The authors would like to thank Wei Zhang from Guanhao Biotech for providing the decellularized scaffolds. We thank Xiao-hong Peng for the helpful discussions. This research was financially supported by the National Natural Science Foundation of China (Project No.31322021).

All experiments were approved by Peking University Institutional Animal Care and Use Committee (IACUC, IACUC no. COE-LuoY-1).
1. Islet Isolation
<ol>
	<li>Preparation of reagents and equipment.
	<ol>
		<li>Reconstitute collagenase P powder (2 U/mg) in HBSS to make a 5 mg/mL solution and filter it through a 0.22 &#181;m filter to remove the bacteria. Prepare 0.6 mL-aliquot solutions of collagenase P in 15-mL conical tubes and store at -20 &#176;C. 
		NOTE: During use, ea...

<ol>
	<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> N Engl J Med. 343, 230-238 (2000).</li><li>Shapiro, A. M. 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=International+Trial+of+the+Edmonton+Protocol+for+Islet+Transplantation.">International Trial of the Edmonton Protocol for Islet Transplantation.</a> N Engl J Med. 355, 1318-1330 (2006).</li><li>Ryan, E. 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=Five-year+follow-up+after+clinical+islet+transplantation.">Five-year follow-up after clinical islet transplantation.</a> Diabetes. 54, 2060-2069 (2005).</li><li>Merani, S., Toso, C., Emamaullee, J., 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=Optimal+implantation+site+for+pancreatic+islet+transplantation.">Optimal implantation site for pancreatic islet transplantation.</a> Br J Surg. 95, 1449-1461 (2008).</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> Nat Biotechnol. 35 (1), (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 Eng. 11, 1323-1331 (2005).</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=Controlled+Release+of+Dexamethasone+from+Organosilicone+Constructs+for+Local+Modulation+of+Inflammation+in+Islet+Transplantation.">Controlled Release of Dexamethasone from Organosilicone Constructs for Local Modulation of Inflammation in Islet Transplantation.</a> Tissue Eng Part A. 21, 2250-2261 (2015).</li><li>Wang, K., 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=From+Micro+to+Macro:+The+Hierarchical+Design+in+a+Micropatterned+Scaffold+for+Cell+Assembling+and+Transplantation.">From Micro to Macro: The Hierarchical Design in a Micropatterned Scaffold for Cell Assembling and Transplantation.</a> Adv Mater. 29, (2017).</li><li>Blomeier, 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=Polymer+Scaffolds+as+Synthetic+Microenvironments+for+Extrahepatic+Islet+Transplantation.">Polymer Scaffolds as Synthetic Microenvironments for Extrahepatic Islet Transplantation.</a> Transplantation. 82, 452-459 (2006).</li><li>Gibly, R. F., 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=Extrahepatic+islet+transplantation+with+microporous+polymer+scaffolds+in+syngeneic+mouse+and+allogeneic+porcine+models.">Extrahepatic islet transplantation with microporous polymer scaffolds in syngeneic mouse and allogeneic porcine models.</a> Biomaterials. 32, 9677-9684 (2011).</li><li>Moskalewski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Culture+of+the+Islets+of+Langerhans+of+the+Guinea+Pig.">Isolation and Culture of the Islets of Langerhans of the Guinea Pig.</a> Gen Comp Endocrinol. 5, 342-353 (1965).</li><li>Lacy, P. E., Kostianovsky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+the+isolation+of+intact+islets+of+Langerhans+from+the+rat+pancreas.">Method for the isolation of intact islets of Langerhans from the rat pancreas.</a> Diabetes. 16, 35-39 (1967).</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> J Vis Exp. (50), (2011).</li><li>Li, D. S., Yuan, Y. H., Tu, H. J., Liang, Q. L., Dai, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+islet+isolation+from+mouse+pancreas.">A protocol for islet isolation from mouse pancreas.</a> Nat Protoc. 4, 1649-1652 (2009).</li><li>Stull, N. D., Breite, A., McCarthy, R., Tersey, S. A., Mirmira, 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=Mouse+Islet+of+Langerhans+Isolation+using+a+Combination+of+Purified+Collagenase+and+Neutral+Protease.">Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease.</a> J Vis Exp. (67), (2012).</li><li>Sakata, N., Yoshimatsu, G., Tsuchiya, H., Egawa, S., Unno, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+diabetes+mellitus+for+islet+transplantation.">Animal models of diabetes mellitus for islet transplantation.</a> Exp Diabetes Res. , 256707 (2012).</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> Nat Biotech. 35, 8-8 (2017).</li><li>Londono, R., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biologic+scaffolds+for+regenerative+medicine:+mechanisms+of+in+vivo+remodeling.">Biologic scaffolds for regenerative medicine: mechanisms of in vivo remodeling.</a> Ann Biomed Eng. 43, 577-592 (2015).</li></ol>

