Liver is a primary site for transplantation in the human islet clinical setting. Mouse intrahepatic islet transplantation is technically challenging, and a high percentage of mice could die from surgical complications, especially bleeding. In this study, the two methods we showed both could effectively prevent bleeding-induced mouse death.
Our method can be applied to research in liver cancer and other liver diseases by allowing injection of cancer cells, mesenchymal stem cells, and hepatocyte directly to the liver. To begin, detach a human islet culture with gentle scratching. Use a 1 cubic centimeter syringe to pick 300 to 350 islets into sterile 1.5 milliliter microcentrifuge tubes on ice.
When all of the islets have been picked, centrifuge the tubes for 10 seconds to sediment the cells. Discard all but a small volume of supernatant to prevent pellet loss and resuspend the islets in 200 microliters HBSS containing 5%BSA. Load the resuspended islets into a 5 milliliter insulin syringe and turn the syringe needle-end up for 1 minute to let the islets settle at the plunger.
Depress the plunger to remove any bubbles, keeping approximately 100 to 150 microliters of solution in the syringe and invert the syringe with gentle tapping to evenly distribute the islets. After confirming a lack of response to pedal reflex, apply ointment to the eyes to prevent drying. Shave the abdomen of the anesthetized mouse to remove the fur and disinfect the surgical area with three alternating wipes of 2%iodine and 75%alcohol.
Next, make a 1 to 1.5-centimeter incision in the abdomen and open the peritoneal cavity. Place a sterile piece of gauze around the incision and use forceps or cotton tips to gently transfer the intestine onto the gauze. The then cover the intestine with a piece of saline-soaked gauze and expose the portal vein.
To stop islet transfer-induced bleeding using gel foam, hold the needle with the bevel facing down and position the needle tip with the opening parallel to the portal vein wall before inserting the needle into the portal vein. Retract the plunger to draw 20 to 50 microliters of blood into the syringe to mix with the islets before infusing the islets into the portal vein while repeatedly depressing and retracting the plunger. When all of the islets have been delivered, place an approximately 5 by 5 centimeter piece of gel foam over the injection site.
Use a cotton tip to press the gel foam down while removing the needle from the portal vein. Hold the gel in place for about 2 minutes, rolling the gel foam to make sure it covers the portal vein. Once the bleeding is stopped, gently return the intestine to the peritoneal cavity in the original position and inject 500 microliters of warm saline into the abdominal cavity.
Use sutures to close the muscle and skin layers. Then place the mouse in a clean cage on a heating pad with monitoring until complete recovery from anesthesia, administering analgesia every 12 hours and heat for 48 hours. To stop islet transfer-induced bleeding using a fat pad, after exposing the portal vein as demonstrated, use two cotton tips to hold the exposed vein on the left and right sides and locate the fat tissue pad between the duodenum and portal vein.
Penetrate the fat pad with the needle tip before insertion into the portal vein. Infuse the islets into the portal vein as demonstrated. When all of the islets have been delivered, press the fat tissue with a cotton tip for about 1 minute while removing the needle from the vein.
Then return the intestine to the abdominal cavity and perform the rest of the postsurgical care procedures as demonstrated. The effects of the islet transplantation are dose-dependent, as normal glycemia is observed 30 days after syngeneic islet transplantation when 500 islets are transferred, but only transitory normoglycemia is observed when 250 islets are delivered, with the mice returning to hyperglycemia by 10 days post-transplant. An increased body weight is observed in both groups and identical responses observed when human islets are transplanted into diabetic NOD-SCID mice, with normoglycemia observed when higher numbers of islet equivalents are transplanted but not smaller numbers.
The majority of the mice that have bleeding after the transfer die after the surgery, while the mice without bleeding survive. At 28 days post-transplantation, insulin staining reveals an even distribution of the human islet throughout the liver, mostly close to blood vessels. In addition to insulin, fibrin and polymorphonuclear cell filtration are also observed within the transplanted human islets.
Intrahepatic islet transplantation will generate a large hole in the portal vein that may cause bleeding. Using our two improved procedures can prevent bleeding and reduce mortality post islet transplantation. The intraportal islet transplantation model can mimic clinical human islet transplantation.
Our refined procedures can lead to higher survival rates for recipients, which allow further islet function study after transplantation.
