This study was funded by a Grant-in-Aid for Scientific Research (C) (19K09839, NS) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

The following procedure is performed in three steps. The first step includes the induction of diabetes in the recipient mice and the isolation of donor islets. The second step involves the preparation of islets before transplantation. In the third step, islet transplantation onto epididymal adipose tissue and covering of the islets using the adipose tissue is performed. After that, the therapeutic effects were assessed. The handling of the mice and the experimental procedures performed in this study comply with the &#39;...

<ol>
	<li>Barton, F. B., 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=Improvement+in+outcomes+of+clinical+islet+transplantation:+1999-2010.">Improvement in outcomes of clinical islet transplantation: 1999-2010.</a> Diabetes Care. 35 (7), 1436-1445 (2012).</li><li>Balamurugan, A. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+product+characteristics+and+factors+related+to+successful+human+islet+transplantation+from+the+Collaborative+Islet+Transplant+Registry+(CITR)+1999-2010.">Islet product characteristics and factors related to successful human islet transplantation from the Collaborative Islet Transplant Registry (CITR) 1999-2010.</a> American Journal of Transplantation. 14 (11), 2595-2606 (2014).</li><li>Collaborative Islet Transplant Registry. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Collaborative Islet Transplant Registry. Annual Report. , (2017).</li><li>Rajab, 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=Total+Pancreatectomy+and+Islet+Autotransplantation+Following+Treated+Hepatitis+C+Infection.">Total Pancreatectomy and Islet Autotransplantation Following Treated Hepatitis C Infection.</a> Cell Transplantation. 27 (10), 1569-1573 (2018).</li><li>Mellgren, A., Schnell Landstrom, A. H., Petersson, B., Andersson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+renal+subcapsular+site+offers+better+growth+conditions+for+transplanted+mouse+pancreatic+islet+cells+than+the+liver+or+spleen.">The renal subcapsular site offers better growth conditions for transplanted mouse pancreatic islet cells than the liver or spleen.</a> Diabetologia. 29 (9), 670-672 (1986).</li><li>Hiller, W. F., Klempnauer, J., Luck, R., Steiniger, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+deterioration+of+endocrine+function+after+intraportal+but+not+kidney+subcapsular+rat+islet+transplantation.">Progressive deterioration of endocrine function after intraportal but not kidney subcapsular rat islet transplantation.</a> Diabetes. 40 (1), 134-140 (1991).</li><li>Yasunami, Y., Lacy, P. E., Finke, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+site+for+islet+transplantation--a+peritoneal-omental+pouch.">A new site for islet transplantation--a peritoneal-omental pouch.</a> Transplantation. 36 (2), 181-182 (1983).</li><li>Kin, T., Korbutt, G. S., Rajotte, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+and+metabolic+function+of+syngeneic+rat+islet+grafts+transplanted+in+the+omental+pouch.">Survival and metabolic function of syngeneic rat islet grafts transplanted in the omental pouch.</a> American Journal of Transplantation. 3 (3), 281-285 (2003).</li><li>Kasoju, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioengineering+a+pre-vascularized+pouch+for+subsequent+islet+transplantation+using+VEGF-loaded+polylactide+capsules.">Bioengineering a pre-vascularized pouch for subsequent islet transplantation using VEGF-loaded polylactide capsules.</a> Biomaterials Science. 8 (2), 631-647 (2020).</li><li>Sakata, N., Yoshimatsu, G., Kodama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=White+Adipose+Tissue+as+a+Site+for+Islet+Transplantation.">White Adipose Tissue as a Site for Islet Transplantation.</a> Transplantology. 1 (2), 55-70 (2020).</li><li>Osama Gaber, A., Chamsuddin, A., Fraga, D., Fisher, J., Lo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+independence+achieved+using+the+transmesenteric+approach+to+the+portal+vein+for+islet+transplantation.">Insulin independence achieved using the transmesenteric approach to the portal vein for islet transplantation.</a> Transplantation. 77 (2), 309-311 (2004).</li><li>Fujita, 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=Technique+of+endoscopic+biopsy+of+islet+allografts+transplanted+into+the+gastric+submucosal+space+in+pigs.">Technique of endoscopic biopsy of islet allografts transplanted into the gastric submucosal space in pigs.</a> Cell Transplantation. 22 (12), 2335-2344 (2013).</li><li>Sakata, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategy+for+clinical+setting+in+intramuscular+and+subcutaneous+islet+transplantation.">Strategy for clinical setting in intramuscular and subcutaneous islet transplantation.</a> Diabetes/Metabolism Research and Reviews. 30 (1), 1-10 (2014).</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=Transplant+Site+Influences+the+Immune+Response+After+Islet+Transplantation:+Bone+Marrow+Versus+Liver.">Transplant Site Influences the Immune Response After Islet Transplantation: Bone Marrow Versus Liver.</a> Transplantation. 101 (5), 1046-1055 (2017).</li><li>White, S. 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=The+risks+of+total+pancreatectomy+and+splenic+islet+autotransplantation.">The risks of total pancreatectomy and splenic islet autotransplantation.</a> Cell Transplantation. 9 (1), 19-24 (2000).</li><li>Itoh, T., Nishinakamura, H., Kumano, K., Takahashi, H., Kodama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Spleen+Is+an+Ideal+Site+for+Inducing+Transplanted+Islet+Graft+Expansion+in+Mice.">The Spleen Is an Ideal Site for Inducing Transplanted Islet Graft Expansion in Mice.