I microchip di silicio per la manipolazione di interazione cellula-cellula

Hi, I am Elliot Hu.I'm a postdoctoral fellow in Tia's lab here at the Massachusetts Institute of Technology. Today we're gonna prepare cell cultures on silicon micro mechanical chips. The devices look like little combs that lock together and they allow us to precisely manipulate the interactions between two different populations of cells.

Today we're gonna study the interaction between liver hepatocytes and supportive stromal cells. Specifically three T three fibroblasts cells are cultured on the top surface of the combs, so that by shifting the comb fingers, you can move the cells and change their spatial arrangement. Each device is divided into two parts which snap together in two possible configurations.

The first brings the two cell populations into contact with each other. The second separates the two populations slightly so that the cells cannot touch. Here, contact interactions are prevented, but interactions through secreted Factors are preserved.

Let's Start by talking About how to handle the parts with tweezers. I like to use both larger Teflon tweezers as well as smaller round tip metal tweezers. With the larger tweezers, you can pick up the parts on the edges like this with the bigger parts with the arms, you have to be careful not to break the arms because they're very fragile.

So you wanna pick them up at the back of the part like this. With the metal tweezers, you can reach into the hole and pick up the chips directly. The larger parts will fit into a wall of a 12 well plate, whereas the smaller parts will fit into either a 24 well plate or can be paired with the larger part.

In a 12 well plate, usually I use the metal tweezer. When I handle the parts in the plates to snap two parts together, usually I put the larger part into the well first and push it all the way to one side. Then I take the complimentary repair and put it on the other side of the well, and I pushed them together by taking two tweezers one into each hole.

So now I can just lock the parts together, either in the gap configuration or the contact configuration. If I want to take the parts apart, I like to pull the parts to the gap configuration and then pull up vertically To remove the part. Now the devices are Made outta silicon, so they need to be coated to allow proper cell adhesion.

You're probably gonna receive the devices already coated with polystyrene and plasma treated, so we'll assume that that's our starting point. First of all, we're gonna use collagen to coat the surface that the hepatocytes will attach to. We're gonna put the hepatocyte combs in a 50 microgram per mill collagen, one solution, and incubate at 37 C for 45 minutes.

The fibroblast combs on the other hand, we do not need to coat. After incubation, we aspirate the collagen solution and wash and water. Now we are gonna lock the ES together in contact with complimentary parts so that we form a flat surface for cell seeding.

First, we'll fill some wells with 70%ethanol. I'll put a pair in each well and then lock them together. After locking together, we wanna look at the parts under the microscope to make sure there are coplanar.

Once in a while the parts can lock together misaligned. And on the microscope you'll see that they're on two different focal planes. In this case, simply separate the parts and push them back together After checking that alignment is fine, we let the parts sit in ethanol for a while to sterilize.

Often I'm in a hurry, so I just soak for 10 minutes. After sterilization, we need to rinse away. The ethanol thoroughly aspirate and wash in water, then aspirate and wash in water again, and finally aspirate the water and replace With your culture media.

Now let's Seed Cells Onto the devices. Typically, we suspend the cells at a concentration of 500, 000 cells per milliliter, and we pipette one milliliter suspension over each comb pair using a 12 well plate. So we seeded the fibroblasts at 500, 000 cells per milliliter in two of the wells.

Similarly, we took a suspension of 500, 000 hepatocytes per well and seeded them in the other two wells. Now, before incubating, we're going to sh shake up the cells thoroughly to make sure they're evenly distributed. And I like to shake three times vertically, three times horizontally, and then repeat this three times.

After shaking, we're gonna place it in the incubator and we're gonna let the cells sit for 15 minutes, at which point we're going to shake them up again. After shaking every 15 minutes for one hour, we're going to then aspirate off the cell suspension and play on a new suspension of cells. And we're gonna repeat this until we have a confluent layer of cells.

Typically with fibroblasts, it takes one or two seatings. And with hepatocytes it can take two to four seatings. To get a confluent monolayer, the cells are seated at a fairly high density, shaking, ensures that the cells are distributed evenly.

