We would like to acknowledge Gundula Hertl for her support.

All procedures in this protocol have been approved by German Animal welfare law and guidelines. Consider using 10- to 12-week-old NMRI nude mice and C57Bl/6 mice (donor: old female; recipient: male) throughout the experiments. Use NMRI nude mice for islet isolation. Keep the mice under defined conditions according to local animal facility regulations.
1. Induction of Diabetes by Streptozotocin
<ol>
	<li>On day -4, measure the body weights of 10 to 12 week-old NMRI nude mice and C57Bl/6 mice using a weighing machine.</li>
	<li>According to the measured body weight, prepare a 180 mg/kg dose of streptozotocin in 0.1 mL of saline per mouse. 
	NOTE: Streptozotocin should be freshly prepared before use and covered with aluminum foil, as it is light-sensitive.</li>
	<li>Inject the syngeneic and allogeneic mice each with a single dose of 0.1 mL of streptozotocin (180 mg/kg) intraperitoneally using a 26-G needle.</li>
	<li>On day -3,-2 and -1 collect 2-4 &#181;L of blood by puncturing the tail veins of non-fasted mice.</li>
	<li>Use 2-4 &#181;L of the blood sample per test strip to measure the blood glucose in NMRI nude mice and C57Bl/6 mice 
	NOTE: At this point, 80% of the mice should become diabetic (20 mmol/L).
	<ol>
		<li>Use mice for islet transplantation when their blood glucose levels reach 20 mmol/L.</li>
	</ol>
	</li>
</ol>
2. Isolation of Pancreatic Islets from Mice
<ol>
	<li>Anesthetize the mice with ketamine (100 mg/kg body weight) and xylazine (20 mg/kg body weight) by intraperitoneal injection with a 26-G needle. 
	NOTE: The volumes of ketamine and xylazine were determined according to the body weights of the animals (0.10 mL/10 g).</li>
	<li>Keep the mice in supine position and disinfect with povidone-iodine solution. Confirm proper euthanasia by checking the pedal reflex. 
	NOTE: In case of C57Bl/6 shave the abdominal region.</li>
	<li>Hold the skin with fine forceps and make a midline incision with scissors to expose the abdominal cavity. Place the intestine situs to the left and outside of the abdominal cavity on a sterile gauze compress.</li>
	<li>Adjust the stomach in another direction to locate the pancreas and other associated tissues.</li>
	<li>Reach the aorta and make a small cut to drain the blood. Use sterile gauze for blood absorption.</li>
	<li>Carefully remove the intestine. Separate the pancreas from the stomach and spleen.</li>
	<li>Excise the pancreas and place it in a fresh Petri dish. Remove all the unwanted materials, such as fat, using forceps.</li>
	<li>Fill a 5 mL syringe with 4 mL of collagenase and inject into the pancreas with a 26-G needle (stock solution: 30 mg of collagenase in 12 mL of Hank&#39;s solution).</li>
	<li>Mince the pancreas with scissors to increase the surface area for an improved digestion and transfer to a 12 mL red cap tube. 
	NOTE: Hank&#39;s balanced salt solution: HBSS 10x (100 mL), Penicillin-Streptomycin (10 mL), Gentamycin (1 mL), Ciprofloxacin (10 mL), HEPES (35 mL), distilled H2O (900 mL)</li>
	<li>Perform a digestion for 10 min at 37 &#176;C in a shaking water bath. Vortex the sample thrice at a regular interval for 15 s.</li>
	<li>Mix the digested pancreas vigorously by hand for 3 min and place it on ice for 10 s.</li>
	<li>Add 6 mL of cold Hank&#39;s solution to stop the digestion reaction and centrifuge at 560 x g for 3 min at room temperature.</li>
	<li>Remove the supernatant and dissolve the pellet in 10 mL of Parker/fetal calf serum (P/FCS) medium at room temperature. 
	NOTE: P/FCS medium: TCM-199 10x (100 mL), FCS (50 mL), Penicillin-Streptomycin (10 mL), HEPES (20 mL), distilled H2O (900 mL)</li>
	<li>Identify the islets based on their morphology (spheroid, golden-brown color) under a light microscope. Avoid islets with dark centers. Check for purity and viability by performing dithizone staining under a light microscope. 
	NOTE: Dithizone binds to zinc ion and provides red-colored stain to the islets.</li>
	<li>Use a 1-mL pipette to count and handpick islets under a light microscope and transfer them to a fresh Petri dish (60 mm x 15 mm) containing 4 mL of P/FCS medium. 
	NOTE: The volume of P/FCS medium in the Petri dish should not exceed 4 mL in order to ensure oxygen availability.</li>
	<li>Keep the isolated islets in 4 mL of P/FCS medium overnight at 37 &#176;C in an incubator.</li>
</ol>
3. Preparation of Animals for Islet Transplantation
<ol>
	<li>Remove the isolated islets from the 37 &#176;C incubator and place them under a flow bench. Center the islets with a circular swinging motion of the Petri dish.</li>
	<li>Fill a 1-mL syringe (blue 23-G needle) with 0.1 mL of P/FCS medium. Then, add 350 islets in 0.3 mL of Hank&#39;s solution. Keep the syringe in a vertical position to allow the islets to settle.</li>
	<li>Decrease the volume to 0.05 mL and replace the blue 23-G needle with a brown 26-G needle. To the same syringe, add 0.15 mL of Ficoll (density gradient medium) to the top to reach a final volume of 0.2 mL in the syringe. Avoid air bubbles.</li>
	<li>Keep the syringe in a vertical position and rotate it slightly to avoid the clumping of islets. The islets will settle at the cone of the syringe within 2-3 min.</li>
	<li>Measure the body weight and blood glucose levels of the recipient mice; the blood glucose levels should be around 20 mmol/L.</li>
	<li>Anesthetize the mice with ketamine (100 mg/kg body weight) and xylazine (20 mg/kg body weight) by intraperitoneal injection with 26-G needle.</li>
	<li>Place the mice in supine position on a warm thermal plate (37 &#176;C). Check the depth of anesthesia by pinching the toe.</li>
	<li>Clean the abdominal region with povidone-iodine solution (skin disinfectant). To prevent the eyes from drying, keep them moist with eye ointment. 
	NOTE: In case of C57Bl/6 shave the abdominal region.</li>
</ol>
4. Islet Transplantation via the Intraportal Route
<ol>
	<li>On day 0, start with a laparotomy by holding the skin with forceps and making a 2-3 cm-long midline incision with a scissor. Place a sterile wet gauze compress around the wound edges to keep it moist.</li>
	<li>Place the intestine situs to the left and outside the abdominal cavity on the sterile gauze compress. 
	NOTE: Keep the intestine moist throughout the procedure with pre-warmed 0.9% NaCl solution or sterile gauze dipped in normal saline.</li>
	<li>Move the duodenum and locate the pancreas. Locate the portal vein situated at the ventral site by pushing the pancreas upward. Use a finger and thumb to expose the portal vein and, once exposed, puncture it near to the liver.</li>
	<li>Within 1 min, slowly and steadily inject the islets (0.2 mL from step 3.4) into the portal vein under the inspection of magnifying glass to avoid any leakage of islets. 
	NOTE: After injecting, always check for remaining islets in the syringe. Take 1 mL of P/FCS media in a syringe, flush into a fresh Petri dish and count under a microscope.</li>
	<li>Place a sterile gauge at the puncture site and apply pressure with the index finger (penetration depth: 0.5-1 cm) for 6 min to stop the bleeding. 
	NOTE: The volume injected should not exceed 0.2 mL. Excessive volume and pressure can damage the islets due to shear stress.</li>
	<li>Carefully place the intestine situs, peritoneum, abdominal muscle layer, and fascia back into position.</li>
	<li>Using forceps, lift the skin, close the muscle incision with absorbable silk suture, and upper skin layer with 2-3 clips.</li>
	<li>Clean the surface with skin disinfectant, apply a wound healer, and place the mice in individual cages. Wound healer provides a water-resistant acrylate layer and protect from maceration and secondary infection. 
	NOTE: Observe the mice until they have regained sufficient consciousness, provide tramadol (0.2 mg per mouse) along with fluids. In this protocol, mice recovered from anesthesia quickly and tramadol administration reduced the pain. After 7 days, the wound should heal.</li>
	<li>Feed the mice with regular food and water. Food and water consumption and urine excretion will increase after the streptozotocin injection.</li>
	<li>Measure the blood glucose levels every day for the first week and then once a week in syngeneic mice. In allogeneic mice, measure the blood glucose levels thrice a week for up to 15 days.</li>
</ol>

