Name: a method for murine islet isolation and subcapsular kidney transplantation
Uploaded: 2011-04-13T11:00:00
Duration: 17 min 42 s
Description: Watch this Scientific Journal Video about A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation at JoVE.com

This work is supported by RO1 DK064938 (to T.H.).

<ol>
	<li>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=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>Weir, G. C., Bonner-Weir, S. <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+as+a+treatment+for+diabetes.">Islet transplantation as a treatment for diabetes.</a> J Am Optom Assoc. 69, 727-732 (1998).</li><li>Gangemi, 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+for+brittle+type+1+diabetes:+the+UIC+protocol.">Islet transplantation for brittle type 1 diabetes: the UIC protocol.</a> Am J Transplant. 8, 10-1111 (2008).</li><li>Zmuda, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deficiency+of+ATF3,+an+adaptive-response+gene,+protects+islets+and+ameliorates+inflammation+in+a+syngenic+transplantation+model.">Deficiency of ATF3, an adaptive-response gene, protects islets and ameliorates inflammation in a syngenic transplantation model.</a> Diabetologia. , (2010).</li><li>Bottino, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+Human+Islets+to+Isolation+Stress+and+the+Effect+of+Antioxidant+Treatment.">Response of Human Islets to Isolation Stress and the Effect of Antioxidant Treatment.</a> Diabetes. 53, 2559-2568 (2004).</li><li>Markmann, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+islet+cell+culture+in+vitro+at+24+degrees+C+on+graft+survival+and+MHC+antigen+expression.">The effect of islet cell culture in vitro at 24 degrees C on graft survival and MHC antigen expression.</a> Transplantation. 49, 272-277 (1990).</li><li>Terasaka, R., Lacy, P. E., Hauptfeld, V., Bucy, R. P., Davie, J. 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+effect+of+cyclosporin-A,+low-temperature+culture,+and+anti-Ia+antibodies+on+prevention+of+rejection+of+rat+islet+allografts.">The effect of cyclosporin-A, low-temperature culture, and anti-Ia antibodies on prevention of rejection of rat islet allografts.</a> Diabetes. 35, 83-88 (1986).</li><li>Ricordi, C., Lacy, P. E., Sterbenz, K., Davie, J. M. <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+culture+of+human+islets+or+in+vivo+treatment+with+L3T4+antibody+produces+a+marked+prolongation+of+islet+human-to-mouse+xenograft+survival.">Low-temperature culture of human islets or in vivo treatment with L3T4 antibody produces a marked prolongation of islet human-to-mouse xenograft survival.</a> Proc Natl Acad Sci U S A. 84, 8080-8084 (1987).</li><li>Schnedl, W. J., Ferber, S., Johnson, J. H., Newgard, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STZ+transport+and+cytotoxicity.+Specific+enhancement+in+GLUT2-expressing+cells.">STZ transport and cytotoxicity. Specific enhancement in GLUT2-expressing cells.</a> Diabetes. 43, 1326-1333 (1994).</li><li>Eckhard, M., Brandhorst, D., Brandhorst, H., Brendel, M. D., 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=Optimization+in+osmolality+and+range+of+density+of+a+continuous+ficoll-sodium-diatrizoate+gradient+for+isopycnic+purification+of+isolated+human+islets.">Optimization in osmolality and range of density of a continuous ficoll-sodium-diatrizoate gradient for isopycnic purification of isolated human islets.</a> Transplantation Proceedings. 36, 2849-2854 (2004).</li><li>Gores, P. F., Rabe, F., Sutherland, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+survival+of+intraportal+versus+subrenal+capsular+transplanted+islet+allografts.">Prolonged survival of intraportal versus subrenal capsular transplanted islet allografts.</a> Transplantation. 43, 747-749 (1987).</li><li>Nasr, I. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testicular+Immune+Privilege+Promotes+Transplantation+Tolerance+by+Altering+the+Balance+between+Memory+and+Regulatory+T+Cells.">Testicular Immune Privilege Promotes Transplantation Tolerance by Altering the Balance between Memory and Regulatory T Cells.</a> J Immunol. 174, 6161-6168 (2005).</li><li>Merani, S., Toso, C., Emamaullee, J., 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=Optimal+implantation+site+for+pancreatic+islet+transplantation.">Optimal implantation site for pancreatic islet transplantation.</a> British Journal of Surgery. 95, 1449-1461 (2008).</li><li>Inoue, M., Maeno, T., Hatchell, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+of+allografted+pancreatic+islets+in+the+subretinal+space+in+rats.">Survival of allografted pancreatic islets in the subretinal space in rats.</a> Ophthalmic Res. 35, 48-53 (2003).</li></ol>

No conflicts of interest declared.