</a> PLoS One. 12 (1), 0170899 (2017).</li><li>Sakata, N., Yoshimatsu, G., Kodama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Spleen+as+an+Optimal+Site+for+Islet+Transplantation+and+a+Source+of+Mesenchymal+Stem+Cells.">The Spleen as an Optimal Site for Islet Transplantation and a Source of Mesenchymal Stem Cells.</a> International Journal of Molecular Sciences. 19 (5), (2018).</li><li>Sakata, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+rat-to-mouse+bioartificial+pancreas+xenotransplantation+on+diabetic+renal+damage+and+survival.">Effect of rat-to-mouse bioartificial pancreas xenotransplantation on diabetic renal damage and survival.</a> Pancreas. 32 (3), 249-257 (2006).</li><li>Nagaya, 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=Effectiveness+of+bioengineered+islet+cell+sheets+for+the+treatment+of+diabetes+mellitus.">Effectiveness of bioengineered islet cell sheets for the treatment of diabetes mellitus.</a> Journal of Surgical Research. 227, 119-129 (2018).</li><li>Weaver, J. 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=Vasculogenic+hydrogel+enhances+islet+survival,+engraftment,+and+function+in+leading+extrahepatic+sites.">Vasculogenic hydrogel enhances islet survival, engraftment, and function in leading extrahepatic sites.</a> Science Advances. 3 (6), 1700184 (2017).</li><li>Dufour, J. 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=Development+of+an+ectopic+site+for+islet+transplantation,+using+biodegradable+scaffolds.">Development of an ectopic site for islet transplantation, using biodegradable scaffolds.</a> Tissue Engineering. 11 (9-10), 1323-1331 (2005).</li><li>Chen, X., 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=The+epididymal+fat+pad+as+a+transplant+site+for+minimal+islet+mass.">The epididymal fat pad as a transplant site for minimal islet mass.</a> Transplantation. 84 (1), 122-125 (2007).</li><li>Sakata, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+Transplanted+Islet+Engraftment+in+Visceral+White+Adipose+Tissue.">Mechanism of Transplanted Islet Engraftment in Visceral White Adipose Tissue.</a> Transplantation. 104 (12), 2516-2527 (2020).</li><li>Navarro-Requena, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PEG+hydrogel+containing+calcium-releasing+particles+and+mesenchymal+stromal+cells+promote+vessel+maturation.">PEG hydrogel containing calcium-releasing particles and mesenchymal stromal cells promote vessel maturation.</a> Acta Biomaterialia. 67, 53-65 (2018).</li><li>Phelps, E. A., Headen, D. M., Taylor, W. R., Thule, P. M., Garcia, 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=Vasculogenic+bio-synthetic+hydrogel+for+enhancement+of+pancreatic+islet+engraftment+and+function+in+type+1+diabetes.">Vasculogenic bio-synthetic hydrogel for enhancement of pancreatic islet engraftment and function in type 1 diabetes.</a> Biomaterials. 34 (19), 4602-4611 (2013).</li><li>Manzoli, V., 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=Immunoisolation+of+murine+islet+allografts+in+vascularized+sites+through+conformal+coating+with+polyethylene+glycol.">Immunoisolation of murine islet allografts in vascularized sites through conformal coating with polyethylene glycol.</a> American Journal of Transplantation. 18 (3), 590-603 (2018).</li><li>Gotoh, M., Maki, T., Kiyoizumi, T., Satomi, S., Monaco, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+for+isolation+of+mouse+pancreatic+islets.">An improved method for isolation of mouse pancreatic islets.</a> Transplantation. 40 (4), 437-438 (1985).</li><li>Brandhorst, D., Brandhorst, H., Hering, B. J., Bretzel, R. G. <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+survival,+morphology+and+in+vitro+function+of+isolated+pig+islets+under+different+culture+conditions.">Long-term survival, morphology and in vitro function of isolated pig islets under different culture conditions.</a> Transplantation. 67 (12), 1533-1541 (1999).</li><li>Noguchi, 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=Low-temperature+preservation+of+isolated+islets+is+superior+to+conventional+islet+culture+before+islet+transplantation.">Low-temperature preservation of isolated islets is superior to conventional islet culture before islet transplantation.</a> Transplantation. 89 (1), 47-54 (2010).</li><li>Itoh, T., 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=Low+temperature+condition+prevents+hypoxia-induced+islet+cell+damage+and+HMGB1+release+in+a+mouse+model.">Low temperature condition prevents hypoxia-induced islet cell damage and HMGB1 release in a mouse model.</a> Cell Transplantation. 21 (7), 1361-1370 (2012).</li><li>Komatsu, 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=Optimizing+Temperature+and+Oxygen+Supports+Long-term+Culture+of+Human+Islets.">Optimizing Temperature and Oxygen Supports Long-term Culture of Human Islets.</a> Transplantation. 103 (2), 299-306 (2019).</li><li>Unger, R. 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The fat-covered islet transplantation method incorporates techniques from two different transplant techniques: intraperitoneal islet transplantation and intra-adipose tissue islet transplantation. As the surface membrane of epididymal white adipose tissue is considered to be the white adipose tissue that is covered by the peritoneum and that is attached to the epididymis, the fat-covered islet transplantation method can be anatomically categorized as a type of intraperitoneal islet transplantation. The technique by which...