<ol>
	<li>McCall, M., Shapiro, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+islet+transplantation.">Update on islet transplantation.</a> Cold Spring Harbor Perspectives in Medicine. 2 (7), a007823 (2012).</li><li>Shapiro, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+transplantation+in+seven+patients+with+type+1+diabetes+mellitus+using+a+glucocorticoid-free+immunosuppressive+regimen.">Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen.</a> New England Journal of Medicine. 343 (4), 230-238 (2000).</li><li>Cantarelli, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+as+an+alternative+site+for+islet+transplantation.">Bone marrow as an alternative site for islet transplantation.</a> Blood. 114 (20), 4566-4574 (2009).</li><li>Echeverri, G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoscopic+gastric+submucosal+transplantation+of+islets+(ENDO-STI):+technique+and+initial+results+in+diabetic+pigs.">Endoscopic gastric submucosal transplantation of islets (ENDO-STI): technique and initial results in diabetic pigs.</a> American Journal of Transplantation. 9 (11), 2485-2496 (2009).</li><li>Korsgren, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimising+islet+engraftment+is+critical+for+successful+clinical+islet+transplantation.">Optimising islet engraftment is critical for successful clinical islet transplantation.</a> Diabetologia. 51 (2), 227-232 (2008).</li><li>Merani, S., Toso, C., Emamaullee, J., Shapiro, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+implantation+site+for+pancreatic+islet+transplantation.">Optimal implantation site for pancreatic islet transplantation.</a> British Journal of Surgery. 95 (12), 1449-1461 (2008).</li><li>Lingwal, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+gelatinase+B+(matrix+metalloprotease-9)+activity+reduces+cellular+inflammation+and+restores+function+of+transplanted+pancreatic+islets.">Inhibition of gelatinase B (matrix metalloprotease-9) activity reduces cellular inflammation and restores function of transplanted pancreatic islets.</a> Diabetes. 61 (8), 2045-2053 (2012).</li><li>Chen, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+intraportal+islet+transplantation+outcome+by+systemic+IKK-beta+inhibition:+NF-kappaB+activity+in+pancreatic+islets+depends+on+oxygen+availability.">Improved intraportal islet transplantation outcome by systemic IKK-beta inhibition: NF-kappaB activity in pancreatic islets depends on oxygen availability.</a> American Journal of Transplantation. 11 (2), 215-224 (2011).</li><li>Wang, Y., Wang, T., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+aspects+are+critical+for+carrying+out+intraportal+islet+transplants+successfully+in+a+diabetes+mouse+model.">Three aspects are critical for carrying out intraportal islet transplants successfully in a diabetes mouse model.</a> Experimental and Clinical Transplantation. 10 (3), 302-304 (2012).</li><li>Neuman, J. C., Truchan, N. A., Joseph, J. W., Kimple, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+mouse+pancreatic+islet+isolation+and+intracellular+cAMP+determination.">A method for mouse pancreatic islet isolation and intracellular cAMP determination.</a> Journal of Visualized Experiments. (88), e50374 (2014).</li><li>Zmuda, E. J., Powell, C. A., Hai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+murine+islet+isolation+and+subcapsular+kidney+transplantation.">A method for murine islet isolation and subcapsular kidney transplantation.</a> Journal of Visualized Experiments. (50), (2011).</li><li>Rother, K. I., Harlan, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+facing+islet+transplantation+for+the+treatment+of+type+1+diabetes+mellitus.">Challenges facing islet transplantation for the treatment of type 1 diabetes mellitus.</a> Journal of Clinical Investigation. 114 (7), 877-883 (2004).</li><li>Su, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+islets+are+essential+for+successful+intraportal+transplantation+in+a+diabetes+mouse+model.">Small islets are essential for successful intraportal transplantation in a diabetes mouse model.</a> Scandinavian Journal of Immunology. 72 (6), 504-510 (2010).</li><li>Rekittke, N. E., Ang, M., Rawat, D., Khatri, R., Linn, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+Therapy+of+Type+1+Diabetes+Mellitus:+From+Pancreatic+Islet+Transplantation+to+Mesenchymal+Stem+Cells.">Regenerative Therapy of Type 1 Diabetes Mellitus: From Pancreatic Islet Transplantation to Mesenchymal Stem Cells.</a> Stem Cells International. 2016, 22 (2016).</li></ol>