The authors have nothing to disclose.

Pancreas perfusion and digestion time are two key parameters that affect islet yield and quality. Moskalewski first reported the use of a crude collagenase mixture to digest minced guinea pig pancreas11. Lacy et al. reported the injection of enzymes into the duct system to perfuse the pancreas, which greatly increased the islet yield12. The ductal perfusion of enzyme allows for the maximal exposure of pancreatic surface area to the enzyme, resulting in a more homog...

Type 1 diabetes mellitus (T1D) is an autoimmune endocrine disorder in which islet cells are ablated by the immune system, rendering patients dependent upon the injection of exogenous insulin for their whole lives. The Edmonton protocol represents a milestone in clinical studies of islet transplantation; islets were infused through the portal vein and transplanted at the intrahepatic site1. However, two main obstacles-inadequate sources of donor islets and poor islet engraftment-prevent the wide success of the islet transplantation2. Usually, islets need to be collected from three cadaveric donors to reverse the hyperglycemic condition of one patient; this is due to the low yield of islet isolation procedures and the islet loss after transplantation. In particular, although the post-transplantation islets were bathed in oxygen-rich blood, the direct contact with blood often evoked the instant blood-mediated inflammatory reaction (IBMIR), which could cause the acute loss of the islets. In the long term, it is thought that the gradual loss of islets in patients accounted for the drop of diabetes reversal rates in the clinical groups, which could reach 90% in the first year and declined to 30% and 10% by 2 and 5 years post-transplantation, respectively3.
Islet transplantation at extrahepatic sites has been an attractive strategy to reduce the direct contact of islets with blood while confining the transplants to more definable locations compared to intrahepatic infusion. Studies have been carried out in the kidney capsule, eye, muscle, fat pads, and subcutaneous spaces over the past years, showing that islets at these sites are able to survive and function to restore normoglycemia4. In addition, the islets at these sites are retrievable, making it possible for biopsy or even for further replacement procedures. Extrahepatic sites therefore demonstrate great potential for clinical transplantation5.
Biomaterial-based scaffolds have been intensively investigated for cell transplantation and tissue engineering. Three-dimensional (3D) scaffolds usually contain porous structures and can serve as cellular templates to generate spatial structure/organization of cells or as reservoirs to provide the controlled release of bioactive cues. Scaffolds have also been fabricated from polymeric materials, such as poly(glycolide-L-lactide)6, poly(dimethylsiloxane)7, and thermoplastic poly(urethane)8, to transplant islets in the EFP. Compared to the direct transplantation of islets, the use of scaffolds was found to reduce islet loss by preventing the leakage of islets into the intraperitoneal cavity9,10, providing mechanical protection and modulating the local inflammatory reaction. The scaffolds thus may be developed to promote islet engraftment at the transplantation sites7.
In this study, we intend to demonstrate a paradigm of islet transplantation in the EFP, carried out in mice models using a DCS. Scaffolds derived from extracellular matrices have attracted great interest in recent years due to the superior biocompatibility and more natural porous structures compared to synthetic products. Here, we describe a robust isolation protocol to obtain pancreatic islets at high yields from C57BL/6J mice. DCSs processed from the bovine pericardium were then seeded with islets, and the grafts were transplanted to the EFP in syngeneic diabetic models. Normoglycemia in mice was achieved within 10 days and was maintained for up to 100 days, until the removal of the grafts.

