La stimolazione meccanica delle cellule staminali Utilizzando ciclica deformazione uniassiale

Hi, I am Song Li.I'm an associate professor in Department of Bio Engineering at uc, Berkeley. And I'm Kyle Kapinsky. I'm a PhD candidate also in bioengineering at uc, Berkeley.

And today we're gonna present a protocol for mechanical stimulation of stem cells using cyclic UNI axial strain. The research in our lab focuses on cell and tissue engineering, especially in the cardiovascular system. And we're interested in the mechanical biology of stem cells because there are a few potential applications.

One is that we want to understand the effect of mechanical factors on development, for example, e embryonic development. And two, we're interested in controlling stem cell differentiation in vitro or in bowel react. And and thirdly, we're interested in how stem cells respond to the cardiovascular mechanical factors in vivo, for example, after transplantation.

So to investigate all these things, we have a custom designed bioreactor in which we can seed cells onto elastic membranes and then stretch these membranes ally for a defined set of time, defined strain, and a defined strain rate. And now we're gonna step you through all the the processes that you need to do to set up this protocol Using some lab soap, any detergent will do, get soap on both sides of the membrane and just give it a good scrub so that if there's any pieces of debris on the membrane, you're trying to clean that off. Anything that would interfere with the cells.

And I just do this by hand. Then once I feel like I've gotten both sides pretty well cleaned off, I rinse it off with water one more time. And from there I wanna make sure that it's really sterile and I get any micro particulates off.

So I'm gonna take this membrane and put it into a beaker with 70%alcohol. And ju just let this go in All the way. And from there you can take the whole beaker and put it in a sonicate and let it sonicate for 10 minutes.

So now that we have the micropattern membrane in a square dish, we wanna verify just to make sure that the pattern side is actually facing us. So I'm gonna take a spray bottle with alcohol and just spray the top side of this membrane. And you can see now the liquid is running down and you see those grooves.

So that does verify that the pattern side is facing up and we have it in the correct orientation. So now we've done the overnight UV step and you can see all the chambers and all the frames for The chambers and the membranes themselves are in the hood and they've been sitting in the hood in UV overnight. And this is our final sterilization step.

So now what I wanna do is plasma treat all the membranes and I'm gonna take one or two membranes out at a time and keep them in their sterile dishes and transport them to the plasma Machine. So now the vacuum pressure has released, I can remove the chamber. And again, this isn't completely sterile, but will u it again later.

So working relatively quickly so that the plasma stays on there, you wanna use forceps. And again, this is very critical. Make sure that you don't drop the membrane or change the orientation.

So right now, only the top surface has been plasma treated and I'm going to put it it back into my relatively sterile dish plasma side up, put the lid back on and then transport it to the hood. So now that the membrane's been plasma treated, I'm going to put it back into the hood and treat it with 2%gelatin. And that's weight per volume.

You can see that the solution is still going out in the in line with the pattern. And it's also very hydrophilic since it's been plasma treated. So the solution spreads out instead of beating up, if you accidentally flipped the membrane over, the solution would be right up and you'd know that you were doing the wrong side.

So make sure that the solution spreads out evenly. Then once you've done this for all your membranes, simply close the hood and turn on the UV for 30 minutes. So at this point, the membrane's been in UV with gelatin on top of the surface for half an hour, and that's long enough to get the gelatin absorbed to the surface.

So at this point I want to wash the membrane twice with sterile PBS and I usually put about 10 mils on in a square dish and just kind of wash around using my hand that way, try to avoid touching the surface of the membrane and then aspirate the PBS off. I'm gonna do this one more time, and after the second time I'm going to leave the membrane in PBS and that helps lubricate the membrane, which makes it easier to assemble into the chamber. If you leave a dry membrane, there's more likelihood that it's going to rip or break somehow.

So this is the second wash. Again, aspirate off one time to make sure that all the gelatin is off the membrane. And then leave your membrane in about 10 milliliters of sterile PBS.

And if you're working with more than one membrane, repeat this process for each one. And after that you'll be ready for assembly. Take one of the frames, and this is actually two pieces, but they're usually preassembled.

So one piece is the T piece here, and the other piece is part of the frame, which is going to hold the membrane itself. So there's a second piece to the frame which looks like this. And you can see it has this groove in it.

That's where the gasket will insert to hold the membrane in place. The second piece has the second groove for another gasket so that it will hold both sides of the membrane. So taking this piece, place it on the frame like this, flip the frame over and grab one of the autoclave screws.

Insert the screw in the top, you can tighten it by hand a little bit first, and then use one of the hex wrenches to tighten it up completely. So at this point I have an assembled frame and the membrane is going to go on the underside this way as if my hand was the membrane. And that means I want the the side that's either patterned and or plasma treated and gelatin treated.

I want that facing up that way. So if I accidentally flip it over and the gelatin side is now down, that's gonna be a major problem for the experiment. So when I assemble it, I want pattern side facing up this way, but I'm gonna assemble it upside down.

So when I take the membrane out, make sure that the pattern side goes face down. So taking one membrane, and remember this is pattern side up right now. I take it out of the PBS and I lay it across the frame like this.