All authors declare that they have no conflict of interests.

In this study, the intraportal route of islet transplantation is explored. This protocol is highly efficient in relieving diabetic stress and provides an in-depth opportunity to explore islet transplantation studies. This protocol confirms that about 350 murine islets are capable of reversing the hyperglycemic state. Moreover, none of the mice died during the procedure, and glycemic control was achieved in all recipients. Body weight and blood glucose levels were regularly recorded to reflect the competence, standard, and functionality of transplanted islets in reversing hyperglycemia. In syngeneically transplanted mice, blood glucose levels were measured every day for the first week and once a week thereafter. After islets transplantation, hypoxic condition and others factors trigger the endothelial cells to secrete pro-angiogenic factors such as VEGF after 14 days and thereby increase the vascular engraftment in the liver as shown in Figure 3. In allogeneic mice, blood glucose levels were measured thrice a week for up to 15 days. After 15 days, transplanted islets were rejected in the allogeneic model due to an immune attack, unless immunosuppressive drugs are administered
There are several islet isolation protocols published in literature10,11. The most straightforward is to pick islets manually on the basis of their size and morphology after collagenase digestion. This is one of the most crucial steps to select only islets and to avoid exocrine cells, debris, etc. Selected islets should be maintained at 37 &#176;C overnight in an incubator to allow them to recover from mechanical stress encountered during the digestion procedure. The selection of the streptozotocin dose and the intraportal islet transplantation are also key to this protocol. The body temperature should be regulated and maintained using a thermal plate throughout the surgical procedure. Sterile gauze should be applied with minimal pressure for 6 min at the site of puncture to avoid bleeding. Mice should be kept under observation until they regain sufficient consciousness.
Certain steps in this protocol can be modified. For example, the optimization of the digestion time and the volume of collagenase may increase the yield of islets in different mice strains. Instead of the rapid induction of diabetes (180 mg/kg streptozotocin), slowly increasing hyperglycemia can be induced using a lower dose over an extended period of time (e.g., 40 mg/kg/day streptozotocin for 5 consecutive days). This could provide better engraftment of transplanted islets. Other sites, such as the bone marrow, omental pouch, intramuscular region, gastric mucosal surface, spleen, and kidney can be explored as alternatives to the intraportal vein.
Poor engraftment of transplanted islets is still a major concern in clinical trials. Others include a shortage of donors, inconsistencies in maintaining blood glucose levels after transplantation, immunosuppression, and blood-mediated reactions12,13. Lack of methods to evaluate proper engraftment is another obstacle. Tracking transplanted islets by magnetic resonance or bioluminescence imaging could possibly replace classical methods such as the glucose tolerance test or c-peptide. In the future, stem cells transplanted together with islets might provide nutritional and immunomodulating support14.

