Name: inguinal subcutaneous white adipose tissue (iswat) transplantation model of murine islets
Uploaded: 2020-02-16T15:00:00
Description: Watch this Scientific Journal Video about Inguinal Subcutaneous White Adipose Tissue ISWAT Transplantation Model of Murine Islets at JoVE.com

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...

<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>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#956;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&#39;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 &#38; 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 ...

Islet transplantation is one of the most promising approaches for treating Type I diabetes mellitus. And the search for a suitable site for islet transplantation is ongoing. Islet transplantation to the inguinal subcutaneous white adipose tissue is a simple yet effective method that may have clinical implications.Extrapolation of this procedure to the clinic may lead to the development of a cure for Type I diabetics. This method could provide insight into clinical islet transplantation as the lingual has been determined to not be an ideal site for islet engraftment. It is important to carefully follow every step of the procedure, especially key steps, such as the pancreas digestion.Visual demonstration will help researchers new to the technique to better determine how to perform the islet isolation and the transmutation. For islet harvest, first spray the fur of a 10 week old C57 black six mouse with 75%ethanol, and fill a five milliliter syringe with freshly prepared collagenase solution. Equip the syringe with a 32-gauge bending blunt pointed perfusion needle, and place the mouse in the supine position on an ice bag.Use straight pointed ophthalmic scissors to make a transverse opening in the skin of the pubic area, and extend the incision toward the head of the animal. Completely open the abdomen via a V-incision from the pubic region to the xiphoid process, and move the liver to expose the gall bladder and the entire length of the common bile duct. Then use a vascular clamp to close the duodenal opening of the common bile duct and cut a small opening in the gall bladder.Cannulate the common bile duct through the opening with the previously prepared perfusion needle, and inject approximately two milliliters of collagenase solution into the pancreas. When all the collagenase has been delivered, use two pairs of noninvasive micro tweezers to separate the perfused pancreas from the intestines, stomach, and spleen, and place the pancreas into a 50 milliliter conical tube on ice. When all of the samples have been collected, add 100 microliters of Dnase I per pancreas to the 50 milliliter tubes and vigorously shake each tube about 40 times over a period of 10 seconds before placing the tissues into a 37 degree Celsius water bath for three to five minutes with moderate shaking.At the end of the incubation, add up to a final volume of 50 milliliters of Stop Solution to arrest the digestion. And pulse centrifuge the tubes to a speed of 750 times G at four degrees Celsius before quickly stopping the centrifuge. After discarding the supernatants, wash the pellets two times in 15 to 25 milliliters of Stop Solution per wash under the same centrifugation conditions as just demonstrated.After the second wash, pool three pellets per tube into new 50 milliliter conical tubes. And resuspend the pancreatic cells in a total volume of ten milliliters of Histopaque 1119. Next carefully add five milliliters of Histopaque 1077 down the side of each tube, followed by five milliliters of HBSS.Separate the cells by centrifugation and use a five milliliter Pasteur pipette to collect the islets from the HBSS Histopaque 1077 interface. Pool the islets in a new 50 milliliter conical tube in a final volume of 50 milliliters of Stop Solution for pulse centrifugation, and discard the supernatant. Resuspend the pellet in 30 milliliters of fresh Stop Solution for a one minute centrifugation, and resuspend the islets in 30 milliliters of culture medium.Decant the islets into one 10 centimeter light-tight culture dish per tube. And place the dish under the stereo microscope. Using 200 microliter gel-loading pipette tips, hand pick the white spheroidal islets from the solution.Placing the islets into an untreated cell culture dish containing 10 milliliters of culture medium as they are harvested. Check the purity of the hand picked islets by dithizone staining under a light microscope. And detect the viability of the islets by fluorescein diacetate propidium iodide staining under a florescent microscope.Then culture the isolated islets in fresh culture medium at 37 degrees Celsius in 95%air and 5%carbon dioxide until transplantation. Starting days three and four after diabetes induction, puncture the caudal vein of each streptozotocin-injected eight week old male C57 black six mouse around 10:00 a.m. to collect a blood sample from each recipient animal.Use a basic blood glucose monitoring instrument to determine the non-fasting blood glucose level of each mouse and then add the blood onto individual test strips. When an animal has demonstrated a blood glucose level of at least 20 millimoles per liter two days in a row, weigh and mark the recipient mouse and confirm a lack of response to pedal reflex in the anesthetized animals. Place an aliquot of hydrogel from minus 20 degrees Celsius storage on ice for thawing.And transfer 450 to 500 islet equivalents per recipient into a sterile 1.5 milliliter centrifuge tube containing 200 microliters of medium per tube on ice. Swab the left inguinal area of the recipient with 75%ethanol and place the mouse in the supine position. Fix the limbs with surgical tape, and use clippers to remove the hair around the surgical site.Swab the surgical area with iodophor and make a vertical incision in the disinfected skin. Identify the inferior epigastric artery and vein within the ISWAT and create a small pocket above the vessels. Sediment the islets by centrifugation and remove as much supernatant as possible.Add 20 microliters of thawed hydrogel to each tube, and gently resuspend the islets, taking care to avoid bubbles. When a homogenous suspension has been obtained, use a 200 microliter pipette tip to carefully deliver the entire islet hydrogel mixture from one tube into the pocket. When the hydrogel has completely solidified, add 20 microliters of cephalosporin to the transplantation site, and close the muscle and skin with a 5-0 continuous surgical suture.Then place the recipient into a clean cage on a thermal pad with monitoring until full recumbency. After murine islet processing as demonstrated, the isolated murine islets will be sufficiently pure for transplantation. Only isolated islets with a high viability should be used for transplantation.After thorough mixing of the islet grafts with hydrogel, the graft can be implanted into the ISWAT site. After about one month of transplantation into the ISWAT, the islet grafts reverse hyperglycemia while the body weight of the recipient mice gradually increases. At 100 days after transplantation, removal of the grafts results in an immediate increase in the 10:00 a.m.non-fasting blood glucose level. Histological analysis at this time point, reveals that the islet grafts have remained intact. And insulin and glucagon immunofluorescent staining indicate that the transplanted islets are also functional at 100 days post transplantation.Be careful not to shake the profused pancreas cells too robustly or else the ability of the obtained islets will be compromised. Isolated islets can be transplanted under the renal capsule as a control to compare the effects of the isolated transplantation into the inguinal subcutaneous white adipose tissue at site. The procedure will add initiation of clinical trials of islet transplantation to other sites besides the liver.

Research

JoVE Journal

Medicine

Mouse Model of Surgically-induced Endometriosis by Auto-transplantation of Uterine Tissue

Endometriosis is a chronic, painful disease whose etiology remains unknown. Furthermore, treatment of endometriosis can require laparoscopic removal of ...

Development of Obliterative Bronchiolitis in a Murine Model of Orthotopic Lung Transplantation

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and ...

Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are ...

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each ...

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 ...

A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology

Stroke affecting white matter accounts for up to 25% of clinical stroke presentations, occurs silently at rates that may be 5-10 fold greater, and ...

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...

A Technique for Subcutaneous Abdominal Adipose Tissue Biopsy via a Non-diathermy Method

Adipose tissue biopsies offer tissue samples that, upon analysis, may provide insightful overviews of mechanisms relating to metabolism and disease. To ...

Transplantation of Adipose Tissue-Derived Stem Cell Sheet to Reduce Leakage After Partial Colectomy in A Rat Model

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of ...