3.2.1. Murine Islet Isolation
Islet isolation techniques differ between laboratories but all rely on collagenase digestion of the pancreas to generate a cell suspension containing exocrine tissue and intact islets of Langerhans (1% of total tissue). Over-digestion results in fragmentation of islets because of disruption of the islet capsule: the function of such islets will be compromised. Under-digestion results in exocrine tissue remaining attached to the islets (referred to as "embedded islets"). We routinely isolate islets using a modification of the method described by Gotoh et al. (8).
2. Sacrifice the donor using a schedule 1 method.
4. Expose and pin back the liver.
6. Aspirate the collagenase solution into a 5-mL syringe and attach a 27-gauge needle, bent at a 90° angle.
7. Cannulate the pancreatic duct and slowly inject 2-3 mL of collagenase solution into the pancreas. It may be necessary to place a bulldog clamp on the shaft of the needle to ensure no reflux of collagenase. When the pancreas is fully distended, release the clamp and dissect the pancreas free.
8. Collect up to three pancreases in a 50-mL tube and add sufficient collagenase P to just cover them completely.
9. Incubate at 37°C for 6-12 min. The time is dependent on the batch of collagenase and has to be optimized for each new batch. The aim is to maximize the number of islets obtained that are free of exocrine tissue, without resulting in fragmented islets due to over-digestion.
10. After digestion, top up to 35 mL with HBSS, 0.01 M HEPES supplemented with FCS to a final concentration of 5% (wash solution) and shake by hand for 15 s to break up the tissue.
11. Pellet the islets by centrifugation at 800g for 1 min and aspirate the supernatant to a volume of 7.5 mL.
12. Wash again by addition of a further 35 mL of wash solution, shake, and centrifuge for 1 min at 800g.
13. Aspirate the supernatant to approx 7.5 mL and resuspend the pellet in 20 mL of wash solution.
14. Pour the cell suspension through a 1-mm sieve into a fresh 50-mL tube to remove any undigested tissue or fat.
15. Add 15 mL of wash solution to the digestion tubes and swirl gently to ensure that all tissue is recovered and pour through the filter.
16. Rinse the filter with a final 15 mL of wash solution.
17. Centrifuge the cell suspension at 800g for 1 min.
18. Discard the supernatant, resuspend the cell pellet in 10 mL of UW solution (Biowhittaker), and leave on ice for 30 min.
19. Centrifuge at 800g for 1 min and carefully pour off the supernatant.
20. Prepare Ficoll solutions (see Table 1) and leave to equilibrate to room temperature for 30 min.
21. Resuspend the digested pancreas pellet from step 19 in 15 mL of 25% Ficoll.
22. Add the next Ficoll layers very slowly by pipetting down the side of the tube: 10 mL of 23% Ficoll; 10 mL of 20% Ficoll; 5 mL of 11% Ficoll.
23. Centrifuge the gradients at 1800g for 10 min with no brake at room temperature.
24. Collect each layer and its interface separately into 50-mL tubes.
25. Top up each tube with wash solution and centrifuge for 2 min at 900g with the brake applied.
26. For each tube, aspirate the supernatant to approx 7.5 mL and wash by addition of 35 mL of wash solution. Pellet by centrifugation for 1 min at 800g and aspirate the supernatant as close to the islet pellet as possible.
27. Resuspend the pellet in 3 mL of RPMI supplemented with 5% FCS and place the islet fractions separately into 60-mm diameter Petri dishes. Top up with RPMI 5% FCS.
28. View under a dissecting microscope using a x10 objective and aspirate the islets using a P200 Gilson pipet (see Fig. 1).
29. Place the aspirated islets into a clean Petri dish containing RPMI 5% FCS. It may be necessary to repeat this process to achieve high degrees of purity, as some exocrine tissue is inevitably aspirated along with the islets during the initial picking.
1. Count the required number of islets into a 1.5-mL tube and centrifuge for 3 min at 800g.
2. Remove the supernatant and resuspend the pellet of islets in 200 |L of PBS. Draw up the islets into a P200 pipet tip and plug the tip by dipping it into molten wax to a depth of 2-3 mm.