<table border="0" cellpadding="0" cellspacing="0" width="364">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:127px;">Dissecting scissor</td>
			<td style="width:63px;">Ningbo Medical</td>
			<td style="width:91px;"></td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:127px;">Forceps</td>
			<td style="width:63px;">Ningbo Medical</td>
			<td style="width:91px;"></td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:127px;">0.5 mm diameter wire mesh</td>
			<td style="width:63px;">Ningbo Medical</td>
			<td style="width:91px;"></td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:127px;">70 &#956;m cell strainer</td>
			<td style="width:63px;">Falcon</td>
			<td style="width:91px;">352350</td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:127px;">Artery hemostatic clamp</td>
			<td style="width:63px;">Ningbo Medical</td>
			<td style="width:91px;"></td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:127px;">Microscopic hemostatic clamp</td>
			<td style="width:63px;">Ningbo Medical</td>
			<td style="width:91px;"></td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:127px;">Hemostatic forceps</td>
			<td style="width:63px;">Ningbo Medical</td>
			<td style="width:91px;"></td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:127px;">Absorbable 6-0 PGLA sutures&#160;</td>
			<td style="width:63px;">JINHUAN</td>
			<td style="width:91px;"></td>
			<td style="width:87px;">With needle</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:127px;">Wound clip</td>
			<td style="width:63px;">Ningbo Medical</td>
			<td style="width:91px;"></td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:127px;">Cotton swab</td>
			<td style="width:63px;">Ningbo Medical</td>
			<td style="width:91px;"></td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:127px;">Gauze</td>
			<td style="width:63px;">Ningbo Medical</td>
			<td style="width:91px;"></td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:127px;">Sterile drapes</td>
			<td style="width:63px;">Ningbo Medical</td>
			<td style="width:91px;"></td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:127px;">10mL syringe</td>
			<td style="width:63px;">JINGHUAN</td>
			<td style="width:91px;"></td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:127px;">1 mL syringe</td>
			<td style="width:63px;">JINGHUAN</td>
			<td style="width:91px;"></td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:127px;">27G intravenous needle</td>
			<td style="width:63px;">JINGHUAN</td>
			<td style="width:91px;">0.45x15 RWSB</td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:127px;">1.5 mL Eppendorf tube</td>
			<td style="width:63px;">Axygen</td>
			<td style="width:91px;"></td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:127px;">15mL conical tube</td>
			<td style="width:63px;">Corning</td>
			<td style="width:91px;">430791</td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:127px;">50mL conical tube</td>
			<td style="width:63px;">Corning</td>
			<td style="width:91px;">430829</td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:127px;">35mm Non-treated&#160; Peri-dishes</td>
			<td style="width:63px;">Corning</td>
			<td style="width:91px;">430588</td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:127px;">Transwell</td>
			<td style="width:63px;">Corning</td>
			<td style="width:91px;">3422</td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:127px;">0.22 &#956;m filter</td>
			<td style="width:63px;">Pall</td>