Islet transplantation is a cellular replacement therapy for patients with severe diabetes mellitus. Recent reports have shown that rates of insulin-independence at three years after transplantation improve up to 44%1 and that approximately 80% of recipients who receive more than 600,000 total islet equivalents achieve insulin independence2. Furthermore, in the most recent Collaborative Islet Transplant Registry report, it was revealed that fasting blood glucose levels were maintained at 60-140 mg/dL for over a period of 5 years in over 70% of patients who underwent islet transplant alone. The study also determined that around 90% of the patients who received islet transplant alone or islet transplantation after kidney transplant did not develop any severe hypoglycemic events for over 5 years3.
Although the clinical outcomes of this treatment have been improving, some limitations must still be addressed, including the necessity of establishing an optimal transplant site. The liver is a typical transplant site for clinical islet transplantation because it is the largest organ that can accommodate a high volume of islets. However, in some patients the liver is unavailable (e.g., due to portal hypertension, hepatitis, and/or cirrhosis4) and therefore other sites, including the renal subcapsular space5,6, omental pouch7,8,9,10, mesentery11, gastrointestinal tract12, skeletal muscle13, subcutaneous tissue13, bone marrow14, and spleen15,16,17, have been considered as alternative transplant sites.
Although intraperitoneal islet transplantation can be performed easily under local anesthesia, making the intraperitoneal cavity an appealing site for clinical islet transplantation, upon transplant, the islets are dispersed throughout the entire intraperitoneal cavity, making islet engraftment detection and successful engraftment confirmation difficult. Therefore, the intraperitoneal cavity is not widely recognized as an ideal clinical transplant site. Instead, it is frequently utilized as a control model for preclinical studies to investigate the effectiveness of transplanted encapsulated18 and bioengineered islets19. However, an exact comparison between bioengineered and control islets is difficult to achieve due to the challenges in performing an accurate engraftment assessment.
In contrast, the use of intraperitoneal white adipose tissue in the omental pouch8, mesentery, and other extrahepatic locations has been well reported10,20,21,22,23 and many of the studies investigating the function of bioengineered islets transplanted using white adipose tissue were able to report promising therapeutic outcomes20,24,25,26. As the use of epididymal adipose tissue facilitates the detection of transplanted islets, the &#34;fat-covered islet transplantation method&#34;, utilizing epididymal adipose tissue, was developed to overcome the limitations of intraperitoneal islet transplantation. In this paper, fat-covered islet transplantation using epididymal adipose tissue is described.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4-0 Nylon</td>