Islet transplantation is a promising approach to treat type 1 diabetes mellitus1. The first attempt to transfer a sheep pancreatic fragment into a diabetic patient was performed by Watson Williams in 1893. However, a major breakthrough in clinical islet transplantation was achieved with the Edmonton protocol, and thereafter, a series of national programs were developed2. In the past, several sites, such as the bone marrow, omental pouch, intramuscular region, gastric mucosal surface, spleen, and kidney capsule, were explored in preclinical models, but the portal venous system is considered as one of the suitable and effective site for clinical programs3,4,5,6.
Exogenous administration of insulin can be substituted by islet infusion into the portal vein. This is considered a preferred site because the oxygen supply is comparable to that in the native pancreas due to the location downstream of the confluence of the portal artery and vein. Moreover, there is a large surface area, so that the three-dimensional structure of the islets may be preserved, and vascularization could be facilitated5. In a recipient diabetic mouse, 2,000 human or porcine islet equivalents or 350 mouse islets are adequate to reverse the hyperglycemic state7,8. Euglycemic levels were reported for 15 days in the mouse recipient model of xenogeneic islets and in the allogeneic mouse-to-mouse model, and for more than 120 days in the syngeneic mouse-to-mouse transplantation.
Factors pertinent to the efficacy of islet transplantation via portal vein are appropriate anesthesia, method of puncture, and hemostasis9. Anesthesia can be induced by inhalation (5% isoflurane) or injection (ketamine, xylazine, or pentobarbital), and the substances are often combined. For control of the depth and time of anesthesia, attention should be paid to the state of the mouse, such as the color of the mucous membrane, eye lid, corneal reflex, respiratory rate, and body temperature. It is important to ensure that the animal is not struggling and that it survives the operation. The temperature can be maintained by different heating devices, such as heating pads, red light bulbs, etc. A warm thermal plate has been used to maintain a temperature of 25-30 &#176;C between the mouse and the operation table, which prevents the occurrence of hypothermia. The dosages of the anesthetics are important, as all of them are metabolized by the liver, and hepatic function may be transiently disordered by the infusion of the islet suspension. The ideal puncture point for the portal vein is the position between the first and second tributary vein.
The number of islets and the intended injection volume also influence the outcome of the transplant, as an excessive volume may increase the shear stress. A 0.2-mL volume is considered appropriate for islet transplantation into the portal vein. The puncturing wound (26-gauge needle) induces bleeding from the portal vein, which needs to be stopped in a timely and effective manner. Sterile gauze or a finger can be applied with minimal pressure at the site of the puncture for about 6 min to stop the bleeding. Taken together, portal islet injection is effective and provides regulation over blood glucose levels in chemically induced diabetic mouse models.

