脑片的刺激，利用微流控网络和标准灌注腔

The main objective of the project that we are going to show you today is to be able to spatially and temporarily control the brain slash stimulation. And another major point is that using a simple modification in the already complicated electrophysiological setup, we can disseminate this microfluidic device among the various labs that actually use this brain life stimulation. Hello, I'm Java Chek Mohammad with the Edington lab in the Department of Bioengineering at University of Illinois at Chicago.

Today I'm going to show you how we are going to apply a simple micro fabricated microfluidic device for brain slice stimulation. The brain slice device that we are going to demonstrate today is very useful because of several reasons and the main reasons are it's so modular that it can fit into the existing electrophysiological setup without any new further modifications. And it avoids the use of tubings and pumpings, which complicates any microfluidic device.

And since we are using the passive pumping method, we do not need any tubings or pumps. And the third thing is that it's just a thin sheet of PDMS membrane that goes in between a cover slip and a standard profusion chamber, which is used in a standard electrophysiology setup. The procedure that I'm going to demonstrate today starts with a a C eight master, which is fabricated using standard SV eight lithography process.

And the master that we have today here is a two level master with one level having the design of the channels, the micro channels, and another design for VR openings that go on top of these channels so that the solutions that are flowing through the channels can come out of the via openings. So we make this master, which is on a silicone wafer, and then we use silicone or the PDMS to pour on the top of this device and mold the structures that are on top of this wafer onto the PDMS. So we are going to make the PDMS pour on top of this device.

And then we are going to take the PDMS membrane that's formed after curing and bonded to a cover slip. And then we have our microfluidic device. And then we are going to modify a standard profusion chamber by quoting a thin layer of PDMS on the bottom of the perfusion chamber.

And once we have this modified perfusion chamber, we are going to take the microfluidic device that we fabricated earlier and bond it to the perfusion chamber. There are two critical points in the fabrication of the microfluidic device. One is that before we put the PDMS, the hot plate should be turned off so that the PDMS doesn't queue instantly when we pour it on the wafer.

And another critical point is the spacers that we use at the four corners of the silicon wafer should have a height that is less than the tallest structure on the silicon wafer. This is to make sure that the VR openings can be obtained when we do the curing Process for the PDMS. Here we Are in this soft wall modular clean room and this is the master that we fabricated using standard a C eight lithography process.

And it is a two level master with the first level having the design for the channels with four inlets and one outlet. And in the middle region of all the four channels, there are vias or pos that are standing on top of the channels. And these are the alignment marks that were created during the fabrication process.

And now I'm going to show you how I'm gonna remove it and do the PDMS curing. Due to the edge bead effect, the thickness in the outer periphery of the wafer is more than the actual devices in the center of the paper. So what we are going to do is remove this alignment marks using the reserv blade and then to accommodate the weights, we are going to use these spacers which are 140 micron height and we are going to put them in four corners of the wafer.

So you need to make sure that the razor blade doesn't reach near the actual devices. Now I am going to remove this SVA particles using the air duster and the wafer is now ready for the PDMS mold preparation. But before doing that, we need to put this spacers which are 140 micron tall.

So I am going to place the four tapes at the four corners of the wafer. Then I'm going to make sure that the tape is adhering to the wafer properly. So the reason why the tape was 140 micron high is that the two level master with the channels and the VS is 150 micron tall.

And when we place the weight on top of the PDMS during the PDMS curing, we want to make sure that the PDMS sheet comes out flat. So we are using four spacers on the corners with equal heights. Now I'm going to show how we are going to code PDMS and cure it to make the devices.

But before I do that, let me show you how we prepared the PDMS solution. So we take 10 parts of the base and one part of the curing agent and mix it thoroughly due due to the mixing process. You see that we generate lots of bubbles in the PDMS solution to remove this bubbles, we are going to put the PDMS in the desiccate after putting in the, after putting the PDMS in the desiccate for 10 minutes, there are no bubbles.

And now the PDMS is ready to be used for the curing. Now I'm going to show you how we are going to put the PDMS on the wafer and cure it to obtain the PDMS membrane. So I'm going to put the PDMS on the wafer and then use this transparency to cover the PDMS.

And this will help us to remove the device sup, separate the device from the transparency, and then I'm going to place the weights on top of the transparency. And the reason why we are using the weights is to get uniform thickness of the PDMS. And another critical issue is to obtain the via openings.

So wherever there are vs, we want no PDMS and the weights will help us to do that. As you can see here, the hot plate is turned off and the reason for that is we don't want the PDMS to cure instantly, so we let it be off. And then you need to make sure that the wafer is flat before you port the PDMS.

