Some of the authors of this work were supported in part by grants from National Key R&#38;D Program of China (2017YFC1103704), 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, JCYJ20160425110110658), Shenzhen Foundation of Health and Family Planning Commission (SZXJ2017021).

All mice used in this study were obtained from the Medical Animal Center of Guangdong Province. The use of animals was approved by the Ethics Review Committee of Shenzhen Second People&#39;s Hospital, in accordance with the principles of animal welfare.
1. Islet Transplantation to the Omentum
NOTE: This protocol requires 2 persons to accomplish.
<ol>
	<li>Assemble surgical materials which are listed in Table 1. Prepare aseptic field in surgical area with sterile materials such as drapes and disposables and maintain aseptic conditions throughout surgery. Sterilize all surgical instruments. Wear sterile gowns.</li>
	<li>Pick the islets using a 200 &#181;L pipette tip under the stereomicroscope. Each mouse will receive 450&#8211;500 Islet equivalent (IEQ) per tansplant. For each animal, place enough islets for a single transplant into a sterile 1.5 mL snap-top tube with 100 &#181;L of CMRL-1066 medium. 
	NOTE: Islets can be isolated on the day of transplantation, but it is better to isolate them the day before to allow recovery for the isolation process.</li>
	<li>Keep the snap-top tubes with islets on ice until ready to transplant.</li>
	<li>Thaw the basement membrane matrix hydrogel and keep it on ice after removing it from the -20 &#176;C freezer. The hydrogel is liquid at 4 &#176;C to 10 &#176;C and solidifies at higher temperatures.</li>
	<li>Weigh and tag all diabetic recipient mice. Inject 60 mg/kg pentobarbital sodium intraperitoneally. Test the the depth of anesthesia by administering a toe pinch. Give an additional 10 mg/kg pentobarbital sodium if the animal reacts to the pinch. If there is no withdraw reflex, the level of anesthesia is correct for surgery.</li>
	<li>Swab the surgical site on the abdomen using 70% ethanol. Shave the hair from the site with a razer blade and disinfect the area with Iodophor. Administer vet ointment to the eyes to prevent dryness while under anesthesia.</li>
	<li>Use the ophthalmic scissors to open the abdomen along the abdomen midline with an incision of 4 to 5 cm. Move the intestines to the left side and cover with saline soaked gauze to prevent excessive dehydration during the surgery procedure.</li>
	<li>Use cotton swabs to fully expose the visual field of the stomach and locate the omentum (located below the stomach). Use two pair of fine forceps to distend the omentum. Take care to prevent damage from tearing.</li>
	<li>The hydrogel is completely thawed at this point. Spin the tube with the islets for 30 s at 200 x g and remove the supernatant. Aspirate 50 &#181;L of hydrogel, add it to the tube containing the islets, and resuspend the mix gently avoiding the formation of bubbles. Keep the tubes on ice during the procedure.</li>
	<li>Use two pair of forceps to pick up the edges of the omentum and gently raise it to form a groove between the gastric wall and intestines that can accommodate a small volume of liquid with the islet graft.</li>