After the first seating, a sparse layer of cells is formed. Notice that cells only attach to the fingers that have been coated with polystyrene and collagen. After seeding fresh cells and incubating for another hour, again with shaking every 15 minutes, the cell layer grows denser.

After the third seeding, we have a good cell density, and now we can Stop after the cells Have been seeded onto the combs. We wanna manipulate the cells into the desired experimental configuration. Devices are now separated from their complementary parts and incubated for 24 hours.

After 24 hours, the cells have spread to form a confluent monolayer over the surface of the combs. Now we wanna form a co-culture. Hepatocyte comb and fibroblast comb are now transferred into the same well and locked together into the desired initial configuration.

If desired. After a certain amount of time, you may change the configuration. For example, we can move to the gap configuration after 18 hours, parts are placed into the same, well pushed together until the gap configuration is reached and then pushed into contact back into the gap configuration.

And now we're gonna Remove one comb. What we're gonna do now Is we're going to go through the process of stripping off the old coating of polystyrene, cleaning the chips, and then putting on a new polystyrene coating and preparing it for cell culture. Again, by stripping off the old polystyrene coating and putting on a fresh coat, we ensure that none of the history of the previous experiment will interfere with a new experiment.

So after you're done with your experiment, first thing you wanna do is bleach your cells and then rinse off your combs and water. So before you put the chips into the toing, you wanna make sure that they're completely dry. So here we have a few combs that I've just left out to air dry for a while and checking that it's completely dry.

And so now we are going to take some toing and put it into this glass speaker. Going to use a glass pipette here so that we don't dissolve it. Halloween dissolves plastic, so we can't use a typical polystyrene pipette.

Okay, that's about enough. And now we'll just take a couple of these parts, place them into the toing, and I'm going to turn the shaker on to agitate and we'll cover it with aluminum foil to keep the toluene from evaporating. So after two hours, the toluene should have dissolved most of the polystyrene on the combs, and we can just pull them out and air dry the combes.

After the Halloween rinse, the parts should be relatively free of polystyrene and should be relatively clean. However, we need the parts to be extremely clean so that the new layer of polystyrene will form good adhesion if they're not extremely clean. The poly ty tends to peel off in the middle of the experiment when the cells start pulling on it.

So what we're gonna do now is a piranha acid clean. I've put the parts into a glass speaker and we're going to heat this up. So we're gonna put on a hot plate.

And here we have sulfuric acid and hydrogen peroxide. I'm gonna pour the sulfuric acid first, and here, make sure that all the chips are immersed underneath the fluid. Sometimes they have a tendency to float.

And also now that we're working with pretty corrosive chemicals, make sure that you're wearing the right protective gear.Here. I'm wearing nitrile gloves. Now we're gonna pour the hydrogen peroxide into the acid and just watch out here because it is an exothermic reaction.

So you can see it's smoking a little bit as it reacts, but we want to add some energy into the system to make it even more reactive. So now we're going to heat it up to 120 degrees Celsius about at one 20 now, so you can rest assured that any remnants of your cell culture are going to be completely cleaned off of your chips. So at this point, you want to just let the reaction run its full course until all of the hydrogen peroxide is consumed.

And at that point, the mixture is gonna stop bubbling. So the reaction takes about half an hour to run its course. And after you stop seeing the bubbles, you can just turn off the hot plate and let it cool down.

And we're gonna transfer them into a BAK curve water. And once again, make sure you're using Teflon tweezers here, not metal, and you can just pick them up, transfer them over. So now we're just going to rinse it under a steady stream of DDH two O.I usually take it straight from the Meine and we'll leave it running for at least 10 minutes here.

And by doing so, we will, we're gonna wash off all the traces of acid after mincing under a stream of water for 10 minutes. The acid should be re removed from the chips, and we're gonna just store them underwater until the time when we're ready to coat the polystyrene 45, 000 Molecular weight polystyrene that we got from Sigma. And we're gonna weigh out a small amount of it here.

We have 400 milligrams and we're gonna dissolve it in toluene at 100 milligrams per milliliter. So toluene needs to be handled in the hood. And the other thing is that since we're gonna use a toluene to dissolve polystyrene, we wanna make sure that we don't use any lab wear that is made from polystyrene.