3. Place the tip in a 15-mL tube and centrifuge for 3 min at 800g to pellet the islets on top of the wax.
4. Carefully pipet off the supernatant and, under a dissecting microscope, cut off the wax tip with a scalpel blade, leaving the islets ready for transplantation.
5. Anesthetize the recipient mouse and make a 5- to 7-mm incision in the abdominal wall on the left side, parallel to, and 5 mm below the line of the rib cage.
6. "Buttonhole" the kidney through the incision. Keep the kidney moist at all times by resting on gauze soaked in PBS and frequently syringing PBS over the surface.
7. Under a dissecting microscope, puncture the capsule of the kidney at one pole using blunted watchmakers' forceps.
8. Insert the pipet tip containing the islets under the capsule and advance it, in the subcapsular space, to the opposite pole of the kidney. Great care must be taken not to tear the kidney capsule.
9. Pipet the islets out into the subcapsular space and remove the pipet tip.
10. Return the kidney to the body cavity and staple the skin with wound clips.
11. Animals should be placed in a cage on a heating pad and observed until fully recovered from anaesthesia.
3.2.3. Induction of Diabetes
1. Select mice weighing between 20 and 30 g and starve overnight, allowing continued access to water.
2. The following day, inject intraperitoneally (i.p.) 100-160 mg of 4% (w/v) STZ (Sigma) dissolved in 0.1 M citrate buffer, pH 4.5.
3. Monitor blood glucose levels using a single drop of blood from a tail vein bleed and a blood glucose monitor (e.g., AccuCheck II, Roche). Mice may be considered diabetic and suitable for transplantation when their blood glucose measures greater than 20 mmol/L.
4. Transplant diabetic mice 3 d after STZ injection.
5. Measure urine glucose levels daily using urinalysis sticks (Roche). We measure blood glucose levels routinely twice weekly and if glycosuria is detected on uri-nalysis. An islet graft is considered to have failed or to have been rejected if the blood glucose levels measure more than 15 mmol/L on two consecutive readings.
1. Grafts that fail in the first 24 h are considered technical failures. Technical competence should be confirmed by performing syngeneic grafts before proceeding to experimental work. Greater than 90% survival of syngeneic grafts for >100 d should be achieved.
2. The gauze strip both keeps the graft and paraffin gauze in place and prevents the plaster of Paris cast sticking to the hair of the thorax, thereby facilitating its removal.
3. In situ perfusion of the pancreas is not essential but greatly increases islet yield.
4. Multiple pancreases will be required to obtain sufficient islets for most experiments. Pancreases may be stored in a 50-mL tube on ice. We routinely retrieve no more than three pancreases at any one time, aiming to complete all surgery within 1 h, to prevent over-digestion of the first pancreas. All isolation steps have been optimized for this number of pancreases.
5. Equilibration of Ficoll solutions to room temperature is essential.
6. It is important to ensure that islets are exposed to Ficoll for the minimum time possible as prolonged Ficoll exposure reduces their viability.
7. STZ must be made up fresh and used immediately. The dose of STZ required varies depending upon the strain of mouse and on the batch of STZ used, the correct dose for each batch being determined by initial titration experiments. In our experiments, the STZ dose range used for C57Bl/6 is between 100 and 120 mg of STZ per kilogram of body weight and for NOD mice is between 150 and 160 mg/kg of body weight.
8. Islet transplants are not carried out until 3 d post-STZ injection to ensure no STZ remains in the animal which might damage the transplant.
9. Watchmakers' forceps are blunted by carefully "flattening" the tip by rubbing on fine emery cloth. If observed under a dissecting microscope, a clear square end to the forceps should be produced with no barbs.
10. Intraperitoneal injection of 100-120 |L of fluanisone and midazolam, made up as described above, is sufficient to anesthetize most mouse strains adequately for islet transplantation.
11. Following STZ injection, mice do not need to be maintained on insulin if transplanted within 3 d. Animals that become diabetic but are not transplanted should be culled.
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