0.13 grams of sodium chloride in each of the wells. Everything else up here, all the syringes, solutions are exactly the same. The only difference is instead of collecting it in a trough that’s linear on the face, I’m going to manually collect drops into the scaffold in a zig zag fashion. I’m gonna start the pumps up now. You can see the polymer solution starting to come down the tip. We’ll let the first drop or two fall and then I’m gonna start collecting. I do two drops per well. Two drops comes to about 30 microliters. That’s about three milligrams of polymer per well. Each scaffold will weigh about three milligrams.
The primary advance of this approach is the transition from 2D to 3D. Previous work, previous methods, made your arrays in a flat format or surface. The cells read more physiologically in 3D and volunteers frequently use a scaffold so it makes sense to do a screening using a 3D scaffold approach. You get more relevant information. The structure of the scaffold influences how the cells respond.
And, that’s it.
This is what one looks like when it’s completed. You mount this on a 96 well adaptor to a centrifuge and spin it and that brings all the solution down to the bottom and makers sure
you get a nice, fully formed scaffold. And, you put it in liquid nitrogen and you freeze dry it overnight to remove all the solvents and the next day you place it into water to leach away the salt and that’ll leave behind these little polymer scaffolds, these little polymer fillings to do your cell screenings.”
Visual: Dr. Simon on camera.
“The combinatorial methods that we’ve been developing for screening how cells interact with materials in 3D provides a systematic approach to developing new materials for tissue engineering and bio-materials application.
This field is significant because the nation spends a
large amount of money on health care and supporting patients with end stage organ failure and on bone grafts and other kinds of tissue engineering applications, bio-materials. This
research will help accelerate and improve the theraputic value and applications of bio-materials in 3D tissue scaffolds.”
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