And now we can port the PDMS that we prepared earlier. Make sure you dispense the PDMS close enough to the wafer so you don't generate bubbles. And the most critical point here is the way you put the transparency on top of the PDMS.

You need to make sure that you do not generate bubbles due to the placing of the sheet. And now I'm going to place one of this glass labs and let the PDMS excess PDMS get out of the way. I am placing three more glass labs and to make sure that the glass labs don't move for this particular master.

And for these particular features, we found out that placing this four glass labs makes the VS.And to let the PDMS excess PDMS come out of the way between the transparency and the wafer. We wait for one to two minutes and then we can start the hot plate for different masters and for different designs, you might need to increase the number of glass labs or decrease the glass labs in order to get the via openings. Now the hot plate is ready to be turned on and I'm going to set a temperature of 75 degrees.

And once the 75 degrees temperature reaches, then we can set the timer for one hour and let the PDMS cure. Now that we have left the PDMS to cure for one hour, we can turn the hot plate off and let it cool down to 50 degrees centigrade. The reason for doing this is so that the SEA doesn't crack.

If we remove it immediately from 75 degrees to room temperature, now we can turn it off and wait for it to come down to 50 degrees centigrade. Now that the hot plate temperature has come down to 50 degrees centigrade, we can remove the weights from the transparency. Now we can remove the wafer from the hot player and it's ready To be cut.

Now the PDMS sheet is ready to be removed from the a C eight master and we need to remove the aluminum foil. Then we need to remove the transparency sheet. We have here the standard perfusion chamber.

We modified it so that we can align it. The holes, inlet and outlet ports of the microfluidic device. So these are the four inlet ports and one outlet port on the PDMS membrane.

And these should match with the four inlets and one outlet on the perfusion chamber. Now I'm going to slide the perfusion chamber and then align it with the holes on the PDMS membrane. Now that they're aligned, it's ready to be cut.

Now you can remove the chamber out and using a probe. Make sure that all the edges of the PDMS membrane are free to be removed. Once we do that, we can use a freezer to remove the PDMS membrane and make sure when you're removing the PDMS from top of the device, you move the PDMS real slowly so you don't tear PDMS membrane.

Now I'm going to place the PDMS membrane with the cavities that are formed with the SC eight masters on top and make sure that it's flat. And the reason why we are doing this is so when we make the holes, the inlet and outlet ports, the PDMS is not rough on the side. That is going to be bonded with the cover slip.

So we need to make sure that the cavities are on the top side. Now we can make the inlet and outlet ports just so we can see the holes clearly. We put a black background.

Now I'm going to make the inlet and outlet ports need to make sure that there is no PDMS left at the port. So is itwe, remove any PDMS. Now we are going to transfer the PDMS membrane onto another transparency sheet.

So we can treat the sheet and cover slip with plasma and laying it down so that again, the cavities are still on the top. So once this surface is treated with plasma and the cover slip is treated with plasma, we can bring these two surfaces together to form the microfluidic network to remove the air bubbles between the PDMS sheet. And the transparency can use a scotch tape.

So this will ensure that the PDMS sheet is flat and the bonding occurs much better. And then again, use the scotch tape on top surface to remove any dust from the PDMS membrane. Now we can put the cover slip that we will bond with the PDMS membrane.

And now these two PDMS and the glass are ready to be plasma treated. We are going to plasma treat the PDMS and the cover slip using this microwave modified plasma system where you can see this glass chamber allows us to create the plasma inside this microwave. And let me place the samples inside creating vacuum inside the chamber.

Now I can flow in oxygen. Now the plasma system is ready to go and I'm going to use 10%power and 10 seconds for this particular system to help visualize the plasma. While the plasma treatment is going on, we crank, cranked up the power to a hundred percent and we turned off the lights.

And now you can see the plasma Immediately after the plasma treatment. You need to bond the both surfaces, the cow slip and the PDMS or else the PDMS surface will lose its hydrophilicity due to the plasma treatment. Once you place the cow slip, make sure the whole surface is bonded together without any air bubbles.

If there are any air bubbles, you can remove it. Now you set it aside for five minutes and let it bond. So here we have the modified perfusion chamber where we modified the base of the chamber with PDMS.

And this was done earlier. So we placed a transparency on hot plate on a hot plate that was turned off. And then we poured the P-D-M-S-P-D-M-S that was prepared in a similar manner as shown earlier.