			<td style="width:91px;">PN4612</td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:127px;">10 mL serological pipet</td>
			<td style="width:63px;">Corning</td>
			<td style="width:91px;">4488</td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:127px;">Pipet filler S1</td>
			<td style="width:63px;">Thermo Scientific</td>
			<td style="width:91px;">9501</td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:127px;">Pipette (2-20&#956;L)</td>
			<td style="width:63px;">Axygen</td>
			<td style="width:91px;">AP-20</td>
			<td style="width:87px;">AXYPETTM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:127px;">Dissecting microscope</td>
			<td style="width:63px;">Olympus</td>
			<td style="width:91px;">SZ61</td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:127px;">Centrifuge</td>
			<td style="width:63px;">Eppendorf</td>
			<td style="width:91px;">5810R</td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:127px;">Hank&#8217;s balanced salt solution&#160;</td>
			<td style="width:63px;">Gibco</td>
			<td style="width:91px;">C14175500CP</td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:127px;">Collagenase P</td>
			<td style="width:63px;">Roche</td>
			<td style="width:91px;">COLLP-RO</td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:127px;">Histopaque 1077</td>
			<td style="width:63px;">Sigma</td>
			<td style="width:91px;">10771</td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:127px;">RPMI 1640</td>
			<td style="width:63px;">Gibco</td>
			<td style="width:91px;">11879-20</td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:127px;">FBS</td>
			<td style="width:63px;">Gibco</td>
			<td style="width:91px;">16000-044</td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:127px;">D-glucose</td>
			<td style="width:63px;">Gibco</td>
			<td style="width:91px;">A24940-01</td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:127px;">Glucose meter</td>
			<td style="width:63px;">Roche</td>
			<td style="width:91px;"></td>
			<td style="width:87px;">ACCU-CHEK</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:127px;">Penicillin-streptomycin</td>
			<td style="width:63px;">Gibco</td>
			<td style="width:91px;">15140-122</td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:127px;">Streptozotocin</td>
			<td style="width:63px;">Sigma</td>
			<td style="width:91px;">V900890</td>
			<td style="width:87px;">VetecTM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:127px;">Chloral hydrate</td>
			<td style="width:63px;">J&#38;K</td>
			<td style="width:91px;">C0073</td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:127px;">Sodium citrate</td>
			<td style="width:63px;">Sigma</td>
			<td style="width:91px;">71497</td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:127px;">Citric acid</td>
			<td style="width:63px;">Sigma</td>
			<td style="width:91px;">C2404</td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:127px;">Iodophors</td>
			<td style="width:63px;">Ningbo Medical</td>
			<td style="width:91px;"></td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:127px;">C57BL/6J, 10-12 weeks old</td>
			<td style="width:63px;">VitalRiver</td>
			<td style="width:91px;"></td>
			<td style="width:87px;">Beijing, China</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:127px;">Decellularized scaffold</td>
			<td style="width:63px;">Guanhao Biotec</td>
			<td style="width:91px;">131102</td>
			<td style="width:87px;">Guangzhou, China</td>
		</tr>
	</tbody>
</table>

