Patterning Cells on Optically Transparent Indium Tin Oxide Electrodes

Hi, I am Isha. I am a second year graduate student in Dr.Zen's lab. We are here at the Department of Biomedical Engineering at University of California Davis.

Today I'm going to show you the controlling of Ian on micro fabricated optically transparent Indium 10 oxide electrodes in tissue engineering and regeneration efforts have been made to mimic the microenvironment of the organ. In order to create this microenvironment in vitro, it is essential to spatially and temporally isolate cells on a substrate. We're interested in designing surfaces where we can control the location of different liver cell types.

In today's experiment, we will use surface modification, microfabrication and electrochemistry to form a surface that can be switched from cell non-adhesive to cell adhesive. The first step is photolithography. Here's a flow diagram of how that process is gonna be worked.

The blue is the glass light, the green is indium 10 oxide that is coated on top of the glass light. First, what we are gonna do is spin coat photo resist red shown here on top of the Indium 10 oxide coated glass light. Then we are gonna pass that through a photo mask to ultraviolet light and the exposed regions will be developed in a developing solution.

The ITO regions that are not protected by photo resist will be removed away in IT oing. And then finally, all the remaining photo resist will be removed in acetone to form our ITO electrodes. Here's the flow diagram of the remaining three steps.

First, we are going to modify the entire surface with peg cylin. This is gonna make the surface non fouling and no cells or proteins will bind to it. Next we're gonna use electrochemistry to specifically absorb peg cylin from the I two electrodes that we made earlier.

And finally, we are going to incubate fibroblasts on top of the surface and they will selectively bind only to the strip regions. So now we're ready to begin our experiment. The first step involves photolithography, which is gonna be carried out in a clean room.

So let's go ahead and get into the clean room. So now here We are in our clean room. Before we start our experiment, what we're gonna do is have our indium tin oxide or ITO coated samples baked at 200 C in our oven.

That way we can dehydrate all our surfaces. And after baking it at 200 C is when we're gonna place it on our spin tech and actually apply the photo resist. So after dehydrating our ITO substrate, we're gonna take it and place it on the chuck of the spin tech.

This chuck is just big enough to hold a quarter of a glass light. Then we are gonna, we're gonna apply vacuum. After that, we apply positive tone, photo resist on the substrate.

After the application of the photo resist, we just go ahead and start. This involves two steps. First is the spreading, and the second is the spin coating in the spreading step.

The chuck rotates at 800 RPM and that just uniformly spreads the photo resist on the entire surface. However, the next one is the spin step, which is at 4, 000 RPN and at 4, 000 RPM, it forms a really thin layer. By thin I mean about one microns.

If you break up the word photo, resist, it turns as photo and resist. It is activated by light. Hence, we work in the clean room and we use filtered light so that our photo door resist doesn't get activated by the light and it is resistant to assets, which we will the property we'll use later on when we do etching.

So now we're done with spin coating, photo resist on the surface, and we're just gonna take it out and move on to our next step. So after baking the sample for one minute and 45 seconds, we are gonna take it from the hot plate and move it over to our cannon mask liner. Here we take a mask that we need and then we place the mask on top on top of our substrate in order to have a contact printing between our substrate with port resist and the mask, we place it with a four inch glass wafer just to add weight to it.

And once we're done with that, we just put the shutter back on and then we expose the sample to UV light. Once the exposure is complete, we just move the lid away in reverse order. We take out our glass wafer, we take out our mask, and then we remove our substrate and take it over for development.

After exposing the sample, we place it in a developer solution. When the sample was exposed to UV light, there were hydrogen ion form, which neutralized with the base and the developer solution and are actually removed from the surface. So now you wanna make sure when you're doing the sample, you're constantly swerving the substrate.

And when you do that, you can see in about the first minute or minute and a half, the ex exposed regions are actually developed and removed from the surface. But for complete development to happen, we let it sit in the developer solution for about five minutes. After the development is complete, we're gonna take it out, we're gonna wash it and di water and then dry it using a nitrogen gun.

Once we've dried our sample, we're gonna take it over and visualize it under our microscope. Here we are looking at the sample under a microscope. We just took it out after developing it for five minutes.

There are two regions that you can see here, a darker region that is covered by photo resist and the lighter region where photo resist has been removed. In relation to the mask that we use earlier, the darker regions on the mask were the darker regions that we see right now under the microscope, whereas the clear regions that we see here, were transparent on the mask. One of the designs that is shown here is just arrays of different sizes going from 500 down to 50 microns, whereas the other design is one 1000 micron circles that are just interconnected.

So here we have a different design that was made using the same procedure. Here we have individually addressable electrodes. What I mean by that is that different regions of ITO are interconnected and they can be activated differently.

For example here, the central circular regions, they are connected to one electric pad here, whereas the outermost rectangular regions, they are connected to a different electric pad. This way we can stimulate these two different surfaces at different time points. After looking at our samples, the next step we move on to is we etch the sample.