	<li>Let the second person assist in the procedure and aspirate the resuspended islet-hydrogel mixture (the entire contents of the tube) with a 200 &#181;L pipette tip, Deliver the content into the groove.</li>
	<li>Ensure that the mixture is well positioned into the groove by gently raising or lowering the edges of the omentum. Complete positioning of the mixture within 3 min before the hydrogel solidifies as an effect of the body temperature. After the hydrogel sets, fold the omentum to cover the graft. 
	NOTE:The omentum will adhere to the surrounding gastric wall as the hydrogel solidifies.</li>
	<li>After the hydrogel is completely solidified, use cotton swabs to reposition the intestines back into the abdominal cavity, taking care not to touch the site of transplantation.</li>
	<li>Add 20 &#181;L of cephalosporin (5~10 mg) into the abdominal cavity to prevent infection, then use 4-0 suture to close the abdomen.</li>
	<li>Return the mouse to its cage and repeat all steps for each mouse recipient. Keep the mice warm and monitor visually until they fully regain sufficient consciousness to maintain sternal recumbency. Keep the mice separate from other animals until they have fully recovered.</li>
	<li>Inject 50 &#181;L of cefazolin sodium (0.05 mg/mL) each day for a week as post-surgery prophylaxis. Administer Bupivicaine + Buprenorphine, i.e. Apply 1-3 drops of 0.25% Bupivicaine at the incision site topically prior to placement of wound clips. Administer Buprenorphine&#65288;0.03 mg/ml with sterile 0.9% saline, 0.05-0.10 mg/kg&#65289;via the intraperitoneal (IP) route.&#160;</li>
	<li>Measure non-fasting blood glucose level from a tail-vein blood sample using a blood glucose meter once a day after the transplant. When transplanting to the omentum, the islet graft may have a delayed function and not reach a completely normal blood glucose level for 2&#8211;3 weeks.</li>
	<li>For histology, remove the omental graft from the mouse at the end of the experiment following the transplantation. Fix the tissue according to the histological protocols. The graft can be analyzed for immunostaining or immunofluorescence studies.</li>
</ol>
2. Alternative Method for Transplantation to the Omentum
NOTE: An alternative fibrin-thrombin hydrogel that is used at room temperature may be substituted for the basement membrane matrix hydrogel. It consists of 2 components, a fibrin sealer protein solution (50 U/mL thrombin and 10 mg/mL fibrinogen). When the components are mixed, they form a clot that holds the islets in place.
<ol>
	<li>Use the fibrin-thombin hydrogel compound at room temperature. As in step 1.2, each mouse will receive 450&#8211;500 islet equivalents (IEQ) per transplant. Place the islets in a sterile 1.5 mL snap-top tube with 100 &#181;L of CMRL-1066 medium. Immediately before transplantation, aspirate the islets into a sterile PE50 tubing for a length of 10 cm and centrifuge gently (30 s at 200 x g) to form a loose pellet.</li>
	<li>Prepare the animal as stated above (steps 1.5-1.8) with the omentum spread out. Mix the hydrogel components (10 &#181;L/each) and place on the omentum. (Figure 3D)</li>
	<li>Immediately expel the islets from the tubing onto the hydrogel. The hydrogel forms a clot around the islets. (Figure 3E)</li>
	<li>Fold the omentum over the hydrogel and islets to form a pouch. (Figure 3F) Continue with steps 1.13 to 1.18.</li>
</ol>

