Hi, My name is Greg Zod. I'm at UCSF Diabetes Center at the Bluestone Laboratory, and today I'm gonna show you how to transplant mirroring eyelets under the kidney capsule of diabetic mice. This procedure involves taking eyelets that have been purified from either syngeneic or allogeneic strain of mice, taking those eyelets, purifying them, and transplanting em under the kidney capsule of diabetic mice.
Mice that could be naturally diabetic like the NOD or mice that are made diabetic artificially with the injection Of strep pitocin. Now we're Ready to handpick the eyelets. I'm going to handpick the eyelets and count them while I'm sucking them up with a pasture with a pipette.
I'm gonna leave behind any a in our tissue, and you can see just by the, the, the way the lights refracted in the tissue, what is an eyelet and what's not an eyelet. And then I count the eyelets. I'll put them in a micro centri tube and, and we'll collect them for transplant.
The eyelets have a more dense, almost reddish look to them, and the ace in R is lighter and fluffier. And it also has like a whitish look. So we, you're going to get some a in r tissue while you do the eyelets, but as long as you grab a majority of eyelets, you'll be okay.
And you can see there's a nice big piece of a in R tissue. And then here's a nice eyelet with another eyelet above it. And then, but there's some ace in r in between you can kind of move things around and capture the individual eyelets.
And if you notice, I'm using a non conical pipette tip. This tip is very thin walled and it's straight. This allows for greater ease of the eyelets to go in and out of the tip.
Be careful not to try to grab a, a lymph node. These are esoteric lymph nodes that are pancreatic lymph nodes that get digested out and purified out. I only try to grab the eyelets if I can and leave the lymph nodes behind.
Once I've hand picked the eyelets, I have 500 I free eyelets in the bottom of this micro centrifuge tube. Now I'm gonna transfer these eyelets into our P one 50 tubing for transplant. And how I do that is I take a pipette tip and I slowly aspirate the eyelets off the bottom of the tube.
I, once the eyelets are in the pipette tip, I take my silicone tubing attached to the B 50 tubing, and I will let them settle now in the tip of the pipette by taping them to the side of the wall of the hood. Once the pellets have settled it within the pipette tip, we'll remove the pipette and slowly advance the pellet into the tubing by using the dial on the pipette, not the plunger. And as I dial the pipette, I move the tubing.
They allow the pellets to enter the P 50 tubing. When the pellets had moved to about an inch from the end of the tubing, I kinked the tubing to keep the pellets in there. I removed the silicone tubing and transferred to the kink.
This is then placed into a centrifuge tube with a five mill pipette that's been cut. And then I fold over the tubing and tape it to the five mill pipette. And now it's ready for Centrifusion.
The mouse has been shaved and anesthetized, And we're gonna check to make sure that it doesn't respond to toe pinch. And I'm gonna clean the, the mouse with propone iodine swabs. The surgery is done aseptically, but all the tips of my instruments are sterile.
Just to the right of the spleen on the left flank of the mouse, I'm going to make a small incision. Now the idea is to keep that incision small so that it holds the, the kidney up. When you isolate the kidney, it will now cut through the peritoneum wall right above that kidney, making a small incision.
And with one move, I lift the skin to the left and push up, push down on the kidney and pop it out just like that. I keep the kidney moist, a little saline and gentamycin an antibiotic. With a 25 gauge needle, I'm going to make a small nick in the kidney for where my tubing's gonna be placed.
You don't want to gouge the kidney, you just wanna make a small nick. You'll see a little line of blood. Then you know you've, You're into the capsule.
Taking my P 50 tubing that's been cut on a bevel, I'm going to catch underneath the lip of that cut that I made and slowly place the tubing under the capsule. Pavel keeps the kidney moist during this procedure because a dry capsule will tear easily. Once the P 50 tubing is underneath the capsule, Pavo will advance the pellet using a Hamilton syringe with the screw drive.
And you can see the pellet going in slowly here slowly. And you direct the, the eyelet delivery based on the screw drive. Once the eyelets are delivered, I take a dry gauze tip, blot the area, and using a cautery iron, I seal The nick and the kidney.
Using five oh silk, I grabbed the peritoneum and sew it up. I do a running stitch to close the mouse. I, we close the skin with skin staples.
Before I wake the mouse up, I give him a subcutaneous injection of bpro, amine and flu nixon anti-inflammatories and a painkiller. The mouse is then returned to a his Cage on a heating pad. So I've just shown you how to transplant eyelets under the kidney capsule Of mice.
This is a great model for looking at transplantation tolerance or ways of treating diabetes. Specifically in our lab, we use this model to look at the effects of tregs on eyelet cell transplantation or inducing tolerance with different costimulatory blockade molecules.
