Een microfluïdische apparaat met Groove patronen voor het bestuderen van Cellular Gedrag

My name is Jiang, I'm a poster fellow Harvard and MIT, health, science and Technology. So my expertise in this lab, I can just generate novel my predict device to study cellular behavior. I can generate very novel gradient device as well as I can just also generate integrate to my predict device.

TC device can precisely manipulate the cell cell interaction and cell interaction as well as the cell solar factor contact. So using the TC system I can study to understand the the basic cell biology as well as apply some the development biology as well as some bio bio stem cell bioengineering. My name is Amir Manchi and I'm undergraduate student professor Haan lab in Harvard, MIT Division of Health Sciences and Technology.

And I've been working on micro fillic devices and we've been trying to as a proof of principle showing that microfluidic devices are great potential devices for culturing cells and do different studies such as toxicity studies and we've also tried to study and optimize the fluid characterization inside these devices as a function of flow rates, geometry and other different sort of parameters. Right now we are starting from the microfabrication room and what we are gonna do is that we're trying to make some microfluidic devices in order to make the micro folic devices to happen we need some silicon wafer patterns and we need some PDMS molds on top of them. This PDMS polymer is actually a mixture of two different things.

It is a mixture of silicon elastomer base and also silicon elastomer current agent. The way we mix these two is that we mix them at to a 10 to one ratio, so 10 of base and one of curing agent. Now I want about 20 gram of my base here.

I need also two gram of the curing agent and the curing agent is much less dense. It'll come much faster and I have to be a little bit careful. So it is about 22 now and I want to mix the base and the curing agent together.

So I'll use pipe as for doing that. I'll try to mix it as much as I can very well together. I want to pour this PDMS polymer on top of the silicon wafers that have actually pattern on top of them and the silicon wafer helps giving pattern to the PDMS molds.

You can actually notice that I have two silicon wafers right here and the reason is that one of them is for the top layer of our micro fillic device and the other one is for the bottom layer. We need two layers in micro fillic devices cause we are trying to have a channel inside and the whole story behind the micro fluidic devices is that we want to have flow inside those channels. So I need to pour this mixture on top of both the silicon wafers because I have lots of bubbles inside this mixture.

I want to take out these bubbles and what we do is that we use vacuum in order to take those bubbles out. We are back in the main laboratory area and as I told you before because inside the PDMS mixture there are lots of bubbles and we want to remove it and I'm gonna place the mixtures on top of the silicon wafers inside the vacuum chamber. I'm gonna close the chamber and I'm gonna open up the vacuum after this is done we are gonna take the PDMS molds inside an oven overnight in order to make them more solid.

Alright, we're in laboratory now and we want to assemble these micro folic devices. What we're gonna do is that we're gonna take the PDMS molds, which have been inside the incubator overnight and they have formed, they have formed the patterns which have been on the silicon wafers and I'm gonna use two different patterns. On the left hand side you can see the micro channel, which is gonna be the top layer and the right one you can see some green line patterns which are gonna be the bottom layer.

And this is, this will form the grooves inside the micro device. I'm starting with cutting these gels in order to be able to have the top layer so there's easily come apart from the silicon wafer. And what I'll do is that I'll transfer it into these Petra dish face up so that the patterns would be facing up.

And I'm gonna try the same thing for the grooves, which were the green line patterns. So these are gonna form the bottom layer. So once the gel is cut, I'm gonna transfer it into the Petra dish and in order to prevent dust on top of the patterns, I'm gonna tape them.

The next step is punching the ends of the channels in order to let cell and media flowing in and out. What I'm gonna do is that because I have a wide surface here and I can't see the patterns myself, I'm gonna use this in order to be able to see the channels and I'm gonna punch the both ends. So I'm gonna punch a big punch here so it has to be able to have a reserve order and on the other side I'm gonna punch a small hole in order to be able to use poly ethanol tubings at the other side.

We are now in the microfabrication room and the next step is to attach the two different surfaces that we have. In order to do that you're using this machine which is called plasma cleaner and the process is called plasma treatment. What happens inside this chamber is that we're gonna have a plasma environment and at the plasma surface interaction what's gonna happen is that the plasm is actually gonna break the weak surface balance and it's gonna replace it with highly reactive chemical groups.

So what's gonna happen now is that I'm gonna take out the tapes that it put here in order to prevent the dust and I am gonna put these molds inside the chamber. I'm gonna close this door all the way, power first and then pump and then we're ready to go. We'll pick it up in five to 10 minutes.

Now I want to turn it off so I turn off the pump and the power then and I'll open the chamber. Actually there's a negative pressure here so you might hear the sound. That's what, because of the negative pressure, so I slowly take my molds out.

Since we have both the pattern surfaces faced up, I'm gonna take the bottom layer, place it in my left hand and I'm gonna take the top layer, invert it and place it on top of my grooves and then push the layers on top of onto each other. Adhesive strength and permanency is the advantage of using this plasma treatment machine. And what you see here right now is that you can see actually the channel on the top layer and you can see the grooved patterns on the bottom layer and it's ready to go for the next step.

And then bring here this culture room, this culture food and then open door dish. And then we can just loading the fibronectin, take out the 60 microliter of fibroin and then loading into the device. And then to generate some flow inside of the device to to induce small coating the vibrate inside of channel we can just gently suction outlet.

After that we can just place on this device on the incubator and it's one hour After one Hour cooling the vibrating inside of the incubator. We just take out the dis sample from the incubator and then we just use five rust, three, three fiber rust. We just send fusion and dis shade and then put the media and then we we just also cell county using the cell counter.

We usually the the city million cells premier sitting inside the device. So after the dissociation we can just one a couple times push shampoo and then take out the sales suspension and then loading the inside of the channel to better to loading the cell inside of the channel. You can just gently flow using the aspirate.

The sum media and the surface suspension aspirate through the outlet cell is generally automatically, you know, seeding the selective, the global channel. After that we can just disassemble put the incubator for one hour, it's until the cell is completely spreading inside of the channel. And then we can just infuse annexing five and the prop iodide as well as hydro peroxide.

And then study your time hypothesis assay. We can just take the sample inside of a tissue culture food and then we can just make the solution two mill DM DMM media as well as 20 microliter annex five 40 microliter prop iodine, 100 milli hydrogen peroxide. And then take the two meter media media with annex five, prop iodide, hydro perside, and then put the syringe after.

Put the two mill media and annex five prop iodide, hydro perside. We can just put the solution inside of the ceilings and then remove the bubble and then connect the C bear. This one is gas tight Hamilton warm mill ceilings.

This one is three bear. This one is disposable ceiling ceilings. This one is 27 gauge needle.

This one is PE 20 poly tubing. After loading the solution inside of a syringe, you can just couple time full and push full and push full and push and then sometimes some tip, tap some syringe and then remove the bubble and also pull the solution. So one more time, push completely, remove the bubble and then gently program again.

So take the solution for one meal and then we can switch the valve. Push, push the solution through the threeway valve and the fully in the tubing. So finally solution, come out through the tip of our tubing and then we can just connect the tubing into the micro device and start infuse one micro per minute.