<ol>
	<li>Guariguata, L., 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=Global+estimates+of+diabetes+prevalence+for+2013+and+projections+for+2035.">Global estimates of diabetes prevalence for 2013 and projections for 2035.</a> Diabetes research and clinical practice. 103 (2), 137-149 (2014).</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=Potent+Induction+Immunotherapy+Promotes+Long-Term+Insulin+Independence+After+Islet+Transplantation+in+Type+1+Diabetes.">Potent Induction Immunotherapy Promotes Long-Term Insulin Independence After Islet Transplantation in Type 1 Diabetes.</a> American Journal of Transplantation. 12 (6), 1576-1583 (2012).</li><li>Moberg, L., 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=Production+of+tissue+factor+by+pancreatic+islet+cells+as+a+trigger+of+detrimental+thrombotic+reactions+in+clinical+islet+transplantation.">Production of tissue factor by pancreatic islet cells as a trigger of detrimental thrombotic reactions in clinical islet transplantation.</a> The lancet. 360 (9350), 2039-2045 (2002).</li><li>Nilsson, B., Ekdahl, K. N., Korsgren, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+instant+blood-mediated+inflammatory+reaction+to+improve+islets+of+Langerhans+engraftment.">Control of instant blood-mediated inflammatory reaction to improve islets of Langerhans engraftment.</a> Current Opinion in Organ Transplantation. 16 (6), 620-626 (2011).</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 (2014).</li><li>Shapiro, A. J., Pokrywczynska, M., Ricordi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+pancreatic+islet+transplantation.">Clinical pancreatic islet transplantation.</a> Nature reviews Endocrinology. 13 (5), 268-277 (2017).</li><li>Cantarelli, E., Piemonti, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+transplantation+sites+for+pancreatic+islet+grafts.">Alternative transplantation sites for pancreatic islet grafts.</a> Current diabetes reports. 11 (5), 364 (2011).</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=Murine+animal+models+for+preclinical+islet+transplantation:+no+model+fits+all+(research+purposes).">Murine animal models for preclinical islet transplantation: no model fits all (research purposes).</a> Islets. 5 (2), 79-86 (2013).</li><li>Beli, 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=Erratum+to:+CX3CR1+deficiency+accelerates+the+development+of+retinopathy+in+a+rodent+model+of+type+1+diabetes.">Erratum to: CX3CR1 deficiency accelerates the development of retinopathy in a rodent model of type 1 diabetes.</a> J Mol Med (Berl). 95 (5), 565-566 (2017).</li><li>Hafner, 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=Regional+Patterns+of+Retinal+Oxygen+Saturation+and+Microvascular+Hemodynamic+Parameters+Preceding+Retinopathy+in+Patients+With+Type+II+Diabetes.">Regional Patterns of Retinal Oxygen Saturation and Microvascular Hemodynamic Parameters Preceding Retinopathy in Patients With Type II Diabetes.</a> Invest Ophthalmol Vis Sci. 58 (12), 5541-5547 (2017).</li><li>Schmidt, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+islets+find+a+new+transplant+home+in+the+omentum.">Pancreatic islets find a new transplant home in the omentum.</a> Nature Biotechnology. 35 (1), 82017 (2017).</li><li>Voigt, 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=Prevalence+and+Progression+Rate+of+Diabetic+Retinopathy+in+Type+2+Diabetes+Patients+in+Correlation+with+the+Duration+of+Diabetes.">Prevalence and Progression Rate of Diabetic Retinopathy in Type 2 Diabetes Patients in Correlation with the Duration of Diabetes.</a> Exp Clin Endocrinol Diabetes. , (2017).</li><li>Baidal, D. 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=Bioengineering+of+an+Intraabdominal+Endocrine+Pancreas.">Bioengineering of an Intraabdominal Endocrine Pancreas.</a> New England Journal of Medicine. 376 (19), 1887-1889 (2017).</li><li>Espes, 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=Rapid+restoration+of+vascularity+and+oxygenation+in+mouse+and+human+islets+transplanted+to+omentum+may+contribute+to+their+superior+function+compared+to+intraportally+transplanted+islets.">Rapid restoration of vascularity and oxygenation in mouse and human islets transplanted to omentum may contribute to their superior function compared to intraportally transplanted islets.</a> American Journal of Transplantation. , (2016).</li><li>Berman, D. 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=Bioengineering+the+endocrine+pancreas:+intraomental+islet+transplantation+within+a+biologic+resorbable+scaffold.">Bioengineering the endocrine pancreas: intraomental islet transplantation within a biologic resorbable scaffold.</a> Diabetes. 65 (5), 1350-1361 (2016).</li><li>Delaune, V., Berney, T., Lacotte, S., Toso, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraportal+islet+transplantation:+the+impact+of+the+liver+micro-environment.">Intraportal islet transplantation: the impact of the liver micro-environment.</a> Transplant International. 30 (3), 227-238 (2017).</li><li>Espes, 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=Rapid+restoration+of+vascularity+and+oxygenation+in+mouse+and+human+islets+transplanted+to+omentum+may+contribute+to+their+superior+function+compared+to+intraportally+transplanted+islets.">Rapid restoration of vascularity and oxygenation in mouse and human islets transplanted to omentum may contribute to their superior function compared to intraportally transplanted islets.</a> American Journal of Transplantation. 16 (11), 3246-3254 (2016).</li><li>Hajizadeh-Saffar, 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=Inducible+VEGF+expression+by+human+embryonic+stem+cell-derived+mesenchymal+stromal+cells+reduces+the+minimal+islet+mass+required+to+reverse+diabetes.">Inducible VEGF expression by human embryonic stem cell-derived mesenchymal stromal cells reduces the minimal islet mass required to reverse diabetes.</a> Scientific Reports. 5, (2015).</li></ol>