			<td>Alfresa</td>
			<td>ER2004NA45-KF2</td>
			<td>Closing abdomen</td>
		</tr>
		<tr>
			<td>Alexa 488-conjugated donkey anti-guinea pig</td>
			<td>Jackson Immunoresearch</td>
			<td>706-546-148</td>
			<td>Secondary antibody for insulin antibody</td>
		</tr>
		<tr>
			<td>Alexa 647-conjugated donkey anti-rabbit</td>
			<td>Jackson Immunoresearch</td>
			<td>711-606-152</td>
			<td>Secondary antibody for von Willebrand factor antibody</td>
		</tr>
		<tr>
			<td>DMEM, low glucose, pyruvate</td>
			<td>ThermoFisher Scientific</td>
			<td>11885084</td>
			<td>Culturing islets, transplanting islets</td>
		</tr>
		<tr>
			<td>Eosin</td>
			<td>Fujifilm Wako Chemicals</td>
			<td>051-06515</td>
			<td>Using for staining tissue by eosin</td>
		</tr>
		<tr>
			<td>Eppendorf Safe-Lock Tubes, 1.5 mL</td>
			<td>Eppendorf</td>
			<td>30120086</td>
			<td>Collecting islets&#160;</td>
		</tr>
		<tr>
			<td>Falcon 15 mL Conical Centrifuge Tubes</td>
			<td>Corning</td>
			<td>352095</td>
			<td>Collecting islets</td>
		</tr>
		<tr>
			<td>Falcon 40 &#181;m Cell Strainer</td>
			<td>Falcon</td>
			<td>352340</td>
			<td>Using for separating islets from other pancreatic tissue</td>
		</tr>
		<tr>
			<td>Falcon 50 mL Conical Centrifuge Tubes</td>
			<td>Corning</td>
			<td>352070</td>
			<td>Discarding excessive medium/buffer</td>
		</tr>
		<tr>
			<td>Guinea pig anti-insulin</td>
			<td>Agilent Technologies Japan, Ltd. (Dako)</td>
			<td>IR002</td>
			<td>Primary antibody for murine insulin</td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>Muto Pure Chemicals Co., Ltd.</td>
			<td>30002</td>
			<td>Using for staining tissue by hematoxylin</td>
		</tr>
		<tr>
			<td>Isodine solution 10%</td>
			<td>Shionogi&#38;Co., Ltd.</td>
			<td>no catalog number</td>
			<td>Using for disinfection</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Fujifilm Wako Chemicals</td>
			<td>095-06573</td>
			<td>Using for anesthesia</td>
		</tr>
		<tr>
			<td>Labcon 1000 &#181;L ZapSilk Low Retention Pipette Tips</td>
			<td>Labcon</td>
			<td>1177-965-008</td>
			<td>Using for separating islets from other pancreatic tissue</td>
		</tr>
		<tr>
			<td>Labcon 200 &#181;L ZapSilk Low Retention Pipette Tips</td>
			<td>Labcon</td>
			<td>1179-965-008</td>
			<td>Using for seeding islets onto epididymal white adipose tissue</td>
		</tr>
		<tr>
			<td>Mintsensor</td>
			<td>Sanwa Kagaku Kenkyusho Co. Ltd.,</td>
			<td>8AEB02E</td>
			<td>Using for monitoring blood glucose</td>
		</tr>
		<tr>
			<td>Pipetteman P-1000</td>
			<td>Gilson</td>
			<td>F123602</td>
			<td>Using for separating islets from other pancreatic tissue</td>
		</tr>
		<tr>
			<td>Pipetteman P-200</td>
			<td>Gilson</td>
			<td>F123601</td>
			<td>Using for seeding islets onto epididymal white adipose tissue</td>
		</tr>
		<tr>
			<td>Rabbit anti-vWF</td>
			<td>Abcam</td>
			<td>ab6994</td>
			<td>Primary antibody for murine von Willebrand factor</td>
		</tr>
	</tbody>
</table>