Whatever regions that are not protected by the photos are gonna be etched away in an ITO etching. Before doing that, we have to make sure that we put appropriate clothing on since ITO etching contains acids. And now after getting into my acid protective gown, putting my face shield on, I'm gonna pour some ITO etching into a glass beaker.

The ITO etching is composed of 20%hydrogen chloride, 5%nitric acid, and the rest is DI water. And then bring my sample over and place it in the etching. Earlier in the microscope, we saw that there were some regions that were not protected by photo raises.

The acids in the ITO etching attacked those regions and caused the ITO from those regions to be etched away. As the ITO slowly degrades from the surface, it changes color. Here you can see that it changed from a bluish change that it originally had to this purplish change and it is now moving on to the yellow exchange.

The change in color indicates that the ITO is being etched away. So now here we are at 15 minutes. All the ITO, it's completely etched away.

And what do you see right now are photos is protected ITO regions. So after the etching is completed, there are still some acids that are remaining on the surface. To remove these acid, we're gonna place it in a sodium carbonate solution, which is gonna neutralize all the acid.

After neutralizing the acids, we're gonna take it out and clean it with DI water and then dry with the nitrogen done. So now we're done with the acid etching. No more acid, I'm out of my hot gown.

We're gonna take it out and transfer it over and visualize under our microscope. So now we're observing the sample under the microscope. This is after we performed the ITO etching.

In this case, the darker regions are glass regions from where ITO was etched away. And the lighter regions are ITO that are protected by photo resist in this case. Once again, these are the arrays from 500 to 50 microns and in the other case we have our interconnected circles of thousand microns each.

So we just looked under the microscope and we saw that there were glass regions and ITO regions, but the ITO regions were protected by photoresist. Now we need to take this photoresist out. So all that we're gonna do is take our substrate, put it in to acetone, and then place this in the sonicate and just sonicate for 10 minutes.

This ensures that all the photo resistance taken off, you could use as tones by itself, but by sonic hitting, which uses sound waves to hit and that ensures that the photo resist is removed correctly. After taking it out of the sonic air, it's washed in DI water, dried again in nitrogen, and now you can see the patterns clearly on glass substrate. These were the patterns that were initially protected by the photo resist.

Now that we've looked at Our samples and found or made our ITO patterns, we are gonna start our second phase of the experiment. This phase involves the modification of the surface with a self-assembled monolayer of PEG cycline. But before doing that, we have to make sure that the surface is reactive.

To do so, we place it in a plasma cleaner. This introduces hydroxyl groups on the surface and thus makes it reactive. To do so, we just place our sample into the plasma cleaner and then we turn on our vacuum pump.

We turn on oxygen, we turn on the main power. This power is used to convert the oxygen gas into plasma, which is then introduced and thus forms the hydroxyl grooves. And once we turn on the power, we go ahead and hit start.

So now after the process is complete, we take our substrate out, store it in a dish and head on outside the clean room de gown and move on to the next step. So now that we are out of the clean room and we finished our ideal patterning, we are gonna modify the surface that we just patterned with polyethylene glycol, CYLIN or PEG Cylin. It is key to know that peg cylin is reactive to moisture.

Hence everything that we use wood has to be moisture free. The glassware that we've used here, the pet dishes, they have been baked at a hundred C for overnight. And the tun that we're going to use as a solvent is also anhydrous.

It doesn't contain moisture. So now I'm going to pour tun, anhydrous tolu into into both these glass speakers, one of which has our sample in it. Now we have to use two petri dishes because one of them is where we're gonna do the modification in.

And the other one we are going to use later on when the modification is over to actually wash our substrate with. So after putting in our in our glass pizza dishes, I'm just gonna take it over and transfer it to our airlock chamber here in the glove box. After closing the airlock chamber, we're gonna introduce nitrogen into the chamber.

Now the main chamber here is already filled with nitrogen. Hence we don't wanna open this chamber unless it is filled with nitrogen. Once both the chambers are nitrogen, we're gonna transfer all of our samples along with a pipet.

We're gonna have our peg and a tweezer that we're gonna use later on to transfer the samples. So we're gonna put our gloves on, open the chamber here and slowly and carefully take out all that we have in our airlock and place it in the main chamber. This is the Petri dish with our substrate and the Petri dish just with toluene that we're going to use for washing.

The pxi that is present was packaged under nitrogen and you should not open this bottle unless you are in an environment of nitrogen. Hence, once when you wanna take out the sample, you put it back in the bag. So you take out you cylin and the concentration that we're gonna use is 2%in toluene.

So you take out, put it in the tall in, we mix it. Well also chamber and ensure that it is mixed well and then let the sample incubate in Toin for two hours. So now that two hours have passed, our sample has been in Pxi, we're gonna take the sample out and transfer it to a fresh toin solution where we're briefly gonna wash it, swerve it around, and move it over to our airlock chamber.

