A Gradient-generating Microfluidic Device for Cell Biology

We describe a protocol for the microfabrication of the gradient-generating microfluidic device that can generate spatial and temporal gradients in well-defined microenvironment. In this approach, the gradient-generating microfluidic device can be used to study directed cell migration, embryogenesis, wound healing, and cancer metastasis.

My name is Jiang. I'm a poster fellow at Harvard and MIT Health, science and Technology. And my name is Amir Manchi and I'm undergraduate student professor ha SANE lab in Harvard, MIT, division of Health Sciences and Technology.

This slide shows the scheme process of how to make the device so I just SUA 50 spin coating on top of silicone vapor and then put a mask further on place over top of a silicone vapor and then uuv exposure and develop. We can finally obtain, put these pad on the silicone vapor. After that we can just cast the PDMS and then crawling and baking and to little punch and then we can just bond deliver over bonding between the PDMS device and glace of straight using the oxygen plasma.

Okay, we are now in the microfabrication room inside the laboratory and we are gonna start by making some PDMS layers. What I mean by PDMS, the actual scientific term is polymethyl suboxane and it is an organic polymer. It is the most widely used silicon-based organic polymer.

It is actually a mixture of two different things is a mixture of silicon elastomer base and also silicon elastomer curing agent. So the ratio is 10 of base and one of curing agent. We need about 10 gram of base and so accordingly we need about one gram of elastomer curing agent and I'll try to mix this base and the curing agent wearable to each other.

I'll use pipettes. So now that the mixture is ready, I'm gonna actually pour it on top of the silicon wafer. So the idea is that these silicon wafers have some patterns on top of them.

They actually help this PDMS polymer to get the pattern and we use those patterns in order to have channels and grooves and or any other pattern that we need on those polymers. So the next step is since we have lots of bubbles inside this mixture, we are gonna try putting it inside a vacuum in order to avoid those bubbles inside our polymer. I'm gonna place the PDMS gels on the silicon wafer inside the vacuum chamber.

I'm gonna close the chamber and I'm gonna open up the vacuum. After the bubbles are gone, maybe about a couple of minutes, we're gonna place it inside the oven overnight, which is about 70 degrees Celsius and this will make them solidify. The pattern I want you to pay attention to is these three different reserv wars in order to make gradients so that we would have zero concentration on one side and we have a specific amount of concentration on this side and we would move uniformly towards that and we'll make a small punch on the other end, which would be our outlet in this case.

The next step is to cut one of these patterns and transfer into a patriot dish, having the pattern surfaces face up. So I hope that you can see the three inlet and one outlet that I explained to you on this patterns. We'll use small punches for our inlet in order to be able to use polyethylene tubing and I'm gonna use big punches for my outlet in order to have a reserve war.

So we're gonna start with the first inlet. I'm gonna make a punch, same thing with the second one and also another small punch for the third one. The last last step is to make a big punch at the outlet.

So once we are done, we are having A-P-D-M-S layer, which is the top layer and I'm gonna use another glass layer at the bottom. And these micro slides are gonna be my bottom layer at the micro folic devices. Now we're in the microfabrication room and I have one glass and one PDMS layer and I want to attach them to each other.

What we are gonna use now is a machine called plasma cleaner. What's gonna happen inside the chamber is that the plasma helps breaking down the weak surface balance and replacing them with highly reactive chemical groups and also the surface would actually turn out to be hydrophilic. What's gonna happen right now is that I'm gonna take the PDMS mold, which was the top layer and had some channels inside and I'll take out the tape that I put before in order to prevent dust attaching to our mold.

I'm gonna place it inside the chamber. The next thing to do is that I have a glass layer, which is a microscope slide, and this is going to be our bottom layer inside the device. So I'm gonna place this one inside the chamber as well.

And I close the chamber all the way once it's closed power first and then pump and then we have to come back to this machine at about five minutes from now. What I'll do is that I want to turn off the machine. So I turn off the pump first and the power next and I'll open up the chamber.

This sound is actually due to the negative pressure that's been applied during the process inside the chamber. So I'll take out the different layers and I want to attach them together. Since the glass is the bottom layer, I'll take it in my left hand and have it fixed and I'll take the PDMS layer and place it on top of my glass layer.

But since the, actually the patterns are faced up right now, I'll kind of invert the PDMS layer. I'll place it on the glass after pressing them together. They're very easily attached and part of the advantage of this plasma treatment process is the adhesive strength and the permanency.

That's how it look like at the end. So again, these are our inlets and this is in my outlet and we are ready to go on to the next step. The fibering cooking inside of the device and then for incubator for one hour and then have take out a SIM sample from the incubator after one hour and then put the fish culture food and then make the solution.

To make the EGF solution, we just put the two mill culture media as Azure as we just put the 15 nanogram per EGF and 10 micromolar 50 D run this solution very hard to visualize EGF gradient across the channel. Then we just take two mill media, virtual media and put here another two mill media. These two sittings only normal cultures, normal, normal culture infusion in the device.

So I remove the bubble, I connect again, this one is also M mobile bubble coming down again. And then I just use 15 nanogram er EGF and 10 micromolar fi dextra and two mil media from the takeout, the sample to and put the syringe and then you know the bubble. And then just solution switch, switch back.

Solution is come into the gas type syringe and push SH for several time to remove the bubble, the all switch syringe solution is getting here first and then switch the bar and the solution through the threeway bar and neither and tubing and the solution coming out the tubing. And then after the mixture, the solution is coming out, we can just insert the tubing into the My Predict device and then all three solutions is the same. You can just gently connect, connect the in net of the infusion channel.

So finally two is normal cultural media. One is cultural media with EGF and to confirm the gradient profile of EGF. This one is the cell in net, cell in net.

This will the cell outlet, so gradient generating using the three hub channel and then generate the grad inside the cell area. So normally what I want do we just do the cell seating, cell suspension, seating the outlet, and then we can just gently suction in net and then cell is flow through the cell area is automatically sitting and they settle down the coating the and then making sure you cell completely spreading. And after make, after making sure cell completely spreading, we can just, we can just start infuse and then study the cell migration using the radio generating device.