This work was supported by grants from National Key R&#38;D Program of China (2017YFC1103704)Special Funds for the Construction of High Level Hospitals in Guangdong Province (2019), Sanming Project of Medicine in Shenzhen (SZSM201412020), Fund for High Level Medical Discipline Construction of Shenzhen (2016031638), Shenzhen Foundation of Science and Technology (JCJY20160229204849975, GJHZ20170314171357556), Shenzhen Foundation of Health and Family Planning Commission (SZXJ2017021SZXJ2018059), Medical Scientific Research Foundation of Guangdong Province of China (A2019218), China Postdoctoral Science Foundation (2018M633218).

All procedures in this protocol followed the principles of animal welfare of the Ethics Review Committee of Shenzhen Second People&#39;s Hospital. Islet graft recipients and donors were 8- to 10-week-old C57BL/6 male mice purchased from the Medical Animal Center of Guangdong Province. The procedure of the harvesting, isolation, culture, or administration of the harvested cells were carried out in aseptic conditions.
1. Islet preparation
<ol>
	<li>Prepare a collagenase Type V working solution. Weigh collagenase Type V and dissolve with D-Hank&#39;s buffer to a final concentration of 1 mg/mL, filter using a 0.22 &#956;m syringe-driven filter unit and a 60 mL syringe and precool in ice. A volume of 5 mL for each donor recipient is required.</li>
	<li>Euthanize a 10-week-old C57BL/6 male mouse (23 &#177; 2 g) by cervical dislocation and spray the external part of the mouse with 75% ethanol for a few seconds. Meanwhile, fill a 5 mL syringe with collagenase solution and change the syringe needle with a bending blunt-pointed perfusion needle (32 G).</li>
	<li>Put the donor mouse in the supine position on an ice bag under the dissecting scope, cut a transverse opening using straight pointed ophthalmic scissors in the skin of the pubic area, and completely incise the skin towards the head. Then completely open the abdomen via a V-incision from the pubic region to the xiphoid process.</li>
	<li>Expose the gall bladder and the entire length of the common bile duct by repositioning the liver. Then clamp the duodenal opening of the common bile duct with a vascular clamp and cut a small opening in the gallbladder. 
	NOTE: Optimal needle placement is important to prevent backflow into the liver and gallbladder.</li>
	<li>Cannulate the common bile duct from the gall bladder opening using the perfusion needle in step 1.2, then inject ~2 mL of collagenase solution into the pancreas. After that, separate the perfused pancreas from the intestines, stomach, and spleen using two pairs of noninvasive microtweezers, and put it into a 50 mL conical tube on ice. 
	NOTE: Repeat the process for all donor mice. Three consecutively perfused pancreases (no more than 40 min apart) can be combined into a 50 mL conical tube prefilled with 3 mL of collagenase solution for each pancreas.</li>
	<li>Add 100 &#956;L DNase I (10 mg/mL) per pancreas into the 50 mL conical tubes, and digest the pancreases in a water bath at 37 &#176;C by shaking the conical tubes for 3&#8211;5 min. 
	NOTE: Shake the tubes vigorously 40x in 10 s intervals to disassociate the tissue prior to digestion in the water bath, and then moderately shake the tubes during digestion.</li>
	<li>Add stop solution (2.5 mg/mL BSA-HBSS solution) up to a final volume of 50 mL to block the digestion, and pulse centrifuge the tubes to a speed of 750 x g at 4 &#176;C and quickly stop.</li>
	<li>Pour off the supernatant and wash the pellets 2x by gently resuspending in 15&#8211;25 mL of BSA-HBSS. Pulse centrifuge the tube with the same speed conditions as outlined in step 1.7 for 1 min.</li>
	<li>Pour off the supernatant and pool the pellets of the three conical tubes into a 50 mL conical tube. Resuspend the pellets in a total volume of 10 mL of histopaque-1119. Mix homogeneously, and sequentially add 5 mL of histopaque-1077 and 5 mL of HBSS by pipetting slowly along the side of the tube.</li>
	<li>Spin the samples in the centrifuge at 750 x g at 4 &#176;C for 10 min without brakes.</li>
	<li>Aspirate the islets from the HBSS/histopaque-1077 interface into a 50 mL conical tube using a disposable 5 mL Pasteur pipette, add BSA-HBSS to a final volume of 50 mL, and pulse centrifuge at 750 x g at 4 &#176;C.</li>
	<li>Pour off the supernatant and wash the islets using 30 mL of BSA-HBSS. Centrifuge for 1 min at 750 x g at 4 &#176;C.</li>
	<li>Resuspend the islets using 30 mL of culture medium (10% FBS-1%P/S-CMRL-1066) per tube and pour in a 10 cm diameter light-tight culture dish. Under a stereomicroscope, using 200 &#956;L gel-loading pipet tips, handpick the islets from the solution based on their morphology (i.e., spheroidal, white). Put into an untreated cell culture dish with 10 mL of culture medium. 
