This protocol makes it possible to assess the mechanism of islet engraftment into white adipose tissue. The main advantage of this fat covered islet transplantation method is that it is technically easy to replicate. The method can be applied to other disease models.
For example it can be used to create a mouse model of cancer. Demonstrating the procedure will be Naoaki Sakata, an Associate Professor at Fukuoka University. Under a dissecting microscope, use a P 200 micro pipette to remove any extra islet components from the pancreas digestions including acinar and fibrous tissues.
Then use a cell strainer to filter out single acinar cells. Transfer the filtered islets to a new culture dish containing the appropriate culture medium or buffer solution supplemented with bovine serum or albumin, and swirl the dish to position the islets in the center. Using a P 200 micro pipette, pick the individual islets into an appropriate collection tube.
Place a new 40 micrometer cell strainer on top of a 50 milliliter plastic tube. Use a 1000 microliter pipette to add the islets to the strainer to separate the islets and the single acinar cells. Use forceps to invert the strainer on a new 60 or 100 milliliter sized non-treated culture dish containing culture medium or an appropriate buffer solution, supplemented with bovine serum or albumin.
Use fresh medium, or buffer to flush the islets into a new culture dish, then add enough medium or buffer to the culture dish to reach a total volume of approximately 20 milliliters. Count the islets under a microscope and divide the number of islets equally between individual 1.5 milliliter plastic centrifuge tubes according to the number of donor animals. Centrifuge the islets at 2, 100 G for one minute at room temperature and discard the supernatant.
Around 20 to 30 microliters of residual solution will typically remain in the tube. After removing the hair from the abdomen to prevent infection place the mouse in a supine position. After disinfecting the abdomen and inguinal region incise the skin at lower median area, creating a skin incision that is approximately two centimeters in length.
Clamp the left abdominal wall with forceps or retractors and pull the tissue to the left side of the mouse to secure the surgical field. Use a cotton swab to mobilize the small and large intestine to the right side of the mouse. The left epidermal white adipose tissue in the abdominal cavity is located in the left inguinal area.
Mobilize the epidermal white adipose tissue and the left testis to the outside of the abdomen and stretch out the tissue. Use a P 200 micro pipette to collect the entire volume of islets from one 1.5 milliliter tube, taking care that no islets are left in the tube upon collection. Allow the collected islets to settle in the tip of the pipette by gravity.
Place the micro pipette tip lightly onto the distended adipose tissue, taking care to prevent excessive flushing of the medium or buffer in the tip. Carefully seed the islets onto the tissue. After seeding, confirm the correct placement of the islets under a dissecting microscope.
Cover the islets with the epidermal white adipose tissue, then place the left testis under the episomal white adipose tissue and return the tissues to the intraperitoneal cavity. Close the skin in two layers using a 4-o suture. When finished, place the mouse under a heat lamp and monitor it until full recovery.
To compare the transplant efficacy of fat covered islet transplantation to that after intraperitoneal islet transplantation, the same number of islets was implanted onto the peritoneum at the left paracolic space of control recipient diabetic animals. The blood glucose levels of mice with fat covered islet transplantation were observed to gradually and significantly decrease compared to intraperitoneal islet transplanted mice. Intraperitoneal glucose tolerance testing was performed one month after transplantation.
The blood glucose in mice with fat covered islet transplantation was maintained at lower levels than that observed in intraperitoneal islet transplanted mice. Histological examination was used to assess islet engraftment into the epidermal white adipose tissue. In fat covered islet transplant recipient animals, hematoxylin eocene staining revealed the presence of islets within the epidermal white adipose tissue.
In addition, fluorescence conjugated antibody staining of insulin positive islets facilitated the detection of von Willebrand factor positive micro vessels within the epidermal white adipose tissue of all recipient mice. To succeed with this procedure make sure to allow the islets to settle at the tip of the pipette completely, and seed them onto the adipose tissue without spilling.
