Hello everyone, I'm Kai Wang from Department of Biomedical Engineering in Peking University at Dr.Lowe's laboratory. Currently, individuals with type one diabetes are treated mainly with insulin injections which do not perfectly simulate the insulin secretion from islets. Replacing the lost islets by islet transportation can show you stable blood glucose control without severe fluctuation.
However, the requirement of producing large numbers of high quality islets and the poor engraftment efficiency compromised the widespread clinical use. Today, I'm gonna show you the process of islet isolation and the transplanting scaffold-supported islets into the epididymal fat pad of diabetic mice. These steps require good knowledge of tissue structures and the skilled hands to perform the surgery.
Spray the whole body with ethanol. Make a V-incision starting from the genital area. Remove the bowel through the right side of the mouse.
Clamp the portal vein and the bile duct with hemostatic clamp. Grip the duodenum carefully with forceps and pull the duodenum until the bile duct is taught. Insert the intravenous needle into the common bile duct through the ampulla.
Clamp the duct segment containing the needle using an ultra hemostatic clamp. Dispense the collagenic solution at a slow and constant rate. After complete inflation of the pancreas, push the bowel through the left side of the mouse.
You can see the pancreatic duct is fully inflated. Remove the pancreas by starting end of descending colon. Use the forceps to lift the bowel and to separate it from the pancreas with another forceps.
Continue to remove the pancreas until it is unattached from the top of the stomach. Finally, lift the pancreas out of the abdomen and cut it free from the remaining spleen. Place a pancreas in the conical tube and deliver on ice.
Incubate the perfused pancreas. Terminate the digestion by adding neutralization solutions. Dissociate the two soup by shaking the tubes vigorously.
Filter the digestive tissue samples through the wire mesh. Collect the islet suspensions into a new conical tube. Centrifuge the tubes.
Pour off the supernatant carefully without disturbing the tissue palettes. Resuspend the palettes with Histopaque by pipetting the suspension up and down for a few times. Slowly pipette HBSS down the side of the tube to the top of the Histopaque solutions.
The two solutions should be well separated layers with a sharp interface. Centrifuge the suspension for 20 minutes. Most of the islets migrate to the interface of the Histopaque and HBSS layers.
Remove the entire supernatant solutions, and pass the solutions through an inverted strainer. The islets will be retained by the strainer. Dip the surface retaining the islets into the solution and gently shake to release the islets.
Hand pick the isolated islets under the microscope. Collect islets into a new culture dish. If the tissue surrounding the duct begins to inflate, it means that the needle passes through the wall of the bile duct.
It is necessary to reposition the needle and try cannulating the bile duct again. As the solutions fill up the pancreas, the tissue near the duodenum inflates first, followed by the region near the pancreatic tail. If the duodenum begins to inflate as shown here with blue stain, adjust the artery clamp and reclamp the bile duct.
Place mouse in supine position. Shave the abdomen. Then swab with iodophors to sterilize the skin.
Cover the mouse with a sterile drape. Make a small incision through the peritoneal wall in the middle close to the genital area. Gently grab and pull out the epididymal fat pad from the abdominal cavity.
Spread the fat pad on a wetted gauze. Place a scaffold containing islets on the fat pad and fold the fat pad to wrap the transplant. Suture the fat pad to secure the islets encapsulated in the fat pad.
Gently place the fat pad back into the abdominal cavity. Close the mouse by suturing the peritoneal wall. Clamp the dermal layer with wound clips.
Inject appropriate doses of pain killers and antibiotics subcutaneously. The fresh isolated islets typically had a rough periphery under optical microscope. Once islets recovered from the isolation process, they looked bright, tight, and acquired a smooth surface.
The calculated mean islet diameter was about 130 micrometers. The decelerized scaffolds are mechanically robust enough to be handled by tweezer. The porous scaffold-supported islets were then transferred onto the surface of the spread fat pad, and then encapsulated by the fat pad.
In a syngeneic transplantation model, typically 500 islets reverse hypoglycemia within 10 days. Normal glycemia was maintained before about 100 days till the retrieval of the grafts. The histological studies show that the islets were revascularized and surrounded by the fat pad tissue and the scaffold.
After watching this video, with practice, you should learn the key steps involved in the islet's isolation and transportation. Firstly, cannulating the common bile duct and dilating the pancreas is the most difficult part of this protocol. Secondly, encapsulate the islet-laden scaffold with a fat pad completely, as the incomplete encapsulation might lead to the failure of the transplantation.