Once it is in our airlock chamber, we're simply just gonna open up our chamber and take our modified surface out and we can drive using the nitrogen gun. We place it in a clean, dry Petri dish again and go over and place it in an oven at a hundred degrees C for two more hours. So now that the samples have been baked in the oven at a hundred C for two hours, we're gonna take and attach our wires to the electrodes that we prepared.

We do so by making a one-to-one mixture of silver epoxy and a curing agent. So this is the silver epoxy, and now we add the same amount of curing into it. We wanna mix it well, and then we're gonna take this and use to attach our wires.

So we place our wire on the electrodes that we want to use and then we code it. Soldering does not work on ITO, hence we have to use silver epoxy and the curing agent. And then we bake so that all the curing agent that we used earlier is removed and it hardens and we can use it as an electrode.

So now that we finish two phases, we're gonna start the third phase of an experiment, which is performing the electrochemistry part. To do so, we're gonna take a sample and place it in our electrochemical cell. After placing it in our electrochemical cell, we're just gonna place an O-ring on top of it.

That's gonna isolate the region where electrochemistry is going to happen, and then we're just gonna seal the cell. So I have placed my sample with my wire attached in the electrochemical cell. Now I'm ready to use the potential stat to apply the voltage.

So the white wire, I'm gonna attach it right here to the reference electrode, the red one, the counter electrode, and then finally the green one that I'm gonna attach to our working electrode. Once all these electrodes are attached, I'm just gonna add one x solution of phosphate phosphate buffer saline or PBS to the electrochemical itself. Now just to clarify this, the way it's gonna work is the entire surface as of now, is modified with pxi.

So all the electrodes, including the surrounding glass regions, are modified with pxi. Once I apply the voltage everywhere, where ITO is present, which are the electrode designs that we made, the peg island is gonna come off only of those surface and it's nothing's gonna be affected in surrounding regions. Hence, we selectively remove peg island from our substrate.

So now I'm gonna press play and that will start and apply a voltage of negative 1.4 volts to our working electrode. When the voltage is applied, there is a change in color that you can see on the electrodes. It changes from the transparent greenish blue ting that we had to brown.

We suspect that there might be an oxidation reaction that might be occurring at the electrodes that causes them to turn brown. This is also one of the indicators that the pxi from the surface was indeed stripped from the surface. And once again, wherever you see brown, that's where peg island has been removed from.

And the surrounding glass regions still have PXI assembled on it. So we apply a voltage of negative 1.4 watts for 60 seconds. This gives enough time to remove all the pxi that were present on the electrodes.

We can also use this method to strip electrodes that are individually addressed. In this case, there were three rows of circles that are going to be stripped. We can select what, which ones we wanna strip by using all three different electrodes.

So now we have successfully finished our third part of electrochemistry where we've removed PG siling selectively from our ITO electrodes. I wash briefly washed it with PBS and DI water, removed the wire and placed the samples in just regular six well plates. Now we're gonna move On and cultured cells on these electrodes.

So now here we are in the tissue culture Room. For the final phase of the experiment, we're gonna use fibroblasts today, three T three cells. The concentration that we use to seed the cells is anywhere between 0.5 to 1.5 million cells per mil.

After seeding the cells, we let the cells onto our sample for approximately 20 minutes. That would allow it ample time to attach it to the stripped regions. So we'll place it in the incubator.

So now it has been 20 minutes and we're ready to look at the cells that have patterned on the stripped regions of ITO, the brown regions that we saw earlier, and they would only specifically bind to those regions and will not attach anywhere outside of those regions. Here you can see the brown regions were ITO regions that were stripped of pxi. By using electrochemistry, the cells have attached to only these brown regions and not the surrounding glass regions.

In this other frame, once again, we do see seltz attaching to the stripped regions. Those are the brown regions, but above those brown regions you can barely see and ITO design PEG was not stripped off of this ITO design, hence no cells attached to it. Okay, so that's it.

We're done with the protocol and cells have pattern on the ITO electrodes and that's exactly what we Wanted. I have Just shown you the controlling of cell adhesion on micro fabricated optically transparent indium 10 oxide electrodes. Today I showed you how to pattern THREET three fibroblasts on these electrodes.

We have also used the same procedure to pattern Hep G two cells, satellite cells, and rat primary cells. Current cell patterning techniques only allow the assembly of two cell types on the surface. The flexibility of this technique will allow the assembly of multiple cell types on the surface that are spatially isolated in the future.

We plan to use this technique to spatially isolate three different liver cell types on the ITO substrate in order to mimic the in vivo microenvironment. By doing this, we can spatially isolate immature cells or stem cells along with mature cells and supporting cells. This will create an instructive environment for the Immature cells to grow and differentiate.