The authors report no conflicts of interest.

Islet transplantation to the liver via the portal vein is the most commonly used method of islet transplantation in humans, but there are still efficiency and safety concerns such as portal vein thrombosis and liver steatosis17. Recent studies show that the omentum may be a suitable alternative to the liver, but more research needs to be conducted prior to clinical translation12,14,18,19.
The mouse is a suitable model for testing the omentum as a site for islet transplantation. In the mouse, the omentum is located under the stomach wall and is generally folded. However, the size of the omentum in mice is small and challenging to access. In order to use it for transplantation, it is necessary to gently spread it out to its full size then fold it over after the islets/hydrogel mix is deposited. In a larger animal, the omental tissue is more extensive and can be sutured to construct one or more pouches into which the islets are deposited. The mouse omentum is fragile and does not easily accept sutures without breaking. For this reason, a hydrogel is used to make the pouch without sutures.
In the first method (steps 1.1&#8211;1.18), the hydrogel used is a basement membrane matrix and is composed of laminin, collagen IV and growth factors. It solidifies as it warms to body temperature. In the second method (steps 2.1&#8211;2.4), a different hydrogel is used composed of 50U/mL thrombin and 10 mg/mL fibrinogen. Two different hydrogels are used to show the versatility of this technique. It may be tailored to fit many circumstances and to establish proof of principle. For clinical applications, whether a sutured pouch or a clinical-grade hydrogel is used, the technique can be combined with a minimally invasive approach, (e.g., an endoscopic method of delivery to the omentum). To this aim, additional studies using larger mammal pre-clinical models are required.
The time to normalize the blood glucose levels in the mouse recipients after transplantation in the omentum in our as well as other studies suggest that the islets in the omentum require slightly longer time to achieve sufficient insulin production. Revascularization efficiency appears to be one important factor, as outlined in the study. Whether the presence of a hydrogel affects the speed of islet revascularization and performance, that remains to be determined.

According to the International Diabetes Federation (IDF), diabetes mellitus currently affects 382 million people, with a projected increase to 592 million people by 20351. In both allogeneic and xenogeneic islet transplantation, systemic immunosuppressive therapy is necessary. Without immunosuppression, immune rejection is a major cause of graft loss2. There is also a significant problem of transplanted islet loss due to the instant blood mediated inflammatory reaction (IBMIR)3,4. However, even in the absence of an immune response such as in syngeneic or auto-transplantation models, islet cells transplanted into the liver via the portal vein are lost due to inflammation and/or to unfavorable environmental conditions, such as poor blood supply with reduced oxygenation and/or nutrients5,6. As a result, in order to ensure long-term metabolic function, higher islet numbers are necessary to compensate for the initial cell loss that reduces engraftment7.
In an attempt to optimize islet engraftment, several alternative anatomical sites have been investigated experimentally as well as clinically, with promising, yet not definitive results8. Whereas some of the alternative sites offer easy and safe access (e.g., skin, kidney capsule, gastric submucosa and anterior chamber of the eye) or a wider surface for larger islet masses (e.g., peritoneal cavity), survival and physiologic metabolic performance of the transplanted islets are still limited and remain a concern9. The search for a more suitable site for islet engraftment is ongoing.
The omentum was among the many anatomical sites that were investigated in the early development of islet transplantation, and proved a successful environment for islets10,11,12,13,14. However, intraportal islet infusion became the clinical choice due in part to the relative simplicity of the procedure and early success in animal models6. Also, in part, the negatives associated with this site, particularly massive early islet loss, were less understood and less constraining in the early days of experimental islet transplantation as the field matured. With more recent compelling evidence indicating that intravascular islet infusion is far from ideal, the omentum is re-emerging as a potentially valuable site for cell transplantation.
The omentum (in the form of an omental pouch) offers relative advantages over the liver15,16. It is well-vascularized and easily accessible. It allows retrieval of the graft (if necessary) and/or biopsy. The ischemic period experienced by the islets is reduced compared to the liver, and the omentum can accept relatively large islet masses which is not possible intraportally, where a rise in portal pressure can cause complications.
A syngeneic mouse model of transplantation was used in the protocol tested in the study, employing C57BL/6 male mice between 6&#8211;8 weeks old with a body weight of 20&#8211;25 g. Islet recipients were rendered diabetic with a single injection of streptozotocin with a dose of 250 mg/kg ip. The induction of diabetes can be considered successful if the blood glucose level of the mouse is greater than 24 mmol/L 48 h after injection and remains above that level for a minimum of 5 days.
Syngeneic islets were isolated from the pancreas of age-matched donors following previously published methods with some modifications. In brief, the collagenase was injected into the gall bladder instead of the bile duct. This was done as an improvement to facilitate the ease of injection. Collagenase infusion was followed by incubation, tissue disruption, density gradient separation and hand-picking to obtain pure islets. Islets were cultured overnight in CMRL-1066 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) in T175 flasks at 37 &#176;C, under 95% air-5% CO2 before transplantation.