	NOTE: A second handpicking can be carried out if the purity of first-purified islets is not optimal after the first picking.</li>
	<li>Check the purity of the handpicked islets by dithizone staining under a light microscope and detect the viability of islets by fluorescein diacetate (FDA)-propidium iodide (PI) staining under a fluorescent microscope.</li>
	<li>Culture the isolated islets in culture medium (as in step 1.13) in an incubator at 37 &#176;C, 95% air-5% CO2 before transplantation.</li>
</ol>
2. ISWAT islet transplantation
<ol>
	<li>Induce diabetes in the 8-week-old male C57BL/6 mice (22 &#177; 2 g) by a single injection of streptozotocin (STZ). On day -4, fast the mice for ~4&#8211;6 h, then prepare a 2% STZ solution using 0.1 M citrate buffer (pH 4.4). Weigh, resuspend in buffer, and inject the mice intraperitoneally at a dose of 180 mg/kg. 
	NOTE: STZ solution needs to be freshly prepared before use and covered with aluminum foil due to its light sensitivity. It should be used immediately, because it will lose activity within 15&#8211;20 min.</li>
	<li>Puncture the caudal veins of the STZ-injected mice to collect some blood at around 10 AM of day -1 and day 0 and measure the non-fasting blood glucose level using a test strip and a basic blood glucose monitoring instrument. The mice will be used as islet transplantation recipients if the blood glucose levels of the 2 consecutive test days are both at least 20 mmol/L.</li>
	<li>On day 0, weigh and mark all the recipient mice. Anesthetize each recipient by injecting 60 mg/kg pentobarbital sodium intraperitoneally. 
	NOTE: Administer a toe pinch to check the depth of anesthesia. If the recipient mouse has no withdrawal reflex, the level of anesthesia is enough for surgery. If not, administer an additional 10 mg/kg pentobarbital sodium. The pentobarbital sodium used was diluted in sterile physiologic saline at the concentration of 2% (m/v).</li>
	<li>Take the hydrogel out from a -20 &#176;C freezer and keep it on ice to allow thawing. The hydrogel is liquid at 4&#8211;10 &#176;C and will solidify at a higher temperature.</li>
	<li>Pick ~450&#8211;500 islet equivalents (IEQ) for each recipient into a sterile 1.5 mL centrifuge tube under the stereomicroscope (as in step 1.13) with 200 &#181;L of culture medium and keep on ice until ready for transplantation.</li>
	<li>Swab the left inguinal area of the recipient using 75% ethanol. Place it in the supine position and fix the four limbs using surgical tape. Shave off the hair around the surgical site with electrical clippers and swab the area with Iodophor.</li>
	<li>Make a vertical skin incision in this area using the ophthalmic scissors and noninvasive microtweezers, identify the inferior epigastric artery and vein in the ISWAT, and create a small pocket above the vessels.</li>
	<li>Spin the islets tube for 30 s at 200 x g and remove the supernatant as much as possible. Aspirate 20 &#181;L of completely thawed hydrogel and load it into the tube with the islets. Resuspend the islets gently, avoiding bubbles.</li>
	<li>Deliver the entire islet-hydrogel mixture in the tube into the pocket (step 2.7) of the recipient with a 200 &#181;L pipette tip. 
	NOTE: This process requires two technicians, one for picking up the edges of the pocket using noninvasive microtweezers, and the other for delivering the islet mixture into the pocket.</li>
	<li>Add 20 &#181;L of cephalosporin (~5&#8211;10 mg) into the transplantation site after the islets-hydrogel mixture is completely solidified, then use a 5-0 surgical suture to close the muscle and skin with the continuous suture method. 
	NOTE: The hydrogel needs about 3 min to solidify due to the body temperature.</li>
	<li>Place the recipient into a clean cage and keep warm using a thermal pad. Keep monitoring until the recipient fully recovers and starts to move autonomously. Then administer 0.03 mg/ml Buprenorphine with sterile physiological saline intraperitoneally, according to 0.1 mg/Kg dose.</li>
	<li>Repeat steps 2.3&#8211;2.11 for each recipient mouse.</li>
	<li>Measure the non-fasting blood glucose levels of the recipient mice (as in step 2.2) 3&#8211;4x a week for the first month, and 1x a week thereafter.</li>
	<li>At the end of the follow-up (100 days after transplantation), under anesthesia (carried out as in step 2.3) excise the ISWAT bearing the graft from the recipients following the sterile steps described in step 2.6. Fix the tissue in formalin and paraformaldehyde according to the histological protocols for hematoxylin and eosin (H&#38;E) staining and immunofluorescence of insulin and glucagon.</li>
	<li>Add cephalosporin and close the incision of the recipients as in step 2.10, then prepare for recovery as in step 2.11.</li>
</ol>