And then we placed the perfusion chamber on top of the PDMS and then placed a single weight as shown here and we let it cure for 30 minutes or 75 degrees centigrade. And now I'm going to show while we are waiting for the PDMS and the cover slip bonding to occur, we can go ahead and prepare the chamber. So we can bond the chamber and the microfluidic device.

So we need to remove excess PDMS from wherever we don't need it. Remove the transparency then PDMS, Then we need to remove the PDMS from the inlet and outlet ports. Now the chamber is ready to be bonded with the microfluidic device that we prepared earlier.

Now that we have waited for five minutes, the microfluidic device is ready to be separated from the transparency, but you need to make sure that you remove the transparency at a slow rate. So the PDMS and cover slip, they do not separate out. Now that the perfusion chamber is ready, we need to plasma treat the bottom surface of the chamber where we just quoted the PDMS and the top surface of the microfluidic device that has the PDMS on it.

So now we are going to put both the, both these pieces into the plasma chamber and do the plasma treatment at 10%power for 10 seconds. Now both the surfaces have been treated with the plasma and they're ready to be bonded. Before you bonded, you need to make sure that the inlet and outlet ports on the chamber align with the microfluidic device.

And since the features that were we are aligning are large enough, we don't need any special equipment and we can do that with naked eye. Once you align and place on top of the chamber, make sure that all the surfaces are in contact and then you can let it sit for five minutes. So the bonding is good enough for the experiments after waiting for five minutes.

Now that the microfluidic device has bonded with the chamber, we are going to make the channels in the channel surface hydrophilic. So when we actually flow the A CFS solution or any other neurotransmitters, the solutions can flow through more easily. So what I'm going to do is put this whole device in the plasma and plasma, treat it for one minute at 10%so the complete inner channel surfaces become hydrophilic.

And then I'm going to fill it up with water and then take it to the electrophysiology setup so we can do the actual experiments. Okay, now we are ready to go to the electrophysiology setup and actually use this device with the brain slice. Hi, my name Is uo.

I'm working with Dr.Arrington and Dr.Fall, the Department of Bioengineering at the University of Illinois. And now I'm going to work with the micro free device that brought over and just ate over the, over this platform. On one side, we can see the profusion tubing.

At the other side, we can see the suction tubing. So the device is going to be perfused with a CSF solution, which is bubbled with 95%of oxygen and 5%of CO2. Now that the micro V device is ready, I'm going to get the brain license and then I'm going to put them over the micro device.

Now I'm going to put the brain slice, eh, on the top of the circular openings, and then I'm going to use the anchor in order to immobilize the brain slice. Now that the brain slice are being immobilized, I'm going to use eh neurotransmitters in order to stimulate it. Right here we have four inlets, so I'm going to put neurotransmitter in each inlet by using the passive pumping from this four inlets until this outlet.

And now Dr.Fall is going to talk about this device. What is so important for, for Us? Hi, I'm Chris Fall and we do brain physiology.

And the reason that we need to use brain slices is for access with micro electrodes and imaging technology. And up until now, the only way that we could change the neurotransmitter environment for these brain slices is to change the flow over the whole slice or to puff neurotransmitters directly on using a very small micro pipette. So we're really thrilled to be working with Eddington's Group.

This new technology will allow us to address large areas of the brain and change locally, the neurotransmitter environment. And then simultaneously we can now go in with our, with our electrodes and our imaging technology and perhaps eventually build multi electrode recording devices into the same unit. Okay, so here you see a, a slice of mouse sprain in the microfluidic device.

And you can notice even by eye that the brain is comprised of many different regions. These regions do different things. And in the alive animal, these different regions of the brain would have different neuromodulatory and neurotransmitter tones.

And we want to be able to replicate this in our slice chamber while we're making electrophysiological and and imaging recordings. And so it's really super to be able to address these different areas with different neurotransmitters in different concentrations and with different time courses. And while we can't really visualize neurotransmitters, we can show you an example here of fluorescent dye that's being pumped into the brain slice in different regions.

Here you see a movie where we're flowing fluorescent dye through the channels of the device and just visualizing it coming out of the pores that have been made. And even in this early prototype, you can see how precise the spatial resolution is with the flow. As we watch the movie, you'll see the dye flowing into the channels and then out of the holes, and then we'll flush that dye out, giving you some idea of what the temporal resolution is of the flow.

Thanks for joining us today. I think that we've been able to demonstrate how microfluidics and, and indeed micro machines can be married to traditional physiological techniques in order to help us understand how the brain works.Thanks.