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.

To compare the transplant efficacy of fat-covered islet transplantation to that after intraperitoneal islet transplantation, the same number of islets was implanted onto the peritoneum at the left paracolic space of control recipient diabetic animals. The blood glucose levels of mice with fat-covered islet transplantation were observed to gradually and significantly decrease compared to intraperitoneal islet transplanted mice (p = 0.0023; Figure 3A). One month after transplantation, the bloo...

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Fat-Covered Islet Transplantation Using Epididymal White Adipose Tissue

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 ...

This protocol makes it possible to assess the mechanism of islet engraftment into white adipose tissue. The main advantage of this fat covered islet transplantation method is that it is technically easy to replicate. The method can be applied to other disease models.For example it can be used to create a mouse model of cancer. Demonstrating the procedure will be Naoaki Sakata, an Associate Professor at Fukuoka University. Under a dissecting microscope, use a P 200 micro pipette to remove any extra islet components from the pancreas digestions including acinar and fibrous tissues.Then use a cell strainer to filter out single acinar cells. Transfer the filtered islets to a new culture dish containing the appropriate culture medium or buffer solution supplemented with bovine serum or albumin, and swirl the dish to position the islets in the center. Using a P 200 micro pipette, pick the individual islets into an appropriate collection tube.Place a new 40 micrometer cell strainer on top of a 50 milliliter plastic tube. Use a 1000 microliter pipette to add the islets to the strainer to separate the islets and the single acinar cells. Use forceps to invert the strainer on a new 60 or 100 milliliter sized non-treated culture dish containing culture medium or an appropriate buffer solution, supplemented with bovine serum or albumin.Use fresh medium, or buffer to flush the islets into a new culture dish, then add enough medium or buffer to the culture dish to reach a total volume of approximately 20 milliliters. Count the islets under a microscope and divide the number of islets equally between individual 1.5 milliliter plastic centrifuge tubes according to the number of donor animals. Centrifuge the islets at 2, 100 G for one minute at room temperature and discard the supernatant.Around 20 to 30 microliters of residual solution will typically remain in the tube. After removing the hair from the abdomen to prevent infection place the mouse in a supine position. After disinfecting the abdomen and inguinal region incise the skin at lower median area, creating a skin incision that is approximately two centimeters in length.Clamp the left abdominal wall with forceps or retractors and pull the tissue to the left side of the mouse to secure the surgical field. Use a cotton swab to mobilize the small and large intestine to the right side of the mouse. The left epidermal white adipose tissue in the abdominal cavity is located in the left inguinal area.Mobilize the epidermal white adipose tissue and the left testis to the outside of the abdomen and stretch out the tissue. Use a P 200 micro pipette to collect the entire volume of islets from one 1.5 milliliter tube, taking care that no islets are left in the tube upon collection. Allow the collected islets to settle in the tip of the pipette by gravity.Place the micro pipette tip lightly onto the distended adipose tissue, taking care to prevent excessive flushing of the medium or buffer in the tip. Carefully seed the islets onto the tissue. After seeding, confirm the correct placement of the islets under a dissecting microscope.Cover the islets with the epidermal white adipose tissue, then place the left testis under the episomal white adipose tissue and return the tissues to the intraperitoneal cavity. Close the skin in two layers using a 4-o suture. When finished, place the mouse under a heat lamp and monitor it until full recovery.To compare the transplant efficacy of fat covered islet transplantation to that after intraperitoneal islet transplantation, the same number of islets was implanted onto the peritoneum at the left paracolic space of control recipient diabetic animals. The blood glucose levels of mice with fat covered islet transplantation were observed to gradually and significantly decrease compared to intraperitoneal islet transplanted mice. Intraperitoneal glucose tolerance testing was performed one month after transplantation.The blood glucose in mice with fat covered islet transplantation was maintained at lower levels than that observed in intraperitoneal islet transplanted mice. Histological examination was used to assess islet engraftment into the epidermal white adipose tissue. In fat covered islet transplant recipient animals, hematoxylin eocene staining revealed the presence of islets within the epidermal white adipose tissue.In addition, fluorescence conjugated antibody staining of insulin positive islets facilitated the detection of von Willebrand factor positive micro vessels within the epidermal white adipose tissue of all recipient mice. To succeed with this procedure make sure to allow the islets to settle at the tip of the pipette completely, and seed them onto the adipose tissue without spilling.

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JoVE Journal

Medicine

1.8K ビュー.  Fukuoka University. このプロトコルは、白色脂肪組織への膵島生着のメカニズムを評価することを可能にする。この脂肪で覆われた膵島移植法の主な利点は、技術的に複製が容易であることです。この方法は、他の疾患モデルにも適用することができる。例えば、癌のマウスモデルを作成するために使用することができる。手順のデモンストレーションは、福岡大学の坂田直明准教授?...