<ol>
	<li>Cho, N. H., 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=IDF+Diabetes+Atlas:+Global+estimates+of+diabetes+prevalence+for+2017+and+projections+for+2045.">IDF Diabetes Atlas: Global estimates of diabetes prevalence for 2017 and projections for 2045.</a> Diabetes Research and Clinical Practice. 138, 271-281 (2018).</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>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), 007823 (2012).</li><li>Pathak, V., Pathak, N. M., O'Neill, C. L., Guduric-Fuchs, J., Medina, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapies+for+Type+1+Diabetes:+Current+Scenario+and+Future+Perspectives.">Therapies for Type 1 Diabetes: Current Scenario and Future Perspectives.</a> Clinical Medicine Insights: Endocrinology and Diabetes. 12, 1179551419844521 (2019).</li><li>Bottino, R., Knoll, M. F., Knoll, C. A., Bertera, S., Trucco, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Future+of+Islet+Transplantation+Is+Now.">The Future of Islet Transplantation Is Now.</a> Frontiers in Medicine (Lausanne). 5, 202 (2018).</li><li>Stokes, R. A., 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=Transplantation+sites+for+human+and+murine+islets.">Transplantation sites for human and murine islets.</a> Diabetologia. 60 (10), 1961-1971 (2017).</li><li>van der Windt, D. J., Echeverri, G. J., Ijzermans, J. N., Cooper, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+choice+of+anatomical+site+for+islet+transplantation.">The choice of anatomical site for islet transplantation.</a> Cell Transplantation. 17 (9), 1005-1014 (2008).</li><li>Addison, P., Fatakhova, K., Rodriguez Rilo, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Considerations+for+an+Alternative+Site+of+Islet+Cell+Transplantation.">Considerations for an Alternative Site of Islet Cell Transplantation.</a> Journal of Diabetes Science and Technology. , (2019).</li><li>Pepper, A. R., Bruni, A., Shapiro, A. M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+islet+transplantation:+is+the+future+finally+now.">Clinical islet transplantation: is the future finally now.</a> Current Opinion in Organ Transplantation. 23 (4), 428-439 (2018).</li><li>Bellin, M. D., 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=Similar+islet+function+in+islet+allotransplant+and+autotransplant+recipients,+despite+lower+islet+mass+in+autotransplants.">Similar islet function in islet allotransplant and autotransplant recipients, despite lower islet mass in autotransplants.</a> Transplantation. 91 (3), 367-372 (2011).</li><li>Bruni, A., Gala-Lopez, B., Pepper, A. R., Abualhassan, N. S., Shapiro, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+cell+transplantation+for+the+treatment+of+type+1+diabetes:+recent+advances+and+future+challenges.">Islet cell transplantation for the treatment of type 1 diabetes: recent advances and future challenges.</a> Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy. 7, 211-223 (2014).</li><li>Smood, B., Bottino, R., Hara, H., Cooper, D. K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+the+renal+subcapsular+space+the+preferred+site+for+clinical+porcine+islet+xenotransplantation?+Review+article.">Is the renal subcapsular space the preferred site for clinical porcine islet xenotransplantation? Review article.</a> International Journal of Surgery and Medicine. 69, 100-107 (2019).</li><li>Luan, N. M., Iwata, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+allogeneic+islet+graft+survival+in+prevascularized+subcutaneous+sites+without+immunosuppressive+treatment.">Long-term allogeneic islet graft survival in prevascularized subcutaneous sites without immunosuppressive treatment.</a> American Journal of Transplantation. 14 (7), 1533-1542 (2014).</li><li>Yasunami, Y., 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=A+Novel+Subcutaneous+Site+of+Islet+Transplantation+Superior+to+the+Liver.">A Novel Subcutaneous Site of Islet Transplantation Superior to the Liver.</a> Transplantation. 102 (6), 945-952 (2018).</li><li>Rajab, A. <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:+alternative+sites.">Islet transplantation: alternative sites.</a> Current Diabetes Reports. 10 (5), 332-337 (2010).</li><li>Ekser, B., Vagefi, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Search+for+the+best+site+in+islet+xenotransplantation.">Search for the best site in islet xenotransplantation.</a> International Journal of Surgery and Medicine. 70, 106-107 (2019).</li><li>Lu, Y., 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=A+Method+for+Islet+Transplantation+to+the+Omentum+in+Mouse.">A Method for Islet Transplantation to the Omentum in Mouse.</a> Journal of Visualized Experiments. (143), e57160 (2019).</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), e2096 (2011).</li><li>Khatri, R., Hussmann, B., Rawat, D., Gurol, A. O., 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=Intraportal+Transplantation+of+Pancreatic+Islets+in+Mouse+Model.">Intraportal Transplantation of Pancreatic Islets in Mouse Model.</a> Journal of Visualized Experiments. (135), e57559 (2018).</li><li>Carter, J. D., Dula, S. B., Corbin, K. L., Wu, R., Nunemaker, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+guide+to+rodent+islet+isolation+and+assessment.">A practical guide to rodent islet isolation and assessment.</a> Biological Procedures Online. 11, 3-31 (2009).</li></ol>

