Hi, my name is Jager Davis and today I'll be showing you how to isolate human umbilical vein endothelial cells, also known as ic. We'll be using a variety of mediums, starting with Hank's balance salt solution, which will be used to clean our cells of any red blood cells or other contaminants that might be in the vein. Secondly, we'll be using collagenase resuspended in DPBS, and that solution will be used to dislodge our ubic from the vein and we'll be plating ourselves in 20%effect medium, which is M1 99 with heat in activated FBS and pen stripp solution.
We will also be supplementing our Resus suspension with endothelial cell growth supplement by BD Biosciences. Okay, I have a pair of dissecting scissors. I'm going to make a cut straight across the cord like this.
The next, I'm gonna go ahead and clean the cord up. So what I'm gonna do is identify the arteries and the vein. So there's two arteries and one vein.
I usually identify the arteries by the two. They're coming out toward you. So we have one right here and another artery right here.
And as you can see, the vein is actually going into the cord, and that's where we're gonna place our butterfly needle. We're using a 21 gauge and three quarter Accutane butterfly needle. So open it up.
Just gonna go in just like this. Again, I'm gonna identify the arteries. We have one artery and the second artery, and then there's our vein.
So I'm gonna take the butterfly needle and insert it into the vein very gently, but you may find some resistance. So I like to just twist the cord a little bit and then push the needle at the same time and kind of this kind of motion and just keep going until the wings of the butterfly needle are flush with the cord, just like that. And we're all set and go ahead and clamp it.
So what I'm going to do right now is fill two 20 mil syringes with 20 mils of inks. Next, I'm gonna fill a 50 mil conical vial with five mils of 20%effect. And next I'm going to pipette out 10 mils of DPBS to one of these 15 mil conical vials.
So this 15 mils of DPBS, I'm going to add 10 milligrams of our collagenase powder, which I'll measure out in a balance. And I'm going to resuspend the collagenase just by inverting the tube a few times until the powder off the walls of the tube. So about five or six times after that, I'm going to sterile filter the collagenous using a 10 mil syringe to add the filter.
And now we're ready to put these medias through the cord. So what I've done is I've poured the remaining DPPS into this one liter baker, and I'm going to place it into the 37 degree water bath and let it warm up. And this will be used to incubate our cord for about 15 minutes.
This is our filtered collagenase solution. I will be placing this conical vial into the 37 degree water bath, allowing it to warm up, and I'll be using this later on in the procedure to dislodge uve from the inside of our vein. So what I'm gonna do next is clamp the cord at the point of the butterfly needle, and I'm going to run 20 mils of hanks through the cord to flush out any blood cells.
After securing the butterfly needle, I'm going to push through 20 mils of our hanks. I'm gonna do this very slowly to prevent any breakage of the vein. And this is about the speed you want it to come out, but you don't wanna stream the hanks.
That means you're pushing it too hard. So I usually do it, you know, drop by drop. And again, I'm pushing the hanks through just to clean out the vein.
And you expose our end cells. So after that, we've pushed through all of our hangs. I'm going to go ahead and lay down the cord, and after that I'm going to clamp the lower end of the cord and remove the syringe, fix it into a biohazard bag.
Next thing, I'm going to attach a new 10 mil syringe. Okay, next I'm going to push collagenase through the cord, and this is to rinse out any residual hangs that might be left over in our vein. And I will push the collagenase through just enough.
So when it comes out of the end over here, we'll see it about the same color as what's in the syringe. And after that I'm going to clamp the lower end of the cord. What I'm gonna do next is I'm going to push through a little bit more collagenase just enough to where we can see the cord expand.
So after it's expanded a little bit, I'm going to go ahead and massage the cord, starting from left to right. I usually use three fingers by somewhat firm pressure. It's almost like rolling thread dough.
You don't want to go and push the cord too hard, but not too soft. If you push it too hard, you have, you're on the risk of smooth muscle contamination. But if you do it too gently, you won't dislodge any of the Q back.
So at about five to six seconds in one spot and kind of work your way down. So I'll go ahead and transfer the cord from the table into the 37 degree water bath, and I will let this sit for 15 minutes and incubate. So one lot you might have to incubate for 15 minutes.
Another lot may be 20 minutes, and a third lot might be 10 minutes. So it's important to optimize the time for incubation for our particular lot. Here is 15 minutes.
We'll go ahead and remove our cords from the DPVS bath. We're gonna lay it down here for a second. We remove the cap from our 15 mil conical vial containing our five mils of 20%feck.
The 20%feck has a trypsin inhibitor, so it'll stop the action of our collagenase. So I'm gonna go ahead and hold the cord above the tube with our scissors. And what I'm going to do now is cut along the line of our clamp.
You move the lower section here. Okay, so now we have a fresh cut at the end of our cord. And what I'm going to do next is push through our collagenase.
And again, just like with the hanks, you don't wanna push too fast. And this is about the speed that you wanna push your immediate through. And that's gonna be it for our collagenase.
Occasionally you'll find that the syringe is secured on a little bit too tight. So what I'll do in these situations is use the clamp that we had on our umbilical cord and basically just grab it right here and screw it gently and just enough to loosen and I can remove it with my hand or remove itself. So next I will take our remaining 20 mil syringe of hangs and attach it to our needle, and I'll push through the entire volume of hangs.
Again, I'm going to push very slowly, just like with the collagenase and the other syringe of hanks here it comes drop by drop. This is about the speed that you wanna push your media through. This contains 20%effect are hanks and our collagenase solution.
So what I'm going to do now is place it into a centrifuge and spin it for five minutes at 1200 RPMs. So I'm gonna double check to make sure the lid is secure. Our two, and I'm going to place it into our centrifuge.
We have spin for five minutes at 1200 RPMs. So we've spun our 50 mil conical vial for five minutes at 1200 RRP M.And what we're left with is a small pellet at the bottom of the tube. What this pellet consists of is some remaining red blood cells and our endothelial cells.
What I'll do next will aspirate the Bernet and reus, suspend our pellet, and five milliliters of 20%fed pipe petting gently up and down a couple of times until it appears that there's little debris matter in the suspension. And I'm going to pipe pet the entire five milliliters and plate it onto a T 25 fla. I'm try and be careful not to generate any bubbles.
And I'm going to add my ECPS. I'll be adding 50 microliters, and I'm going to just gently rock the flask just to make sure all the ECPS distributes to go in the class. And that's it.
Go ahead. The flask and incubated 37 degrees.