<table><tbody><tr><td>C57Bl/6 mice&amp;nbsp;</td><td>Charles River (Sulzfeld, Germany)</td><td></td><td>(10-12 weeks)</td></tr><tr><td>BALB/c mice</td><td>Charles River (Sulzfeld, Germany)</td><td></td><td>(10-12 weeks)</td></tr><tr><td>NMRI nude mice</td><td>Charles River (Sulzfeld, Germany)</td><td></td><td>(10-12 weeks)</td></tr><tr><td>Ketamine</td><td>Bela-pharm</td><td></td><td></td></tr><tr><td>Xylazine</td><td>CEVA TIERGESUNDHEIT GmbH</td><td></td><td></td></tr><tr><td>Syringe, 23G and 26G needle</td><td>BD</td><td></td><td></td></tr><tr><td>Petri Dish</td><td>Falcon</td><td>303800</td><td></td></tr><tr><td>NaCl</td><td>Carl Roth</td><td>351007</td><td></td></tr><tr><td>Sterile Gauze</td><td>Fuhrmann</td><td>7647-14-5</td><td></td></tr><tr><td>Shaking Water bath</td><td>Kotterman</td><td>1501</td><td></td></tr><tr><td>Scissors</td><td>Braun</td><td>1501</td><td></td></tr><tr><td>Glucose Meter&amp;nbsp;</td><td>One Touch</td><td></td><td></td></tr><tr><td>Warm Thermal Plate</td><td>Thermo Fisher</td><td></td><td></td></tr><tr><td>Braunol (povidone-iodide solution)</td><td>B.Braun Melsungen AG</td><td>3864154</td><td></td></tr><tr><td>Liposic Edo - Sprinkled Ointment (eye ointment)</td><td>Dr. G Mann Chem.-Pharm.&amp;nbsp; GmbH</td><td></td><td></td></tr><tr><td>Incubator</td><td>Fa. Roth</td><td></td><td></td></tr><tr><td>Flow Bench</td><td>Herdus</td><td></td><td></td></tr><tr><td>Ficoll</td><td>Sigma-Aldrich</td><td>10771</td><td></td></tr><tr><td>5-0 Silk Suture Vicryl</td><td>Braun</td><td></td><td>(7.5 *1.75mm)</td></tr><tr><td>Michel Clamps (clips)</td><td>Aesculap&amp;nbsp;</td><td>BN507R</td><td></td></tr><tr><td>P/FCS Solution</td><td>Gibco</td><td>21180-021</td><td></td></tr><tr><td>TCM-199 (10x)</td><td>Gibco</td><td>21180-021</td><td></td></tr><tr><td>FCS</td><td>Biowest</td><td>S1810-500</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>HEPES (1 M)</td><td>Biochrom</td><td>L 1613</td><td></td></tr><tr><td>Gentamycin</td><td>Ratiopharm</td><td></td><td></td></tr><tr><td>Ciprofloxacin</td><td>Fresenius Kabi</td><td></td><td></td></tr><tr><td>Hank&#039;s Solution</td><td>Gibco</td><td>14060-040</td><td></td></tr><tr><td>Collagenase</td><td>Roche</td><td>11213865001</td><td></td></tr><tr><td>Streptozotocin</td><td>Calbiochem</td><td>572204</td><td></td></tr><tr><td>OPSITE-spray (wound healer)</td><td>Smith and nephew</td><td>66004978</td><td></td></tr><tr><td>Reugular food</td><td>Altromin</td><td></td><td></td></tr><tr><td>Head light LED 16042 (magnifying glass)</td><td>Eschenbach</td><td>16042</td><td></td></tr><tr><td>Rat anti-mouse PECAM-1(CD31) monoclonal primary antibody</td><td>Millipore, Chemicon</td><td>CBL1337</td><td>Dilution (1:100)</td></tr><tr><td>Donkey anti-rat secondary antibody</td><td>Dianova</td><td>712095153</td><td>Dilution (1:400)</td></tr><tr><td>Polyclonal guinea pig anti-insulin primay antibody</td><td>Dako</td><td>A0564</td><td>Dilution (1:500)</td></tr><tr><td>Donkey anti-guinea pig secondary antibody</td><td>Dianova</td><td>706-259-148</td><td>Dilution (1:400)</td></tr></tbody></table>

intraportal transplantation of pancreatic islets in mouse model

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation site in experimental islet transplantation is the kidney capsule. However, as less is known about the interaction of pancreatic islets with blood constituents, it also makes sense to utilize the portal vein approach in experimental islet transplantation.
This protocol demonstrates an intraportal islet transplantation technique in NMRI nude mice. Streptozotocin (180 mg/kg) is injected intraperitoneally to induce hyperglycemia in recipient mice. They are considered as diabetic at a non-fasting blood glucose level greater than 20 mmol/L. One day prior to transplantation, mouse pancreatic islets are isolated from the donor pancreas by collagenase digestion; a minimum of 350 islets are utilized per diabetic recipient. Depending upon the islet isolation yield, two or more donor mice are utilized per recipient. After overnight culture at 37 &#176;C, islets are administered into the recipient liver via the portal vein. After surgery, the mice are protected in red Makrolon houses and observed until are awake. This protocol maintains glycemic control for 120 days in syngeneic mice and 15 days in allogeneic mice.

