Name: A Method for Islet Transplantation to the Omentum in Mouse (Video) | JoVE Journal
Uploaded: 2019-01-07T14:00:00.000+00:00
Duration: 6 min 25 s
Description: Learn a method for islet transplantation to the omentum in diabetic mice using hydrogel support. This technique enables researchers to study islet survival and advance type I diabetes therapy.

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5 minutes

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/pubmed?term=((Global+estimates+of+diabetes+prevalence+for+2013+and+projections+for+2035%5BTitle%5D)+AND+%22Diabetes+research+and+clinical+practice%22%5BJournal%5D)">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/pubmed?term=((Potent+Induction+Immunotherapy+Promotes+Long-Term+Insulin+Independence+After+Islet+Transplantation+in+Type+1+Diabetes%5BTitle%5D)+AND+%22American+Journal+of+Transplantation%22%5BJournal%5D)">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/pubmed?term=((Production+of+tissue+factor+by+pancreatic+islet+cells+as+a+trigger+of+detrimental+thrombotic+reactions+in+clinical+islet+transplantation%5BTitle%5D)+AND+%22The+lancet%22%5BJournal%5D)">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/pubmed?term=((Control+of+instant+blood-mediated+inflammatory+reaction+to+improve+islets+of+Langerhans+engraftment%5BTitle%5D)+AND+%22Current+Opinion+in+Organ+Transplantation%22%5BJournal%5D)">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/pubmed?term=((Similar+islet+function+in+islet+allotransplant+and+autotransplant+recipients%2C+despite+lower+islet+mass+in+autotransplants%5BTitle%5D)+AND+%22Transplantation%22%5BJournal%5D)">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/pubmed?term=((Islet+cell+transplantation+for+the+treatment+of+type+1+diabetes%3A+recent+advances+and+future+challenges%5BTitle%5D)+AND+%22Diabetes%2C+metabolic+syndrome+and+obesity%3A+targets+and+therapy%22%5BJournal%5D)">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="https://www.ncbi.nlm.nih.gov/pubmed/27834384">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/pubmed?term=((Alternative+transplantation+sites+for+pancreatic+islet+grafts%5BTitle%5D)+AND+%22Current+diabetes+reports%22%5BJournal%5D)">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/pubmed?term=((Murine+animal+models+for+preclinical+islet+transplantation%3A+no+model+fits+all+%28research+purposes%29%5BTitle%5D)+AND+%22Islets%22%5BJournal%5D)">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/pubmed?term=((Erratum+to%3A+CX3CR1+deficiency+accelerates+the+development+of+retinopathy+in+a+rodent+model+of+type+1+diabetes%5BTitle%5D)+AND+%22J+Mol+Med+%28Berl%29%22%5BJournal%5D)">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/pubmed?term=((Regional+Patterns+of+Retinal+Oxygen+Saturation+and+Microvascular+Hemodynamic+Parameters+Preceding+Retinopathy+in+Patients+With+Type+II+Diabetes%5BTitle%5D)+AND+%22Invest+Ophthalmol+Vis+Sci%22%5BJournal%5D)">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/pubmed?term=((Pancreatic+islets+find+a+new+transplant+home+in+the+omentum%5BTitle%5D)+AND+%22Nature+Biotechnology%22%5BJournal%5D)">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/pubmed?term=((Prevalence+and+Progression+Rate+of+Diabetic+Retinopathy+in+Type+2+Diabetes+Patients+in+Correlation+with+the+Duration+of+Diabetes%5BTitle%5D)+AND+%22Exp+Clin+Endocrinol+Diabetes%22%5BJournal%5D)">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/pubmed?term=((Bioengineering+of+an+Intraabdominal+Endocrine+Pancreas%5BTitle%5D)+AND+%22New+England+Journal+of+Medicine%22%5BJournal%5D)">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/pubmed?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%5BTitle%5D)+AND+%22American+Journal+of+Transplantation%22%5BJournal%5D)">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/pubmed?term=((Bioengineering+the+endocrine+pancreas%3A+intraomental+islet+transplantation+within+a+biologic+resorbable+scaffold%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">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="https://www.ncbi.nlm.nih.gov/pubmed/28109023">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/pubmed?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%5BTitle%5D)+AND+%22American+Journal+of+Transplantation%22%5BJournal%5D)">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/pubmed?term=((Inducible+VEGF+expression+by+human+embryonic+stem+cell-derived+mesenchymal+stromal+cells+reduces+the+minimal+islet+mass+required+to+reverse+diabetes%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">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,<sup clas...

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

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...

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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Biology

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.

Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice

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.

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

This video describes the protocol for grafting human pancreatic islets into the anterior chamber of the mouse’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.

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

In this video, we describe a procedure to infuse pancreatic islet cells into the hepatic portal vein, followed by application of gel foam to successfully prevent bleeding. Postoperative bleeding is a common cause of high mortality during islet transplantation.

Murine Model of Islet Transplantation: A Surgical Method to Infuse Pancreatic Islets in Portal Vein and Prevention of Postoperative Bleeding

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.

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

Intraportal Transplantation of Pancreatic Islets in Mouse Model

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.

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

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

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.

Fat-Covered Islet Transplantation Using Epididymal White Adipose Tissue

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

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

In this protocol, we discuss the implementation of a model of successful orthotopic liver transplantation (OLT) in mice. Additionally, adjuvants to further analyze allograft patency after successful OLT in a mouse are discussed as well, specifically utilizing microcomputed tomography (microCT) scans.

Successful Orthotopic Liver Transplantation in Mice Utilizing Microcomputed Tomography Angiography

Learn a method for islet transplantation to the omentum in diabetic mice using hydrogel support. This technique enables researchers to study islet survival and advance type I diabetes therapy.

A Method for Islet Transplantation to the Omentum in Mouse

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