So here we're using polypropylene, conical and a glass pipette. So since I have 400 milligrams of polystyrene, I am going to put in four milliliters of of toing, And now we're going to vortex it for half an hour until the poly styrine dissolves. So now we're just gonna vortex the tube at the lowest speed for about 20 minutes to half an hour.

So I'm going to attach the tube to the vortexes. I don't have to hold it there the whole time. After 20 to 30 minutes, the polystyrene should be dissolved and we're ready to coat it.

Now we're gonna spin coat the polystyrene in our facility. Our spin coder is in a clean room facility. And so here we have to wear a cap goggles, a gown, booties, and gloves, but that might not be the same in your facility.

Before we use this chuck, I wanna protect it from the polystyrene. So we're gonna cover it in aluminum foil, and we wanna get this as flat as possible to the surface so that the chip can sit flush against the surface. We're gonna poke a small hole right in the center so that we can keep a vacuum on the chip.

The spin coder is set to 2, 400 RPM and 30 seconds. Now we can take one of our chips, place it so the center of the chip covers up the hole, and I'm gonna have the vacuum off at first when I put the polystyrene on, turn the vacuum on, and then start the spin. So for the smaller parts, we can put down just less than 10 drops.

And for the larger parts, it takes a little more than 10 drops of polystyrene. And we're gonna spin it for 30 seconds at 2, 400 RPM. Now we're gonna take the poly styrene coated chips and put them in the in the oven at 120 degrees C.That's above the glass transition point of the polystyrene.

So it's gonna reflow the surface to smooth it out a little bit, and it's also going to densify and harden the plastic. So usually I leave it in the oven for overnight. It probably takes at least a few hours for the process to complete.

So after an overnight bake, the chips are ready for plasma treatment. Now we're gonna expose the polystyrene to an oxygen plasma, and that's gonna change the surface energy so that proteins and cells will stick to it better. So we're just gonna load the chips into the plasma chamber and pump it down to a vacuum.

A good setting to use is 200 mil, tour of vacuum and 200 watts of power for one minute under a flow of oxygen gas. Now that the system is pumped down, we're gonna introduce the oxygen. Okay, now we can turn the RF power on, strike the plasma for one minute.

So when the plasma's running, it actually glows. It's like a fluorescent light bulb. After one minute of plasma exposure, we're going to bring the pressure back up the atmospheric and we can pull the parts out.

Now the parts are ready for protein coating and cell feeding. The goal in designing this device was for us to be able to manipulate the micro architecture of tissue in a dynamic fashion. So the micro organization of tissue in the body, specifically where the cells are placed in respect to one another, is very important in determining the function of each particular cell in laboratory culture.

Until recently, we haven't been able to do a good job in replicating that, but over the past five or 10 years, people have started being able to micro pattern cells onto a tissue culture substrate, thereby putting cells exactly where we want to have them in a tissue culture plate. And in doing so, have been able to discover some important aspects of the basic biology of cell cell interactions at the micro scale. Now, what we haven't been able to do up to this point is to do that dynamically.

That is to change the organization of a cell culture in the middle of your, of the experiment. And that's important because in the body, so the tissue is not actually a static environment. We have cells that move around and reorganize, particularly in wound healing or development.

But even in a, a normal organ in homeostasis, there's A lot of stuff moving around. The Most difficult technical aspect to learn is just how to physically ate the parts. And initially when you start working with the system, you may be intimidated by how fragile the parts are or how small the emotions you need to make are.

But I think that if you just practice with it for a little while, you Shouldn't have too much trouble. Some of the places Where we see this device having a role, for example, in in embryonic development, cells will come into contact with a series of different cell environments as the cells are budding through various embryonic layers. And it's known that the interactions with each of the layers as they come into contact sequentially, push us all further along a specific differentiation pathway.

And so we think that this type of device might be good for trying to replicate that type of event In the laboratory. One of the aspects Of micro organization that we particularly wanted to look at was a difference between cells communicating through contact interactions where the cell membranes can touch up against each other, versus soluble factors that are secreted into the surrounding media that diffuse over to a cell that's a little bit farther away. And a lot of times when cells are in co-culture and they have an effect on each other, it hasn't been clear whether or not these are contact interactions or secreted factors That are playing the role.