<table><tbody><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>5 inch (12-13 cm) Scissors</td><td>RWD Life Science&amp;nbsp;</td><td>S12030-11</td><td></td></tr><tr><td>Fine Forceps</td><td>RWD Life Science</td><td>F11010-13</td><td></td></tr><tr><td>Small wound clips</td><td>RWD Life Science</td><td>R33003-01</td><td></td></tr><tr><td>Acutenaculum</td><td>RWD Life Science</td><td>F31044-13</td><td></td></tr><tr><td>2 pair tissue forceps</td><td>RWD Life Science</td><td>&amp;nbsp;F13023-10</td><td></td></tr><tr><td>4-0 Suture with needle</td><td>Chenghe, China</td><td>17094</td><td></td></tr><tr><td>200 &amp;mu;L Pipette and tips</td><td>Gilson</td><td>PN11</td><td></td></tr><tr><td>One Touch ultraeasy Basic blood glucose monitoring system&amp;nbsp;&amp;nbsp;</td><td>Johnson &amp;amp; Johnson</td><td>33391713</td><td></td></tr><tr><td>razor blade</td><td>Philips</td><td>HC1099/15</td><td></td></tr><tr><td>&lt;strong&gt;Material and animals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Pentobarbital Sodium</td><td>Sigmaaldrich.com</td><td>P3761</td><td>For anesthesia&amp;nbsp;</td></tr><tr><td>Hydrogel&amp;nbsp;</td><td>bdbiosciences.com</td><td>356234</td><td>Basement Membrane Matrix</td></tr><tr><td>Fibrin-Thrombin Hydrogel&amp;nbsp;</td><td>Baxter.com</td><td>1501250</td><td>Components clot when mixed</td></tr><tr><td>70% Ethanol</td><td>Yingniu medical, Anhui, China</td><td>23170608</td><td></td></tr><tr><td>Iodophor</td><td>Lierkang medical technology, Shangdong, China</td><td>170521</td><td></td></tr><tr><td>Normal saline</td><td>Baxter.com</td><td>2B1324</td><td></td></tr><tr><td>Cephalosporin</td><td>Lukang medical, Shangdong, China</td><td>150303</td><td></td></tr><tr><td>Cefazolin</td><td>Baxter.com</td><td>2G3508</td><td></td></tr><tr><td>lubricant eye ointment</td><td>Major Pharmaceuticals</td><td>203964</td><td></td></tr><tr><td>streptozotocin</td><td>Sigmaaldrich.com</td><td>S0130</td><td></td></tr><tr><td>collagenase Type V</td><td>Sigmaaldrich.com</td><td>C9262</td><td></td></tr><tr><td>CMRL-1066 media</td><td>celltrans, Wenzhou, China</td><td>X018D1</td><td></td></tr><tr><td>histopaque</td><td>Sigmaaldrich.com</td><td>10771</td><td>density gradient</td></tr><tr><td>PE50 tubing</td><td>Braintreesci.com</td><td>PE50 100 FT</td><td>Polyethylene .023&quot; x .038</td></tr><tr><td>Calcein AM</td><td>Sigmaaldrich.com</td><td>C1359</td><td></td></tr><tr><td>Propidium iodide</td><td>Sigmaaldrich.com</td><td>P4864</td><td></td></tr><tr><td>optimal cutting temperature compound (OCT)</td><td>Tissue-Tek; Miles, Naperville, IL</td><td>4583</td><td>embedding medium</td></tr><tr><td>insulin antibody</td><td>Cell Signaling Technology, Danvers, MA 01923</td><td>8138S</td><td></td></tr><tr><td>hematoxylin staining media</td><td>Cell Signaling Technology, Danvers, MA 01923</td><td>14166S</td><td></td></tr><tr><td>eosin staining media</td><td>Beyotime Biotech, China</td><td>C0109</td><td></td></tr><tr><td>DAPI</td><td>Thermo Fisher Scientific Inc.&amp;nbsp;</td><td>D1306</td><td></td></tr><tr><td>C57Bl/6 Mice</td><td>Medical Animal Center of Guangdong Province</td><td>/</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>GE Healthcare Life Sciences</td><td>SH30084</td><td></td></tr><tr><td>T175 flasks</td><td>Falcon</td><td>353112</td><td></td></tr><tr><td>1.5 mL Snap-top tubes</td><td>Axygen</td><td>MCT-150-C</td><td></td></tr></tbody></table>