So both sides are about even right now. And it's okay to touch the edges of the membrane with your fingers, but try not to touch the opposite side. So now that I've flipped it over this way, the pattern side is now facing down, which is what I want to make the assembly easier, take two gaskets and place it into the PBS.

I usually roll it back and forth. So I wet the whole gasket and this lubricates the gasket just like I lubricated the membrane in PBS. If you use dry gaskets in a dry membrane, it's a lot more likely that something's going to roll.

So the way I start this is to actually pull one side a little bit farther than the other side because when you insert the gasket, it's going, if I insert the gasket on this side, it's going to pull the membrane this way, and then this side becomes a little bit shorter. So start with sort of a shortened end here. Take one gasket, lay it across so that it's pretty even on both sides.

And using even pressure, press down, use your thumbs or the the tips of your thumbs, but not your filler nails at this point because you're more likely to accidentally cut into the membrane and rip it. So once I've got it inserted, I actually take the backside of my forceps and use this to press down. And again, be careful that you don't push real hard or slip and touch the membrane because you can accidentally cut it or rip it at this point and you'll have to get a new membrane and start over.

So now one side's inserted, but the other side is still loose. So flip it around, take the other gasket, make sure it's wet and place it on top just like before. Then I typically use the forceps to actually grab that membrane and stretch it out a little bit because if it's too loose, you're gonna have a lot of slack in the membrane and it's gonna be impossible to see the cells.

So you want it tau, but not overly taught so that it's stretched. If it's pre-stretch, it will affect the experiment because the membrane itself is already tensed up and you might rip it, you will add a little bit of tension as you press the gasket down. So at this point it looks pretty taut.

It's maybe a little bit loose on one side, but I'll fix that in a moment. And again, using pretty even pressure, just press in with the tips of your thumbs or your fingertips. And at this point you can see that one side is pretty taut.

It's straight all the way across the other side. There's a little bit of slack in the membrane right there, and it kind of curls up. So what I'm gonna do is I'm gonna press this side all the way in, just like before, do it slowly and evenly all the way across the gasket.

And you can see that took most of the slack out of it. So we just finished the 30 minute UV of the back sides of the frames and the membranes, and now we want to flip all the membranes over so that they can be uvd on the front side so that everything's completely sterile. And at this point, we're also going to designate certain chambers as either stretch or control.

So you can see I've actually already labeled my chambers going control stretch, control stretch. And the stretch chambers require one extra screw. So I've placed the extra screw for the stretch chamber on top of the underside of the lid here.

So just take the frame and flip it over. And if it's a stretch chamber, that means I need to change a couple of the screws. So if it's a control chamber, everything stays the same.

And when this bar moves back and forth, the membrane itself doesn't get stretched, it just moves with the bar. If I want the membrane to stretch, I need to fix the second part of the frame at this end into the bottom of the chamber so that it's stationary. And then when the bar moves only at this part of the frame, we'll move back and forth.

So to do that, take the additional screw and place it into the screw hole in this piece of the frame. Use the hex wrench and tighten the screw in. Don't overtight it because these poly su one chambers are a little bit fragile, so you don't want to crack a chamber just tight enough that that piece of frame isn't gonna move anywhere.

Then once you've got that first screw in, you can unscrew this screw, which was holding the two pieces of the frame together, and be careful not to drop the screw onto the membrane as it could mess up some of the gelatin that you seeded on there. So once I've got it pretty loose, get the hex wrench out, grab the screw with the forceps, and place it into the second screw hole. Then use the hex wrench again and tighten the second screw.

So once that piece of frame is secured with the two screws, now I have a chamber that's set up for stretch. So when this bar moves back and forth through the stretch machine, you can see the membrane itself is actually going to stretch. And I like to actually do this a few times to make sure that there's nothing wrong with my membrane.

If for some reason there was a crack and then the membrane rips, now I'm gonna know that before the experiment starts and I can put a different membrane in. So you move this off to the side, get your next one, and in this case it's labeled as a control chamber. So all I have to do for the control chamber is just flip it over.

Now when it's put into the machine, it will move back and forth without stretching. Then just repeat the process for however many chambers and membranes you have. Each one either is stretcher control, then close up the hood and UV the top sides of the membranes for 30 minutes.

So we just finished uv all the chambers and the top sides of the frames in the membranes for 30 minutes. So now everything's sterilized and everything's assembled and we're ready to seed the cells. In this case, I'm seeding mesenchymal stem cells and I'm taking them from 10 centimeter dishes.

So for each membrane, especially if I'm using a pattern membrane as in this case, you can sort of seed the square area where the pattern is. I wanna seed only on that patterned area. If you're using an un pattern membrane, you still wanna seed in approximately the same area, so as not to get the fluid too close to the edges.

If the fluid's too close to the edges, it can spill off and then your cells will die before you can really get them seeded on. And that area that you're seating in, you wanna put the appropriate amount of cells on. So when I do the experiment, I want it to be pretty confluent on that area.

So if I use a 10 centimeter dish, I want about a third of a confluent plate on that area. And in this case, for meson stem cells, that's about 250, 000 cells. It's gonna change depending upon what cell type you use.

