This method can help answer key question in the cell therapy field, such as, can islet transplantation be optimized by the hOMING surgical technique? The main advantages of this technique are that it is simple, fast, and tunable, and the biomaterial validation is also very rapid. Visual demonstration of the needle placement is critical to ensure a proper graft injection inside the omentum, as an incorrectly-placed needle can result in islet leakage.
To induce diabetes, inject 75 milligrams per kilogram of streptozotocin intraperitoneally into each six-week-old, 150 to 190 gram Lewis recipient rat, and check the diabetes status by daily blood glucose measurement for the first four days after injection. When the rats exhibit a glycemia over two grams per liter, subcutaneously inject six units of long-acting insulin per day to prevent diabetes complications and weight loss. Include rats in the experimental cohort when two measures of the tail vein blood glucose level are greater than four to five grams per liter for two consecutive days, with a C-peptide level under 200 picomolar.
For pellet implantation, confirm a lack of response to toe pinch in the anesthetized diabetic recipient animal and clean the neck with povidone iodine. Shave the implantation area and clean the exposed skin with additional povidone iodine. After three minutes, sterilize one 1/2 insulin pellets in a one to five povidone iodine solution and use a 16 gauge trocar to pierce the exposed neck skin.
Then, use the guide and stylet to insert the pellets and suture the opening with a single point. Clean the stitch with more povidone iodine and allow the rat to recover in a cage with monitoring and food to avoid hypoglycemia. To measure the efficiency of the pellets, measure the glycemia decrease every week for one month after implantation, including rats in the experimental cohort when a measure of tail vein blood C-peptide level is maintained under 200 picomolar at one month after insulin pellet implantation.
For intra-omental matrix islet filling, count the number of islets isolated from healthy donor rats in islet equivalents, and prepare one 7, 660 islet equivalent per kilogram of recipient in individual 1.5-milliliter tubes. Wash each aliquot with 500 microliters of CMRL medium without fetal bovine serum and resuspend the islet pellets in 150 microliters of freshly-prepared alginate hydrogel carrier per tube with careful mixing before placing the tubes on ice. Next, load one-milliliter syringes equipped with an atraumatic 21-gauge needle with 150 microliters of empty alginate without dead volume, followed by 150 microliters of alginate plus islets.
Place the syringes on ice and prepare the neck of the first recipient animal as just demonstrated. Use a scalpel to make an incision over the old wound and use forceps to remove the pellets. Close the stitch with one or two single stitch points and place the rat in the supine position.
Sterilize the peritoneal area and shave. Use a scalpel to create a one 1/2 centimeter laparotomy just under the sternum. Place wet sterilized gauze around the incision and identify the omental fat pad located next to the stomach.
Use forceps to gently pull the fat pad out of the peritoneal cavity, spreading it on the gauze. Hydrate the omental tissue well with two milliliters of pre-warmed, 37-degree-Celsius sterile saline, and, holding the tissue with small curved forceps in one hand, penetrate the omental edge between the omental layers with the needle of an islet-loaded syringe with the other. When inserting the needle, make sure that it's surrounded by a thin layer of omental tissue before starting the injection.
Insert the needle entirely and slowly begin injecting the islet preparation while retracting the needle to allow the injected islets to be spread throughout the tissue. During the injection, retract the needle to the edge of the tissues, stopping the injection when the needle is almost out. Then, wait five seconds before starting a new injection in the next location.
And inject another three to four aliquots as demonstrated. When all of the hydrogel has been dispensed, withdraw the needle carefully to avoid the loss of the injected islets, checking that the islets are not clustered in the syringe at the end of the injection. When all of the islets have been delivered, hydrate the omentum and the wall of the laparotomy before carefully returning the omental tissue to the abdominal cavity.
Inject two milliliters of pre-warmed sterile saline into the abdominal cavity to rehydrate the rat. Then close the muscle wall with a continuous thread suture and stitch the cutaneous layer with single stitch point. After one to two months of metabolic follow-up, open the surgical incision of each recipient as demonstrated, and use forceps to carefully spread the omenta onto saline-soaked pieces of gauze laid out next to the incisions.
Starting from the region adherent to the pancreatic tail, use scissors to excise the omental tissue and inject two milliliters of pre-warmed saline before closing each animal as demonstrated. Fix the retrieved omenta in 4%paraformaldehyde before embedding in paraffin. Then, acquire four-micrometer-thick sections of the tissues on a microtome and apply hematoxylin and eosin stain for morphological evaluation of the transplants.
Dextran beads transplanted using the hOMING method as demonstrated were frequently observed close to blood vessels and were well implanted within the fat tissue. In this representative isogenic study, glycemia was first controlled by implantation of one 1/2 insulin pellets as demonstrated and then by implanted islets as demonstrated by a glycemia of approximately two grams per liter and a C-peptidemia of greater than 500 picomolar in the recipient animals. In addition, histological analysis of the explanted omenta revealed highly-revascularized islets, most likely as a result of their proximity to blood vessels.
After its development, this technique paved the way for researchers in cell therapy fields to explore bioengineered cell microenvironment in preclinical models.