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.

Post-digestive state of the pancreas is shown in Figure 1A. Purified islets are shown in Figure 1B. Dithizone staining and viability testing of the islets are shown in Figure 2. The main steps of the islet transplantation to the omentum are shown in Figure 3. The blood glucose levels of the recipients after omental transplantation are shown in Figure 4. Histological analysis of grafts is shown in Figure 5.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57160/57160fig1.jpg" /> 
Figure 1: Pancreas digestion and islet isolation. (A) The pancreas following the digestion. Note the sand-sized particles in the suspension. (B) Light microscopy image of the islets after isolation. <a href="/files/ftp_upload/57160/57160fig1large.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/57160/57160fig2.jpg" /> 
Figure 2: Islet dithizone staining and fluorescence viability dyes. (A) Islets are stained in red by dithizone. (B) Light microscopy. (C) Propidium Iodide fluorescence red staining (dead cells). (D) Calcein-AM green staining (viable cells). <a href="/files/ftp_upload/57160/57160fig2large.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/57160/57160fig3.jpg" /> 
Figure 3: Transplantation in the omentum. (A) After the recipient laparotomy, the stomach is exposed and the omentum located. (B) Islets are mixed in hydrogel and slowly placed onto the omental tissue. (C) Islet graft at higher magnification. The hydrogel is a semi-solidified state. (D) Alternative method. Hydrogel is placed on the omentum without islets. (E) Islets are placed on top of the hydrogel. (F) The omentum is folded around the islets. <a href="/files/ftp_upload/57160/57160fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57160/57160fig4.jpg" /> 
Figure 4: Blood glucose levels post-transplant. Non-fasting blood glucose levels of recipients (n = 7) up to 35 days after transplantation. The graft recipients did not all receive the same batch of islets so there are differences in quality and function resulting in large fluctuations in blood glucose level.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57160/57160fig5.jpg" /> 
Figure 5: Histology. Graft sections (graft retrieval 14 days after transplantation) were stained with DAPI (nuclei), anti-mouse insulin antibodies, and hematoxylin. <a href="/files/ftp_upload/57160/57160fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Method for Islet Transplantation to the Omentum in Mouse at JoVE.com

A Method for Islet Transplantation to the Omentum in Mouse

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 ...