The authors report no conflicts of interest.

&#160; &#160;

Pancreas islet transplantation is a promising therapy to treat T1DM. The effect of this therapy is affected by many factors and choosing an optimal site for islet implantation is extremely important. The ideal anatomical site for islet transplantation should have the following characteristics: accessibility for simple transplantation, biopsy, and graft retrieval procedures; reduced complications; high success rate of blood glucose control; and long-term survival of the islet grafts15,<s...

The worldwide incidence and prevalence of type 1 diabetes mellitus (T1DM) are quickly rising, according to the statistical data of the International Diabetes Federation (IDF)1. Islet transplantation is one of the most promising approaches for treating T1DM4. Since the great breakthrough made in clinical islet transplantation using the Edmonton protocol2 was reported, functioning islet graft survival in T1DM recipients after 5 years now reaches about 50%3.
In the past, several transplantation sites, such as the liver, kidney capsule, spleen, intramuscular region, subcutaneous space, bone marrow, and omental pouch were explored for experimental islet transplantation5,6,7. Some of the above sites have been tested in clinical settings8. Although islet transplantation into the liver remains the most widely used method in clinical application at present9, there are several important problems to address when using this site. For instance, how to reduce early loss of the transplanted islets caused by the instant blood mediated inflammatory reaction (IBMIR) and poor oxygenation supply10,11 and how to retrieve the islet grafts if necessary, because they diffusely localize in the liver. The renal capsule may be an ideal site for rodent recipients. However, the tight kidney capsule limits the transplantation of sufficient allogeneic islets in humans, although it may be a better fit for islet xenotransplantation due to the highly purified porcine islet preparations used clinically5,12. Therefore, the search for a more suitable site for islet transplantation is in progress.
The subcutaneous space may be used as a clinically applicable site for islet transplantation due to its accessibility. However, the efficiency of islet transplantation into the subcutaneous space is extremely low, thus requiring a relatively large number of islets to reverse hyperglycemia13. Recently, a Japanese research team found the ISWAT, a novel subcutaneous site superior for islet transplantation in a murine model when compared to the liver14. The ISWAT contains the epigastric artery and vein, so the rich blood supply may ensure islet graft revascularization. In this manuscript, we propose an easy implantation method using a basement membrane matrix hydrogel to fix syngeneic murine islets in the ISWAT. This protocol proves effective for islet transplantation.

