This method can help answer key questions in the islet transplantation field such as islet transplantation to the omentum. The implications of this technique extend toward therapy of type I diabetes because the omentum is re-emergent as a potential value site for islet transplantation. The main advantage of this technique is that the isolated islets are mixed with hydrogel and the mixture is placed into the omentum pouch of diabetic mouse.
Begin by preparing an aseptic field in the surgical area with sterile material such as drapes and disposables. Place the sterilized surgical instruments in the aseptic field. Pick the donor islets under the stereo-microscope using a 200 microlitre pipette tip.
For each animal, collect 450 to 500 islets and transfer to a sterile 1.5 milliliter snap top tube with 100 microlitres of CMRL 10 66 medium. Place the tube on ice until ready to transplant. Once the islets have been harvested, thaw basement membrane matrix hydrogel on ice.
Weigh and tag all diabetic recipient mice. After anesthetizing the first mouse according to an institutionally approved protocol, test the depth of anesthesia by administering a toe pinch. If there is no withdrawal reflex, the level of anesthesia is correct for surgery.
Administer vet ointment to the eyes to prevent dryness while under anesthesia. Swab the abdomen with 70%ethanol. Then shave the hair from the surgery site.
Cover the animal with a sterile drape, and disinfect the area with an iodine scrub. After using ophthalmic scissors to create a 4 to 5 centimeter incision along the midline of the abdomen, move the intestines to the left side and cover with saline soaked gauze to prevent dehydration during the surgery procedure. Then use cotton swabs to fully expose the stomach and locate the omentum below the stomach.
At this point, the hydrogel should be completely thawed. Spin the tube containing the islets at 200 times g for 30 seconds. After the spin, remove the supernatant.
Then aspirate 50 microliters of the hydrogel add it to the tube containing the islets and mix gently avoiding the formation of bubbles. Next use two pairs of forceps to pick up the edges of the omentum and gently raise it to form a groove between the gastric wall and the intestines. At this point in the surgery, a second person is needed to aspirate the resuspended islet hydrogel mixture with a 200 microliter pipette tip and deliver it into the groove.
Ensure that the mixture is well positioned in the groove by gently raising or lowering the edges of the omentum. Complete the positioning of the mixture within three minutes before the hydrogel solidifies. After the hydrogel sets, fold the omentum to cover the graft.
The omentum will adhere to the surrounding gastric wall as the hydrogel solidifies. When the hydrogel is completely solidified, use cotton swabs to reposition the intestines in the abdominal cavity taking care not to touch the site of transplantation. Next dispense 200 microliters of an appropriate antibiotic into the abdominal cavity to prevent infection.
Then use 4 O suture to close the abdomen. Generally individuals new to this method will struggle because the sites of the omentum in mice is small and tiring to assess. Place the mouse into a warmed recovery cage until it fully regains external recumbency.
The next day and every day thereafter for one week, inject antibiotic as post-surgery prophylaxis. Also, measure non-fasting blood glucose level from a tail vein blood sample using a blood glucose meter once a day after the transplant. When transplanting to the omentum, the islet graft may have a delayed function and not reach a completely normal blood glucose level for two to three weeks.
The grafted tissue was retrieved from the euthanized mouse 14 days after transplantation. Here the graft is visible as a cluster of nuclei after DAPI staining. The functionality of the grafted tissue is revealed by immunofluorescence staining with anti-insulin antibodies.
H&E staining shows the grafted tissues surrounded by the adipocytes of the omentum. Once mastered, this technique can be done in one hour if it is performed properly. While attempting this procedure, it is important to remember too the mouse omentum is fragile.
Following this procedure, other methods like used in different high school, MB performance in all that will answer particular questions like the spread of islet in vascuolization and the performance. After this development, this technique paved the way for researchers in their and transplantation to explore treatment for type I diabetes in more than one excerpt. After watching this video, you should have a good understanding of how to transplant the islets to the omentum in the mouse.