In the sections above, we described detailed steps for isolating and transplanting the mouse pancreatic islets. Here, we briefly highlight the steps that are critical for the success of the procedures.
1. Islet isolation
The key steps are identifying an optimal enzymatic digestion time (1.4), positioning the hemostat clamp over the duodenal opening of the common bile duct (2.2.3), cannulating the common bile duct (2.3), vigorous shaking of the pancreas after the 37&deg;C digest (3.2), and removal of exocrine cell contamination (3.3.7 and 3.3.16). These steps have a dramatic influence on the quality, yield and purity of islets. Perhaps the most technically challenging step is the cannulation of the common bile duct. In many mice, the common bile duct can be difficult to visualize due to surrounding fascia. Therefore, it is common that attempts to cannulate the common bile duct result in penetration of the fascia tissue only. In these instances, the Liberase begins to fill the surrounding connective tissue and does not perfuse the pancreas. If this is observed, stop the flow of Liberase and reposition the needle so that it penetrates into the lumen of the bile duct. With practice, it is possible to quickly identify the optimal position for needle placement and to complete the cannulation without damaging the friable duct.
2. Islet transplantation
The critical steps are the STZ induction of diabetes (5.1) and the efficient delivery of islets to the subcapsular pouch (5.3.14-5.3.16). STZ is a naturally occurring glucose analogue that is selectively toxic to the insulin secreting cells &beta;-cells of islets 9. However, this selective toxicity occurs in a relatively narrow concentration range. This is evidenced by the fact that high doses of STZ can result in rapid (2-3 days) lethality in the absence of hyperglycemia. Therefore, care should be taken to identify the optimal dose of STZ for the strain and age of mice being used before any large scale experiments are initiated. The physical delivery of the islets to the subcapsular pouch is also a key step. To achieve consistent results, it is imperative to minimize the loss of islets during their delivery. This can occur if there are defects or tears in the kidney capsule covering the pouch or if the injected volume is too large. Damage to the capsule can be avoided by gentle and careful manipulation, and by using smooth glass probe to prepare the subcapsular pouch. Additionally, the injection volume can be minimized with careful removal of the supernatant in step 5.3.6.3. If the supernatant is removed down to the level of the sedimented islets, there is typically more than enough room in the subcapsular pouch to place 400 to 500 islets. However, if the injection volume is too large, the islets are more likely to leak out of the pouch during the transplant process, compromising the reproducibility of the experiment.
Within this protocol, it is possible to modify some of the steps and still achieve satisfactory outcomes. These include the following: (a) the use of a different enzyme to digest the pancreas, (b) a different method for delivering the enzyme to the pancreas, (c) the use of a continuous Ficoll-sodium-diatrizoate (FSD) gradient instead of the single density Histopaque1077 step, and (d) islet transplantation to locations other than the kidney subcapsule.
<ol class="lalpha">
	<li>In this protocol, Liberase is used to weaken the cell-cell contacts within the pancreas. Liberase is essentially a mixture of highly purified collagenases that have proven useful in releasing islets from the pancreas. However, less purified collagenases are capable of accomplishing a similar result. Therefore, it is possible to use different commercially available enzyme preparations for digesting the pancreas, as long as the digestion condition is optimized to achieve high islet quality, yield and purity.</li>
	<li>It is also possible to digest the pancreas without ductal perfusion of enzyme. Delivery of Liberase through the common bile duct allows for maximal exposure of pancreatic surface area to the enzyme. This results in more even digestion of the pancreas and greater release of intact islets. However, other steps to increase the surface area of the pancreas to the enzyme can be used. For example, mincing the pancreas before incubating it in Liberase or directly injecting Liberase through the pancreatic capsule can be used to isolate islets. However, no other method of Liberase delivery is as effective as perfusion through the common bile duct. Therefore, mincing or direct injection should be reserved as a last resort, if the common bile duct is damaged and not perfusable.</li>
	<li>An alternative method for separating the islets from the exocrine cells involves the use of a FSD gradient instead of Histopaque1077 10. The efficient separation of islets from exocrine cells in this protocol is in part due to a density differential between the two cell types. Histopaque1077 has a density of 1.0771 g/ml at 25&deg;C. At this temperature, Histopaque is denser than the islets but less dense than the exocrine cells. Therefore, under the force of centrifugation, Histopaque1077 pellets the exocrine cells while lifting the islets to the surface. In principle, any other substance with a density that that can separate islets from exocrine cells can be used in this step and the FSD gradient is one such substance.</li>
	<li>With respect to the location for islets transplantation, the kidney capsule used in this protocol is one of the few possible sites. While the kidney subcapsule is the most commonly reported site of transplantation in mice, other transplantation sites have been found to be effective and they include the hepatic portal vein, subretinal space, testis, epididymal fat pad, spleen, and even pancreas 11-14. Each of these locations has their own advantages and challenges. Therefore, the selection of a site for islet transplantation should be determined by the questions addressed and specific circumstances of the experiments.</li>
</ol> 
It is believed that strategies that can overcome the limitations on islet transplantation will dramatically enhance its therapeutic potential. Therefore, the use of a murine model as detailed by this protocol offers an attractive approach for identifying such strategies.

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<td>RPMI 1640</td>
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<td>Roche</td>
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<td>Gibco</td>
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<td>Sigma Aldrich</td>
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<td>Fisher Scientific</td>
<td>22362574</td>
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<td>Insulin Syringe</td>
<td>Equipment</td>
<td>BD Falcon</td>
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<td>BD Falcon</td>
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<td>Fisher Scientific</td>
<td>NC9083963</td>
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<td>BD Falcon</td>
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<td>BD Falcon</td>
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<td>BD Falcon</td>
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<td>0300241</td>
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<td>Fisher Scientific</td>
<td>15-462-5</td>
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<td>Dissecting Microscope</td>
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<td>Inverted Microscope</td>
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<td>Nikon</td>
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<td>Tissue Culture Incubator</td>
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<td>Napco</td>
<td>6300</td>
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<td>Kent Scientific</td>
<td>INS14393</td>
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<td>McPherson-Vannas Scissors</td>
<td>Equipment</td>
<td>Kent Scientific</td>
<td>INS500260</td>
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<td>Equipment</td>
<td>Kent Scientific</td>
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<td>Kent Scientific</td>
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<td>Fisher Scientific</td>
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<td>Equipment</td>
<td>USA Scientific</td>
<td>111-0730</td>
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a method for murine islet isolation and subcapsular kidney transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Watch this Scientific Journal Video about A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation at JoVE.com

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

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