<table><tbody><tr><td>0.22 &amp;mu;m Syringe-driven Filter Unit</td><td>Merck Millipore</td><td>SLHV033RB</td><td></td></tr><tr><td>1.5 mL centrifuge tube</td><td>Axygen</td><td>MCT-150-C</td><td></td></tr><tr><td>5 mL Pasteur pipette</td><td>JingAn Biological, China</td><td>J00085</td><td></td></tr><tr><td>5 mL syringe</td><td>Szboon, China</td><td>20170829</td><td></td></tr><tr><td>50 mL conical tube</td><td>Corning</td><td>430829</td><td></td></tr><tr><td>5-0 surgical suture</td><td>sh-Jinhuan, China</td><td>CR537</td><td></td></tr><tr><td>60 mL syringe</td><td>Szboon, China</td><td>20170623</td><td></td></tr><tr><td>75% Ethanol</td><td>LIRCON, China</td><td>9180527</td><td></td></tr><tr><td>Alexa Fluor 488 donkey anti-mouse IgG(H+L)</td><td>Invitrogen</td><td>A21202</td><td>Dilution (1:200)</td></tr><tr><td>anti-mouse Glucagon antibody</td><td>Abcam</td><td>ab10988</td><td>Dilution (1:100)</td></tr><tr><td>anti-mouse insulin antibody</td><td>Cell Signaling Technology</td><td>3014s</td><td>Dilution (1:100)</td></tr><tr><td>blunt-pointed perfusion needle</td><td>Oloey, China</td><td>005</td><td>32G, yellow</td></tr><tr><td>BSA</td><td>Meilune, China</td><td>MB4219</td><td></td></tr><tr><td>C57BL/6 Mice</td><td>Medical Animal Center of Guangdong Province</td><td></td><td>8~10 weeks</td></tr><tr><td>cell culture dish</td><td>BIOFIL, China</td><td>TCD000100</td><td>General,Non-treated,87.8 mm diameter</td></tr><tr><td>centrifuge</td><td>Thermo Scientific</td><td>ST16R</td><td></td></tr><tr><td>cephalosporin</td><td>Lukang medical, China</td><td>150303</td><td></td></tr><tr><td>CMRL-1066</td><td>Sigma-Aldrich</td><td>C0422</td><td></td></tr><tr><td>Codos Pet Clipper</td><td>Szcodos, China</td><td>CP-8000</td><td></td></tr><tr><td>collagenase Type V</td><td>Sigma</td><td>C9262</td><td></td></tr><tr><td>DAPI</td><td>Thermo Fisher</td><td>D1306</td><td></td></tr><tr><td>D-hank&#039;s buffer</td><td>Coolaber, China</td><td>PM5140-10</td><td></td></tr><tr><td>dithizone</td><td>Sigma-Aldrich</td><td>D5130</td><td></td></tr><tr><td>Dnase I</td><td>Sigma-Aldrich</td><td>D4263</td><td></td></tr><tr><td>Eosin staining media</td><td>Beyotime Biotech, China</td><td>C0109</td><td></td></tr><tr><td>FBS</td><td>GE Healthcare Life Sciences</td><td>SH30084</td><td></td></tr><tr><td>fluorescein diacetate (FDA)</td><td>Thermo Fisher</td><td>F1303</td><td></td></tr><tr><td>fluorescent microscope</td><td>Leica</td><td>DMIL</td><td></td></tr><tr><td>gel-loading pipet tips</td><td>Corning</td><td>CLS4884</td><td></td></tr><tr><td>HBSS</td><td>Coolaber, China</td><td>PM5150-10</td><td></td></tr><tr><td>hematoxylin staining media</td><td>Cell Signaling Technology</td><td>14166S</td><td></td></tr><tr><td>HISTOPAQUE-1077</td><td>Sigma-Aldrich</td><td>RNBG0522</td><td></td></tr><tr><td>HISTOPAQUE-1119</td><td>Sigma-Aldrich</td><td>RNBG0536</td><td></td></tr><tr><td>Hydrogel</td><td>BD Biosciences</td><td>356234</td><td>Basement Membrane Matrix</td></tr><tr><td>Iodophor</td><td>LIRCON, China</td><td>5190313</td><td></td></tr><tr><td>light-tight culture dish</td><td>DVS, China</td><td>AN-5058548</td><td>self-made, glass dish sprayed with black paint</td></tr><tr><td>Medical Adhesive Tape</td><td>Cofoe, China</td><td>K12001</td><td></td></tr><tr><td>non-invasive microtweezers</td><td>RWD Life Science</td><td>F11033-11 and F12016-15</td><td></td></tr><tr><td>One Touch ultraeasy Basic blood glucose monitoring system</td><td>Johnson &amp;amp; Johnson</td><td>33391713</td><td></td></tr><tr><td>ophthalmic scissors</td><td>RWD Life Science</td><td>S12012-12 and S11001-08</td><td></td></tr><tr><td>P/S (penicillin / streptomycin)</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>pentobarbital sodium</td><td>Sigma-Aldrich</td><td>P-010</td><td></td></tr><tr><td>Propidium iodide</td><td>Sigma-Aldrich</td><td>P4864</td><td></td></tr><tr><td>STZ (streptozotocin)</td><td>Sigma-Aldrich</td><td>S0130</td><td></td></tr><tr><td>Test Strip</td><td>GenUltimate</td><td>100-50</td><td></td></tr><tr><td>TRITC-conjugated Goat anti-Rabbit IgG(H+L)</td><td>proteintech</td><td>SA00007-2</td><td>Dilution (1:200)</td></tr><tr><td>vascular clamp</td><td>RWD Life Science</td><td>R31006-04</td><td></td></tr></tbody></table>