The competency of transplanted islets was studied in chemically induced diabetic mice. The islets were transplanted via the portal venous system in diabetic mice model. The donor-recipient ratio was 2:1. In the syngeneic mouse model, two donor mice were utilized to obtain 350 islets. The islets were then transplanted into diabetic mice. A reduction in blood glucose level was observed on day 1 after transplantation, suggesting a role of transplanted pancreatic islets in reducing the blood glucose level. Streptozotocin increased the blood glucose level up to about 22 mmol/L. A drop in the blood glucose level to 4.6 mmol/L (day 1) was observed after islet transplantation (Figure 1A). Normoglycemia was maintained for up to 121 days, as shown in Figure 1A. The body weight and blood glucose level roughly followed the same pattern. After the streptozotocin injection and surgery, the body weight decreased. From day 2 onward, it tended to rise towards normal, from 25.3 g (day 4) to 29.7 g (day 121), as shown in Figure 1B.
In case of major histocompatibility (MHC) mismatched mice, 350 islets from two C57Bl/6 (H2d) mice were transplanted into diabetic BALB/c (H2b) mice. After streptozotocin injection, the blood glucose increased up to 26.6 mmol/L. After islet transplantation, the blood glucose level dropped to 4.4 mmol/L (day 1; Figure 2A). Normoglycemia (4.44 to 7.2 mmol/L) was observed for up to 10 days, but not after 15 days (20.1 mmol/L). The body weight of diabetic mice dropped from 26 g (day -3) to 23 g (day 0) and further to 21.5 g (day 1). After transplantation, the body weight gradually recovered from 23 g (day 2) to 25 g (day 10). Due to MHC-mismatch, the blood glucose level increased and the body weight decreased 10 days after islet transplantation (Figure 2B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57559/57559fig1.jpg" /> 
Figure 1: Effect of syngeneic islet transplantation. (A) The blood glucose level was measured at different time points in streptozotocin-induced diabetic mice (n = 3). A single dose of streptozotocin (180 mg/kg) was injected before the transplantation of 350 islets to each recipient (day 0). The blood glucose level (mmol/L) was measured for up to 121 days (day -3: 17 &#177; 2.3; day 0: 22.6 &#177; 1; day 1: 4.6 &#177; 0.3; day 5: 7.5 &#177; 0.4; and day 121: 5.3 &#177; 0.09). (B) Increase in body weight after islet transplantation. The data above represents the mean &#177; the standard deviation. <a href="/files/ftp_upload/57559/57559fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57559/57559fig2.jpg" /> 
Figure 2: Effect of allogeneic islet transplantation. (A) The blood glucose level was measured at different time points in streptozotocin-induced diabetic mice (n = 2). A single dose of streptozotocin (180 mg/kg) was injected before the transplantation of 350 pancreatic islets (day 0). The blood glucose level (mmol/L) was measured for up to 15 days (day -1: 25.5 &#177; 5.3; day 0: 26.6 &#177; 1.4; day 2: 8 &#177; 0.9; day 10: 6.2 &#177; 0.08; and day 15: 20.1 &#177; 1.6). Normoglycemic state was maintained for up to 10 days, and subsequently, blood glucose concentrations increased. (B) Follow-up of body weight before and after islet transplantation. The data represent the mean &#177; the standard deviation. <a href="/files/ftp_upload/57559/57559fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57559/57559fig3.jpg" /> 
Figure 3: Representative immunostaining picture of transplanted pancreatic islets into liver: Wash the liver section (5 &#181;m) with PBS, block with 10% donkey serum followed by overnight incubation at 4 &#176;C with insulin primary antibody (1:500) and platelet endothelial cell adhesion molecule PECAM-1 primary antibody (1:100). Next day, stain with secondary antibodies for 1 h, wash with PBS, counter stain with Hoechst and capture the images with Leica fluorescent microscope. <a href="/files/ftp_upload/57559/57559fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Intraportal Transplantation of Pancreatic Islets in Mouse Model at JoVE.com

Intraportal Transplantation of Pancreatic Islets in Mouse Model

Pancreatic islet transplantation is a way to achieve normoglycemia in type 1 diabetes. This article focuses on the transplantation technique through the intraportal route to maintain normal glucose levels in diabetic mice.

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation ...

intraportal-transplantation-of-pancreatic-islets-in-mouse-model

Nanyang Technological University

Research

JoVE Journal

Medicine

9.5K Views.  Clinical Research Lab. Alvleesklier islet transplantatie is een manier om normoglycemia bij type 1 diabetes. Dit artikel richt zich op de transplantatie-techniek via de intraportal route om normale glucoseniveaus in diabetesmuizen te handhaven.

Video: Intraportal Transplantation of Pancreatic Islets in Mouse Model

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice. We found that it was easier to concentrate the islets or cells into pellets in the final delivery tubing (PE50) used to transplant the cells under the kidney capsule. This technique provides both speed and ease while reducing any undue stress to the cells or to the mouse.

