Islet transplantation is one of the most promising approaches for treating Type I diabetes mellitus. And the search for a suitable site for islet transplantation is ongoing. Islet transplantation to the inguinal subcutaneous white adipose tissue is a simple yet effective method that may have clinical implications.
Extrapolation of this procedure to the clinic may lead to the development of a cure for Type I diabetics. This method could provide insight into clinical islet transplantation as the lingual has been determined to not be an ideal site for islet engraftment. It is important to carefully follow every step of the procedure, especially key steps, such as the pancreas digestion.
Visual demonstration will help researchers new to the technique to better determine how to perform the islet isolation and the transmutation. For islet harvest, first spray the fur of a 10 week old C57 black six mouse with 75%ethanol, and fill a five milliliter syringe with freshly prepared collagenase solution. Equip the syringe with a 32-gauge bending blunt pointed perfusion needle, and place the mouse in the supine position on an ice bag.
Use straight pointed ophthalmic scissors to make a transverse opening in the skin of the pubic area, and extend the incision toward the head of the animal. Completely open the abdomen via a V-incision from the pubic region to the xiphoid process, and move the liver to expose the gall bladder and the entire length of the common bile duct. Then use a vascular clamp to close the duodenal opening of the common bile duct and cut a small opening in the gall bladder.
Cannulate the common bile duct through the opening with the previously prepared perfusion needle, and inject approximately two milliliters of collagenase solution into the pancreas. When all the collagenase has been delivered, use two pairs of noninvasive micro tweezers to separate the perfused pancreas from the intestines, stomach, and spleen, and place the pancreas into a 50 milliliter conical tube on ice. When all of the samples have been collected, add 100 microliters of Dnase I per pancreas to the 50 milliliter tubes and vigorously shake each tube about 40 times over a period of 10 seconds before placing the tissues into a 37 degree Celsius water bath for three to five minutes with moderate shaking.
At the end of the incubation, add up to a final volume of 50 milliliters of Stop Solution to arrest the digestion. And pulse centrifuge the tubes to a speed of 750 times G at four degrees Celsius before quickly stopping the centrifuge. After discarding the supernatants, wash the pellets two times in 15 to 25 milliliters of Stop Solution per wash under the same centrifugation conditions as just demonstrated.
After the second wash, pool three pellets per tube into new 50 milliliter conical tubes. And resuspend the pancreatic cells in a total volume of ten milliliters of Histopaque 1119. Next carefully add five milliliters of Histopaque 1077 down the side of each tube, followed by five milliliters of HBSS.
Separate the cells by centrifugation and use a five milliliter Pasteur pipette to collect the islets from the HBSS Histopaque 1077 interface. Pool the islets in a new 50 milliliter conical tube in a final volume of 50 milliliters of Stop Solution for pulse centrifugation, and discard the supernatant. Resuspend the pellet in 30 milliliters of fresh Stop Solution for a one minute centrifugation, and resuspend the islets in 30 milliliters of culture medium.
Decant the islets into one 10 centimeter light-tight culture dish per tube. And place the dish under the stereo microscope. Using 200 microliter gel-loading pipette tips, hand pick the white spheroidal islets from the solution.
Placing the islets into an untreated cell culture dish containing 10 milliliters of culture medium as they are harvested. Check the purity of the hand picked islets by dithizone staining under a light microscope. And detect the viability of the islets by fluorescein diacetate propidium iodide staining under a florescent microscope.
Then culture the isolated islets in fresh culture medium at 37 degrees Celsius in 95%air and 5%carbon dioxide until transplantation. Starting days three and four after diabetes induction, puncture the caudal vein of each streptozotocin-injected eight week old male C57 black six mouse around 10:00 a.m. to collect a blood sample from each recipient animal.
Use a basic blood glucose monitoring instrument to determine the non-fasting blood glucose level of each mouse and then add the blood onto individual test strips. When an animal has demonstrated a blood glucose level of at least 20 millimoles per liter two days in a row, weigh and mark the recipient mouse and confirm a lack of response to pedal reflex in the anesthetized animals. Place an aliquot of hydrogel from minus 20 degrees Celsius storage on ice for thawing.
And transfer 450 to 500 islet equivalents per recipient into a sterile 1.5 milliliter centrifuge tube containing 200 microliters of medium per tube on ice. Swab the left inguinal area of the recipient with 75%ethanol and place the mouse in the supine position. Fix the limbs with surgical tape, and use clippers to remove the hair around the surgical site.
Swab the surgical area with iodophor and make a vertical incision in the disinfected skin. Identify the inferior epigastric artery and vein within the ISWAT and create a small pocket above the vessels. Sediment the islets by centrifugation and remove as much supernatant as possible.
Add 20 microliters of thawed hydrogel to each tube, and gently resuspend the islets, taking care to avoid bubbles. When a homogenous suspension has been obtained, use a 200 microliter pipette tip to carefully deliver the entire islet hydrogel mixture from one tube into the pocket. When the hydrogel has completely solidified, add 20 microliters of cephalosporin to the transplantation site, and close the muscle and skin with a 5-0 continuous surgical suture.
Then place the recipient into a clean cage on a thermal pad with monitoring until full recumbency. After murine islet processing as demonstrated, the isolated murine islets will be sufficiently pure for transplantation. Only isolated islets with a high viability should be used for transplantation.
After thorough mixing of the islet grafts with hydrogel, the graft can be implanted into the ISWAT site. After about one month of transplantation into the ISWAT, the islet grafts reverse hyperglycemia while the body weight of the recipient mice gradually increases. At 100 days after transplantation, removal of the grafts results in an immediate increase in the 10:00 a.m.
non-fasting blood glucose level. Histological analysis at this time point, reveals that the islet grafts have remained intact. And insulin and glucagon immunofluorescent staining indicate that the transplanted islets are also functional at 100 days post transplantation.
Be careful not to shake the profused pancreas cells too robustly or else the ability of the obtained islets will be compromised. Isolated islets can be transplanted under the renal capsule as a control to compare the effects of the isolated transplantation into the inguinal subcutaneous white adipose tissue at site. The procedure will add initiation of clinical trials of islet transplantation to other sites besides the liver.