So I've already sized my cells off of the plate and I've resuspended them in the appropriate amount of volume. And I wanna put one mill of solution of cell solution onto each membrane. So in this case, I have about 250, 000 mesenchymal stem cells per mill of solution.

So I use a P 1000, make sure that the solution is. So I pipe up and down a few times and get one mill in the pipette, and I wanna make sure that I seed only on that area. So I seed very slowly and I use little droplets on that patterned area.

And you can actually see that because this is a pattern, it makes little ovals in this direction because it's actually sensing those grooves. So they're not complete circles. In this case, they're more oval shaped.

And then I see all around this area, but not getting too close to the edges just yet. I don't want to go too far and I get all the solution out of my pipette. Then I use the pipette to actually spread the solution around close to the edges, trying not to go over.

I don't want any cells on the unpassed area, and it's a little hard to get the edge that's closest to you. So normally I carefully rotate this around and get that last edge. And this should spread the cells out pretty evenly.

And now they're all seated on. But you wanna be careful not to spill any of the cells off. So be careful not to pass your hand over and also not to move it too quickly.

And then I move it back out of the way. And I wanna cover this up and just lift the cells seed on top. So to cover this up, you use this lid and there's actually two different holes on the lid.

There's a deeper hole on this side and one that's more shallow on this side that lets gases in. You wanna make sure that the deeper one lines up with this T part here. Otherwise it'll be too tight if you use the shallow one.

So placing it on this way, that will cover up the whole chamber. Then you wanna repeat the process with all your remaining chambers, and you let this sit in the hood for 30 minutes so that the cells can settle to the bottom and start to the attach to the membranes. After that, we're gonna tape all the chambers and transfer them to the hood or to the incubator so that they can sit for another hour and completely see on the membranes.

Okay, so we just finished letting the cells sit on the membranes for half an hour. And now I want to expose the cells to the 37 degrees and the 5%CO2, that's in the incubator itself. So I need to transfer from the hood to the incubator.

And to do this, you just have to make sure that you're really, really careful and don't tilt the chambers in any direction or else the cell solution can slip off. And basically you're gonna have to start over again. So now that the cells have been in the chamber in the incubator for an hour, the cells are firmly attached onto the surface.

So at this point, I've taken the chamber back out of the incubator and carefully put it back into the hood, transporting it very carefully, just like before. So you don't spill any media off of the membrane at this point. You need to add 20 mils of media to the stretch chamber.

And in this case, I use a special combination of media because it's slightly prone to contamination, it's perfectly designed to keep contamination out. I'm using DM plus 10%FBS plus 1%penicillin, streptomycin, and then an additional 1%of fungi zone just to keep out fungus. So taking 20 mils of media, I'm going to carefully add this to the stretch chamber.

We can see that the media has not fallen off of the membrane itself and added to the end of the chamber here, so that the media goes under the membrane first and then over the top of the membrane. And do it slowly so that you're not completely disturbing the cells. Then cover it back up and you can firmly put that cover in place.

And at this point you don't have to worry about disturbing the cells or letting them fall off the membrane anymore. So you can handle it a little less carefully. You have to pick the appropriate amount of strain.

And the way that this machine works is that we have a rotating wheel to which an arm is attached, and this arm can change lengths depending upon the insert of the prefabricated piece that you put into the center of the wheel. So when it rotates, this arm moves back and forth, which then moves this platform back and forth. And the chambers are attached to that.

So they in turn move back and forth as well. So if I want to do 5%strain, I have a prefabricated piece that says five on it, and it's at a specific precalculated length to make this arm go to the correct position. So I insert that into the center of the wheel, then attach the arm, make sure it's firmly in place, and tighten that up using the hex wrench to get the final fastening.

So once this is all set into place, it's at a zero position, everything's locked in. Now we're ready to put the chambers in. So these are the chambers that I just transported.

They have the cells on top, carefully put it into a single position and make sure the bar locks into place. You wanna make sure that it's pushed this direction so that it's locked in its zero position. Don't push it all the way back, or you may pre-train the membrane.

Then lock it into place with this fastener here so that it doesn't move. And then you flip the switch to get it going. You could see there was a little jump at the beginning.

Sometimes that happens, it gets stuck, but then it usually starts going pretty smoothly after that. And all the bars are now moving back and forth and it's 60 cycles per second. So down here it says 600.

That should actually be 60.0. And you can change the frequency if you so desire. So once the cells have actually been mechanically stressed in your system, we typically do it for about two to five days, but you can do it shorter or longer if you want.

Once you're done and you have some sort of defined endpoint, you can take the chambers out and then use the cells on those membranes to investigate different things. So what we normally do is a variety of techniques, including microarray 2D gel electrophoresis. We also look for gene expression with PCR or western blotting for protein expression.

In addition to just lysing the cells, you can also stain them directly on the membrane looking for maybe immunofluorescence of particular proteins, or you can even release the cells from the membrane and then stain them and run them through a fax machine. So there's a lot of different things you can do, a lot of different things you can look for. And of course, there's also different variations of the machine if you wanted to use different types of micro pattern membranes or just different materials, different coatings, all sorts of things that you can do to modify this.