Loading: Settled, hand picked, islets or pelleted cells are carefully aspirated off the bottom of a 1.5 mL microcentrifuge tube using a p200 pipetteman and a straight, thin-wall pipette tip. A length of PE50 tubing is attached to the pipette tip using a small silicone adapter tubing. Cells are allowed to settle, in the tip, and then are transferred to the PE50 tubing by slowly dialing the pipetteman. Once the cells are near the end of the PE50 tubing, a kink is made and the silicone adaptor tubing is placed over the kink. The PE50 tubing is transferred to a 15 mL conical containing a cut 5 mL pipet, and the PE50 tubing is taped over the side of the 5 mL pipet to prevent curling during centrifuging. Cells are allowed to reach 1,000 rpm and stopped.
Transplantation: Recipient mice are anesthetized, shaved, and cleaned. A small incision is made on the left flank of the mouse and the kidney is exposed. The kidney, fat, and tissue are kept moist with normal saline swab. The distal end of the PE50 is attached to a Hamilton screw drive syringe, containing a pipette tip, using the silicone adaptor tubing. A small nick is made on the right flank side of the kidney, not too large nor too deep. The beveled end of the PE50 tubing, nearest the cells, is carefully placed under the capsule, the tubing is moved around gently to make space while swabbing normal saline; a dry capsule can tear easily. A small air bubble is delivered under the capsule by slowly dialing the syringe screw drive. Islets are then slowly delivered behind the air bubble. Once the islets have been delivered kidney homeostasis is maintained and the knick is cauterized with low heat. The kidney is placed back into the cavity and the peritoneum and skin are sutured and stapled. Mice are immediately treated with Flunixin and Buprenorphine s.q. and placed in a cage on a heating pad.

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of mice. Cells are concentrated into pellets in the final tubing used for transplanting the cells under the kidney capsule. The ease of this technique reduces stress to the cells and the mouse.

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice.  We found that it was easier ...

Biology

Scaffold-supported Transplantation of Islets in the Epididymal Fat Pad of Diabetic Mice

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy may incur acute whole blood reactions and result in poor islet engraftment. Here, we report a robust protocol for the transplantation of islets at the extrahepatic transplantation site-the epididymal fat pad (EFP)-in a diabetic mouse model. A protocol to isolate and purify islets at high yields from C57BL/6J mice is described, as well as a transplantation method performed by seeding islets onto a decellularized scaffold (DCS) and implanting them at the EFP site in syngeneic C57BL/6J mice rendered diabetic by streptozotocin. The DCS graft containing 500 islets reversed the hyperglycemic condition within 10 days, while the free islets without DCS required at least 30 days. The normoglycemia was maintained for up to 3 months until the graft was explanted. In conclusion, DCS enhanced the engraftment of islets into the extrahepatic site of the EFP, which could easily be retrieved and might provide a reproducible and useful platform for investigating the scaffold materials, as well as other transplantation parameters required for a successful islet engraftment.

This protocol demonstrates murine islet isolation and seeding onto a decellularized scaffold. Scaffold-supported islets were transplanted into the epididymal fat pad of streptozotocin (STZ)-induced diabetic mice. Islets survived at the transplantation site and reversed the hyperglycemic condition.

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy ...

Bioengineering

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the availability of the widest range of molecular probes and reagents to perform in vivo as well as in vitro studies. Based on our experience with various murine transplantation models, we developed a heterotopic pancreas transplantation model in mice with the intent to analyze mechanisms underlying severe ischemia reperfusion injury-associated early graft damage. In contrast to previously described techniques using suture techniques, herein we describe a new procedure using a non-suture cuff technique. 
In recent years, we have performed more than 300 pancreas transplantations in mice with an overall success rate of &gt;90%, a success rate never described before in mouse pancreas transplantation. The backbone of this non-suture cuff technique for graft revascularization consists of two major steps: (I) pulling the recipient vessel over a polyethylene/polyamide cuff and fixing it with a circumferential ligature, and (II) placing the donor vessel over the everted recipient vessel and fixing it with a second circumferential ligature. The resultant continuity of the endothelial layer results in less thrombogenic lesions with high patency rates and, finally, high success rates.
In this model, arterial anastomosis is achieved by pulling the abdominal aorta of the donor graft over the everted common carotid artery of the recipient animal. Venous drainage of the graft is achieved by pulling the portal vein of the graft over the everted external jugular vein of the recipient. This manuscript provides details and crucial steps of the organ recovery and organ implantation procedures, which will allow researchers with microsurgical skills to perform the transplantation successfully in their laboratories.

Among abdominal solid organ transplantation, pancreatic grafts are prone to develop severe ischemia reperfusion injury-associated graft damage, leading eventually to early graft loss. This protocol describes a model of murine pancreas transplantation using a non-suture cuff technique, ideally suited for analyzing these early, deleterious damages.

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the ...

Inguinal Subcutaneous White Adipose Tissue (ISWAT) Transplantation Model of Murine Islets

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for islet transplantation in rodent models. However, the tight kidney capsule limits the transplantation of sufficient islets in large animals and humans. The inguinal subcutaneous white adipose tissue (ISWAT), a new subcutaneous space, was found to be a potentially valuable site for islet transplantation. This site has better blood supply than other subcutaneous spaces. Moreover, the ISWAT accommodates a larger islet mass than the kidney capsule, and transplantation into it is simple. This manuscript describes the procedure of mouse islet isolation and transplantation in the ISWAT site of syngeneic diabetic mouse recipients. Using this protocol, murine pancreatic islets were isolated by standard collagenase digestion and a basement membrane matrix hydrogel was used for fixing the purified islets in the ISWAT site. The blood glucose levels of the recipient mice were monitored for more than 100 days. Islet grafts were retrieved at day 100 after transplantation for histological analysis. The protocol for islet transplantation in the ISWAT site described in this manuscript is simple and effective.