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.

Two procedures are introduced in this protocol: murine islet preparation and islet transplantation into the ISWAT site. In the first procedure, after perfusing and digesting with Type V collagenase solution, purifying with Histopaque-1119 and Histopaque-1077 and an additional hand-picking step, the isolated murine islets will be sufficiently pure for transplantation (as shown in Figure 1) and the isolated islets that have a high viability will be used for transplantation (as shown in <strong...

Watch this Scientific Journal Video about Inguinal Subcutaneous White Adipose Tissue ISWAT Transplantation Model of Murine Islets at JoVE.com

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.

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 ...

inguinal-subcutaneous-white-adipose-tissue-iswat-transplantation

Research

JoVE Journal

Medicine

6.3K Views.  First Affiliated Hospital of Shenzhen University, Health Science Center. Eilandje transplantatie is een van de meest veelbelovende benaderingen voor de behandeling van type I diabetes mellitus. En de zoektocht naar een geschikte locatie voor eilandjestransplantatie is aan de gang. Eilandjetransplantatie in het inguinale onderhuidse witte vetweefsel is een eenvoudige maar effectieve methode die klinische implicaties kan hebben.Extrapolatie van deze procedure naar de kliniek kan leiden tot de ontwikkeling van een remedie voor type I diabetici. Deze methode ka...

Video: Inguinal Subcutaneous White Adipose Tissue ISWAT Transplantation Model of Murine Islets

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 ...

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

Localization, Identification, and Excision of Murine Adipose Depots

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 reached a staggering sum of $190.2 billion in direct medical costs alone. Obesity is a major risk factor for a wide host of diseases. Historically, little was known regarding adipose and its major and essential functions in the body. Brown and white adipose are the two main types of adipose but current literature has identified a new type of fat called brite or beige adipose. Research has shown that adipose depots have specific metabolic profiles and certain depots allow for a propensity for obesity and other related disorders. The goal of this protocol is to provide researchers the capacity to identify and excise adipose depots that will allow for the analysis of different factorial effects on adipose; as well as the beneficial or detrimental role adipose plays in disease and overall health. Isolation and excision of adipose depots allows investigators to look at gross morphological changes as well as histological changes. The adipose isolated can also be used for molecular studies to evaluate transcriptional and translational change or for in vitro experimentation to discover targets of interest and mechanisms of action. This technique is superior to other published techniques due to the design allowing for isolation of multiple depots with simplicity and minimal contamination.

Due to the drastic and negative connection between obesity and other comorbidities, research on the role adipose plays in disease and overall health is warranted. We present a protocol for the isolation and excision of adipose depots allowing for the study of adipose using in situ and in vitro methods.

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 ...

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular weight metabolites in tissues of the treated host to achieve long-term survival. The semipermeable membrane allows engrafted encapsulated cells to avoid rejection by the immune system. The encapsulation procedure was designed to enable a controlled release of bioactive compounds, such as insulin, other hormones, and cytokines. Here we describe a method for encapsulation of catabolic cells, which consume lipids for heat production and energy dissipation (thermogenesis) in the intra-abdominal adipose tissue of obese mice. Encapsulation of thermogenic catabolic cells may be potentially applicable to the prevention and treatment of obesity and type 2 diabetes. Another potential application of catabolic cells may include detoxification from alcohols or other toxic metabolites and environmental pollutants.

Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular ...

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.

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 ...

Investigation of Beige Fat Biology and Metabolism Using the CRISPR SunTag-p65-HSF1 Activation System

Clustered regularly interspaced short palindromic repeats (CRISPR) technology has prompted a revolution in biology, and recent tools have been applied far beyond the originally described gene editing. The CRISPR activation (CRISPRa) system combines the catalytically inactive Cas9 (dCas9) protein with distinct transcription modules to induce endogenous gene expression. SunTag-p65-HSF1 (SPH) is a recently developed CRISPRa technology that combines components of synergistic activation mediators (SAMs) with the SunTag activators. This system allows the overexpression of single or multiple genes by designing a customized single-guide RNA (sgRNA). In this study, a previously developed SPH mouse was used to generate a conditional mouse expressing SPH in adipocytes (adiponectin Cre lineage), named AdipoSPH. To induce a white-to-beige fat (browning) phenotype, an adeno-associated virus (AAV) carrying sgRNA targeting the endogenous Prdm16 gene (a well-established transcription factor related to brown and beige fat development) was injected into the inguinal white adipose tissue (iWAT). This mouse model induced the expression of endogenous Prdm16 and activated the thermogenic gene program. Moreover, in vitro SPH-induced Prdm16 overexpression enhanced the oxygen consumption of beige adipocytes, phenocopying the results of a previous Prdm16 transgenic mouse model. Thus, this protocol describes a versatile, cost-effective, and time-effective mouse model for investigating adipose tissue biology.

This protocol presents the use of CRISPR SunTag-p65-HSF1 (SPH) in adipocytes (AdipoSPH) as an alternative strategy to adeno-associated virus (AAV) for investigating beige fat biology. In vivo injection of AAV-carrying sgRNA targeting the endogenous Prdm16 gene is sufficient to induce beige fat development and enhance the thermogenic gene program.

Clustered regularly interspaced short palindromic repeats (CRISPR) technology has prompted a revolution in biology, and recent tools have been applied far ...

Fat-Covered Islet Transplantation Using Epididymal White Adipose Tissue

Islet transplantation is a cellular replacement therapy for severe diabetes mellitus. The intraperitoneal cavity is typically the transplant site for this procedure. However, intraperitoneal islet transplantation has some limitations, including poor transplant efficacy, difficult graft detection ability, and a lack of graftectomy capability for post-transplant analysis. In this paper, "fat-covered islet transplantation", an intraperitoneal islet transplantation method that utilizes epididymal white adipose tissue, is used to assess the therapeutic effects of bioengineered islets. The simplicity of the method lies in the seeding of islets onto epididymal white adipose tissue and using the tissue to cover the islets. While this method can be categorized as an intraperitoneal islet transplantation technique, it shares characteristics with intra-adipose tissue islet transplantation. The fat-covered islet transplantation method demonstrates more robust therapeutic effects than intra-adipose tissue islet transplantation, however, including the improvement of blood glucose and plasma insulin levels and the potential for graft removal. We recommend the adoption of this method for assessing the mechanisms of islet engraftment into white adipose tissue and the therapeutic effects of bioengineered islets.

This fat-covered islet transplantation method is suitable for the detection of engrafted islets in the intraperitoneal cavity. Notably, it does not require the use of biobinding agents or suturing.

Islet transplantation is a cellular replacement therapy for severe diabetes mellitus. The intraperitoneal cavity is typically the transplant site for this ...

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 ...

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 ...

Biology

Semi-Automated Isolation of Murine White Adipose Tissue

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their mechanisms. Immortalized white preadipocyte cell lines are publicly available and widely used. However, the emergence of beige adipocytes in white adipose tissue in response to external cues is difficult to recapitulate to the full extent using publicly available white adipocyte cell lines. Isolation of the stromal vascular fraction (SVF) from murine adipose tissue is commonly executed to obtain primary preadipocytes and perform adipocyte differentiation. However, mincing and collagenase digestion of adipose tissue by hand can result in experimental variation and is prone to contamination. Here, we present a modified semi-automated protocol that utilizes a tissue dissociator for collagenase digestion to achieve easier isolation of the SVF, with the aim of reducing experimental variation, reducing contamination, and increasing reproducibility. The obtained preadipocytes and differentiated adipocytes can be used for functional and mechanistic analyses.

Author Spotlight: Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator  

This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases reproducibility.

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their ...

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

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video