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.

Our clamping method, performed using a microscopic hemostatic clamp, is straightforward and time-saving compared with the suture ligation technique. It took roughly 4 h to isolate and purify about 1,200 islets from 6 mice. The freshly isolated islets typically had a rough periphery under an optical microscope (Figure 3A). Once the islets recovered from the isolation process, they looked bright and tight and acquired a smooth surface. However, the stressful is...

Watch this Scientific Journal Video about Scaffold-supported Transplantation of Islets in the Epididymal Fat Pad of Diabetic Mice at JoVE.com

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

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 ...

Hello everyone, I'm Kai Wang from Department of Biomedical Engineering in Peking University at Dr.Lowe's laboratory. Currently, individuals with type one diabetes are treated mainly with insulin injections which do not perfectly simulate the insulin secretion from islets. Replacing the lost islets by islet transportation can show you stable blood glucose control without severe fluctuation.However, the requirement of producing large numbers of high quality islets and the poor engraftment efficiency compromised the widespread clinical use. Today, I'm gonna show you the process of islet isolation and the transplanting scaffold-supported islets into the epididymal fat pad of diabetic mice. These steps require good knowledge of tissue structures and the skilled hands to perform the surgery.Spray the whole body with ethanol. Make a V-incision starting from the genital area. Remove the bowel through the right side of the mouse.Clamp the portal vein and the bile duct with hemostatic clamp. Grip the duodenum carefully with forceps and pull the duodenum until the bile duct is taught. Insert the intravenous needle into the common bile duct through the ampulla.Clamp the duct segment containing the needle using an ultra hemostatic clamp. Dispense the collagenic solution at a slow and constant rate. After complete inflation of the pancreas, push the bowel through the left side of the mouse.You can see the pancreatic duct is fully inflated. Remove the pancreas by starting end of descending colon. Use the forceps to lift the bowel and to separate it from the pancreas with another forceps.Continue to remove the pancreas until it is unattached from the top of the stomach. Finally, lift the pancreas out of the abdomen and cut it free from the remaining spleen. Place a pancreas in the conical tube and deliver on ice.Incubate the perfused pancreas. Terminate the digestion by adding neutralization solutions. Dissociate the two soup by shaking the tubes vigorously.Filter the digestive tissue samples through the wire mesh. Collect the islet suspensions into a new conical tube. Centrifuge the tubes.Pour off the supernatant carefully without disturbing the tissue palettes. Resuspend the palettes with Histopaque by pipetting the suspension up and down for a few times. Slowly pipette HBSS down the side of the tube to the top of the Histopaque solutions.The two solutions should be well separated layers with a sharp interface. Centrifuge the suspension for 20 minutes. Most of the islets migrate to the interface of the Histopaque and HBSS layers.Remove the entire supernatant solutions, and pass the solutions through an inverted strainer. The islets will be retained by the strainer. Dip the surface retaining the islets into the solution and gently shake to release the islets.Hand pick the isolated islets under the microscope. Collect islets into a new culture dish. If the tissue surrounding the duct begins to inflate, it means that the needle passes through the wall of the bile duct.It is necessary to reposition the needle and try cannulating the bile duct again. As the solutions fill up the pancreas, the tissue near the duodenum inflates first, followed by the region near the pancreatic tail. If the duodenum begins to inflate as shown here with blue stain, adjust the artery clamp and reclamp the bile duct.Place mouse in supine position. Shave the abdomen. Then swab with iodophors to sterilize the skin.Cover the mouse with a sterile drape. Make a small incision through the peritoneal wall in the middle close to the genital area. Gently grab and pull out the epididymal fat pad from the abdominal cavity.Spread the fat pad on a wetted gauze. Place a scaffold containing islets on the fat pad and fold the fat pad to wrap the transplant. Suture the fat pad to secure the islets encapsulated in the fat pad.Gently place the fat pad back into the abdominal cavity. Close the mouse by suturing the peritoneal wall. Clamp the dermal layer with wound clips.Inject appropriate doses of pain killers and antibiotics subcutaneously. The fresh isolated islets typically had a rough periphery under optical microscope. Once islets recovered from the isolation process, they looked bright, tight, and acquired a smooth surface.The calculated mean islet diameter was about 130 micrometers. The decelerized scaffolds are mechanically robust enough to be handled by tweezer. The porous scaffold-supported islets were then transferred onto the surface of the spread fat pad, and then encapsulated by the fat pad.In a syngeneic transplantation model, typically 500 islets reverse hypoglycemia within 10 days. Normal glycemia was maintained before about 100 days till the retrieval of the grafts. The histological studies show that the islets were revascularized and surrounded by the fat pad tissue and the scaffold.After watching this video, with practice, you should learn the key steps involved in the islet's isolation and transportation. Firstly, cannulating the common bile duct and dilating the pancreas is the most difficult part of this protocol. Secondly, encapsulate the islet-laden scaffold with a fat pad completely, as the incomplete encapsulation might lead to the failure of the transplantation.

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Research

JoVE Journal

Bioengineering

9.9K vues.  Peking University. Hello everyone, I'm Kai Wang from Department of Biomedical Engineering in Peking University at Dr.Lowe's laboratory. Currently, individuals with type one diabetes are treated mainly with insulin injections which do not perfectly simulate the insulin secretion from islets. Replacing the lost islets by islet transportation can show you stable blood glucose control without severe fluctuation.However, the requirement of producing large numbers of high quality islets and the poor engraftmen...