Inguinal Subcutaneous White Adipose Tissue ISWAT Transplantation Model of Murine Islets

In this protocol, a method of murine islet isolation and transplantation into the inguinal subcutaneous white adipose tissue is described. Isolated syngeneic murine islets are transplanted into a murine recipient using a basement membrane hydrogel. The blood glucose level of the recipients is monitored, and histology analysis of the islet grafts is performed.

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for ...

Minimizing Post-Infusion Portal Vein Bleeding during Intrahepatic Islet Transplantation in Mice

Although the liver is currently accepted as the primary transplantation site for human islets in clinical settings, islets are transplanted under the kidney capsule in most rodent preclinical islet transplantation studies. This model is commonly used because murine intrahepatic islet transplantation is technically challenging, and a high percentage of mice could die from surgical complications, especially bleeding from the injection site post-transplantation. In this study, two procedures that can minimize the incidence of post-infusion portal vein bleeding are demonstrated. The first method applies an absorbable hemostatic gelatin sponge to the injection site, and the second method involves penetrating the islet injection needle through the fat tissue first and then into the portal vein by using the fat tissue as a physical barrier to stop bleeding. Both methods could effectively prevent bleeding-induced mouse death. The whole liver section showing islet distribution and evidence of islet thrombosis post-transplantation, a typical feature for intrahepatic islet transplantation, were presented. These improved protocols refine the intrahepatic islet transplantation procedures and may help laboratories set up the procedure to study islet survival and function in pre-clinical settings.

Here we present refined surgical procedures on successfully performing intraportal islet transplantation, a clinically relevant but technically challenging surgical procedure, in mice.

Although the liver is currently accepted as the primary transplantation site for human islets in clinical settings, islets are transplanted under the ...

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging study, with an abdominal imaging window, enabled visualization of the cellular dynamics in abdominal organs in vivo. However, due to the soft sheet-like architecture of the mouse pancreas that can be easily influenced by physiologic movement (e.g., peristalsis and respiration), it was difficult to perform stabilized longitudinal in vivo imaging over several weeks at the cellular level to identify, track, and quantify islets or cancer cells in the mouse pancreas. Herein, we describe a method for implanting a novel supporting base, an integrated pancreatic intravital imaging window, that can spatially separate the pancreas from the bowel for longitudinal time-lapse intravital imaging of the pancreas microstructure. Longitudinal in vivo imaging with the imaging window enables stable visualization, allowing for the tracking of islets over a period of 3 weeks and high-resolution three-dimensional imaging of the microstructure, as evidenced here in an orthotopic pancreatic cancer model. With our method, further intravital imaging studies can elucidate the pathophysiology of various diseases involving the pancreas at the cellular level.

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

In vivo high-resolution imaging of the pancreas was facilitated with the pancreatic intravital imaging window.

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

A Method for Islet Transplantation to the Omentum in Mouse

Islet transplantation has been proposed to be a potential treatment for type 1 diabetes. Recent compelling evidence indicates that intravascular islet infusion is far from ideal and therefore, the omentum is re-emerging as a potentially valuable site for islet transplantation. This experiment requires the isolation of high quality islets and the implantation of the islets to the diabetic recipients. Transplantation to the omentum requires surgical steps that can be better demonstrated visually. Here, the detailed steps for this procedure are presented. Two methods of mixing the isolated islets with hydrogel before placing the mixture into the omental pouch of diabetic mice are described here. Different hydrogels are used for the different conditions. Blood glucose levels of diabetic mouse recipients of syngeneic islets in the omentum were monitored for up to 35 days. Some animals were sacrificed after 14 days to perform immuno-histochemical analysis. This pre-clinical transplantation approach can be used as preliminary data leading up to translation to clinical transplantation.

A method for the omental transplantation of islets in a mouse is introduced. The isolated islets are mixed with hydrogel and the mixture is placed into the omental pouch of a diabetic mouse. Then, the blood glucose is monitored, and immuno-histochemical analysis is performed.

Islet transplantation has been proposed to be a potential treatment for type 1 diabetes. Recent compelling evidence indicates that intravascular islet ...

Grafting Pancreatic Islets: A Method to Implant Pancreatic Islets into the Anterior Chamber of the Mouse Eye for In Vivo Imaging

Source: Nilsson, J.et. al.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/v/61234/" target="_blank">A Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the Anterior Eye Chamber</a>. J. Vis. Exp. (2020).
This video describes the protocol for grafting human pancreatic islets into the anterior chamber of the mouse&#8217;s eye to monitor revascularization of human islet grafts in vivo. It also helps to understand the contribution of recipient versus donor cells in promoting the encapsulation and vascularization of the graft.

Source: Nilsson, J.et. al.&#160;A Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the ...

Intraportal Transplantation of Pancreatic Islets in Mouse Model

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