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Universidad San Sebastian

Research

JoVE Journal

Biology

10.9K Views.  First Affiliated Hospital of Shenzhen University, Shenzhen University School of Medicine, Shenzhen University Health Science Center. 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.

Video: A Method for Islet Transplantation to the Omentum in Mouse

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 ...

Medicine

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 ...

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

Intra-Omental Islet Transplantation Using h-Omental Matrix Islet filliNG (hOMING)

Regenerative medicine based on cell therapy represents a new hope for curing disease. Current obstacles include proper in vivo validation of the efficiency of the therapy. For transfer to the recipient body, cells often need to be combined with biomaterials, especially hydrogels. However, validation of the efficacy of such a graft requires the right environment, the right hydrogel, and the right recipient site. The omentum might be such a site. Based on the example of islet transplantation, we developed the hOMING (h-Omental Matrix Islet filliNG) technique, which consists of the injection of the graft inside the tissue, in between the omental layers, to improve islet implantation and survival. To achieve this, islets have to be embedded in a hydrogel with a viscosity that enables its injection using an atraumatic needle. Syringes are loaded with a combination of hydrogel and islets. Several injections are performed inside the omental tissue at different entry points, and the deposition of the islet/hydrogel mixture is made along a line. We tested the feasibility of this innovative approach using dextran beads. The beads were well spread throughout the omental tissue, in close proximity to blood vessels. To test the efficacy of the graft, we transplanted islets into diabetic rats and perform a metabolic follow-up over two months. The transplanted islets exhibited a high rate of re-vascularization around and inside islets, and reversed diabetes. The hOMING technique could be applicable for other types of hydrogel or cell therapy, for cells with high metabolic activity.

Intra-Omental Islet Transplantation Using h-Omental Matrix Islet filliNG hOMING

Here, we present a protocol for in vivo validation of hydrogel-based cell therapy, illustrated by the example of islet transplantation. h-Omental Matrix Islet filliNG (hOMING) implantation allows implantation of a cell-hydrogel mixture between the omental layers, near to blood vessels, to maximize engraftment in a proper metabolic environment.

Regenerative medicine based on cell therapy represents a new hope for curing disease. Current obstacles include proper in vivo validation of the ...

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 ...

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 ...

Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the Anterior Eye Chamber

Imaging beta cells is a key step towards understanding islet transplantation. Although different imaging platforms for the recording of beta cell biology have been developed and utilized in vivo, they are limited in terms of allowing single cell resolution and continuous longitudinal recordings. Because of the transparency of the cornea, the anterior chamber of the eye (ACE) in mice is well suited to study human and mouse pancreatic islet cell biology. Here is a description of how this approach can be used to perform continuous longitudinal recordings of grafting and revascularization of individual human islet grafts. Human islet grafts are inserted into the ACE, using NOD.(Cg)-Gt(ROSA)26Sortm4-Rag2-/-mice as recipients. This allows for the investigation of the expansion of recipient versus donor cells and the contribution of recipient cells in promoting the encapsulation and vascularization of the graft. Further, a step-by-step approach for image analysis and quantification of the islet volume or segmented vasculature and islet capsule forming recipient cells is outlined.

The goal of this protocol is to continuously monitor the dynamics of the human pancreatic islet engraftment process and the contributing host versus donor cells. This is accomplished by transplanting human islets into the anterior chamber of the eye (ACE) of an NOD.(Cg)-Gt(ROSA)26Sortm4-Rag2-/-mouse recipient followed by repeated 2-photon imaging.

Imaging beta cells is a key step towards understanding islet transplantation. Although different imaging platforms for the recording of beta cell biology ...

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 ...

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.bdigitaluss.remotexs.co/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 ...