Name: Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice (Video) | JoVE Journal
Uploaded: 2007-10-31T23:37:26.000+00:00
Duration: 10 min 43 s
Description: Learn islet transplantation under the kidney capsule in diabetic mice using purified syngeneic or allogeneic islets. This method minimizes stress on cells and recipients to improve transplantation outcomes.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

playerTrialDesktop

UCSF Diabetes Center. NIH UCSF DERC Islet Core. JDRF. LIfeScan Inc. Johnson &amp; Johnson Company.

Preparation of Islets for Transplant (Tx)
<ol>
<li>Under an inverted microscope, hand-pick islets using a P200 pipetman and straight pipet tip from the cultured islets in a 100mm plate.</li>
<li>Count islets 100 at a time and transfer into each microcentrifuge tube (~500 islets/tube/mouse).</li>
<li>Allow islets to settle to the bottom of the microcentrifuge tubes.</li>
<li>Draw one islet pellet into a P200 pipetman (set at 130ul) using a straight thin-wall pipet tip.</li>
<li>Place a silicone tube adapter over the syringe tip. Insert a length of PE50 tubing into the silicone adapter.</li>
<li>Hang the pipetman to the side of the hood and tape the PE50 tubing to the hood wall higher than the islets in the tip. This will allow the islets to settle only in the tip of the pipette.</li>
<li>Transfer the islets into the PE50 tubing by slowly dialing the pipetman and moving the islets into the PE50 tubing,&nbsp;being careful&nbsp;not to expel the islets from the end.</li>
<li>Make a kink in the beveled end of the PE50 tubing. While maintaining the kink, disconnect the silicone adapter tubing from the pipetman and secure the kink with the silicone adapter.</li>
<li>Place the islets, in the PE50 tubing, kinked silicone adaptor tubing side down, into a 15 mL conical with a cut 5 mL pipet and tape the PE50 tubing over the side of the 5 mL pipet to prevent curling of the PE50 tubing while centrifuging.</li>
<li>Centrifuge the PE50/15mL conicals to 1000 rpm and turn off centrifuge. (Never prepare more than 10 islet preparations at one time).</li>
<li>Place tube on ice.</li>
</ol>
Preparation of Mouse for Transplant
<ol>
<li>Anesthetize the mice with isofluorane or with Ketamine/Xylexene (see preparation protocol).</li>
<li>After anesthetic has taken affect, shave the left flank of the mouse.</li>
<li>Swab skin of mouse, center-out, with Povidone Iodine swab and then wipe off with an ETOH swab.</li>
<li>Locate the left kidney (just right of spleen). Make a small incision in the skin, exposing the peritoneum.</li>
<li>Make a small incision in the peritoneum exposing the kidney. Keeping the incision small will help in keeping the kidney raised and exposed.</li>
<li>Apply a slight pressure to both sides of the incision, raise or pop the kidney out of the mouse.</li>
<li>Keep the kidney moist by applying normal saline with a cotton tipped swab.</li>
<li>Using a syringe 23 or 25 gauge needle, make a small scratch on the right flank of the kidney, creating a nick in the kidney capsule; not too deep or too large.</li>
</ol>
Transplantation of Islets
Note: While the mouse is being prepared for Tx, the second person should prepare the Hamilton screw-drive islet transplant syringe.
<ol>
<li>Slowly remove the silicone adaptor tubing from the PE50 tubing while keeping the kink in the tubing. </li>
<li>Place the opposite end of the PE50 tubing into the silicone adaptor tubing and place the silicone adaptor tubing onto the pipette tip attached to the "screw-drive" glass Hamilton syringe. Slowly release the kink in the PE50 tubing, making sure that the isles do not leak out.</li>
<li>Advance the islets slowly to the tip of the PE50 tubing using the "screw" mechanism, but keep a small air bubble in the front of the islets in the PE50 tubing.</li>
<li>Into the nick made in the kidney, carefully slide the PE50 tubing under the capsule, making a small pocket. Be very careful not to gouge the kidney or puncturing through the capsule. </li>
<li>It helps to keep the area and capsule moist with normal saline-Gentamycin soaked cotton tipped swab; a dry capsule will tear easily.</li>
<li>Gently move the tubing in all directions, creating a "pocket" for the transplanted islets to rest. Remember to reapply additional normal saline to keep the area moist.</li>
<li>Under the direction of the person who has opened the mouse and who has placed the PE50 tubing under the kidney capsule, the second person, who prepared the mouse and Hamilton transplant syringe, will slowly advance islets under the capsule, inside the "pocket", behind a small air bubble delivered by the PE50 tubing, until all the islets are transplanted.</li>
<li>Slowly remove the PE50 tubing, dry the area with a dry swab and carefully cauterize the nick with low heat.</li>
<li>Using a dry cotton tipped swab, make sure all bleeding has stopped. Once bleeding has stopped, re-moisten the kidney with sterile saline, and&nbsp;gently replace the kidney into the peritoneum prior to closing the mouse with suture and skin staples.</li>
</ol>
Closing/Revival of Mouse
<ol>
<li>Close the peritoneum with a running stitch using 5-0 silk sutures w/ a C-6 19mm needle.</li>
<li>Using forceps draw both side of the skin incision together.</li>
<li>Staple the skin together with 2 or 3 staples.</li>
<li>Clean the skin of the mouse of any blood, using a cotton tipped swab and saline.</li>
<li>Immediately treat the mouse with a subcutaneous injection of Flunixin and Buprenorphine.</li>
<li>Place the mouse in a cage, that&nbsp;is placed on a heating pad or below a heating lamp, until the mouse is fully active.</li>
<li>Remove the skin staples in 2 weeks.</li>
</ol>

<ol><li>Tang, Q., Henriksen, K. J., Bi, M., Finger, E. B., Szot, G., Ye, J., Masteller, E. L., McDevitt, H., Bonyhadi, M., Bluestone, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro-expanded+antigen-specific+regulatory+T+cells+suppress+autoimmune+diabetes%5BTitle%5D)+AND+%22J+Exp+Med%22%5BJournal%5D)">In vitro-expanded antigen-specific regulatory T cells suppress autoimmune diabetes.</a> J Exp Med. 199, 1455-1465 (2004).</li>
<li>Rulifson, I. C., Szot, G. L., Palmer, E., Bluestone, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inability+to+induce+tolerance+through+direct+antigen+presentation%5BTitle%5D)+AND+%22Am+J+Transplant%22%5BJournal%5D)">Inability to induce tolerance through direct antigen presentation.</a> Am J Transplant. 2, 510-519 (2002).</li>
<li>Lenschow, D. J., Zeng, Y., Hathcock, K. S., Zuckerman, L. A., Freeman, G., Thistlethwaite, J. R., Gray, G. S., Hodes, R. J., Bluestone, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhibition+of+transplant+rejection+following+treatment+with+anti-B7-2+and+anti-B7-1+antibodies.+Transplantation%5BTitle%5D)+AND+%22Transplantation%22%5BJournal%5D)">Inhibition of transplant rejection following treatment with anti-B7-2 and anti-B7-1 antibodies. Transplantation.</a> Transplantation. 60, 1171-1178 (1995).</li>
<li>Lenschow, D. J., Zeng, Y., Thistlethwaite, J. R., Montag, A., Brady, W., Gibson, M. G., Linsley, P. S., Bluestone, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term+survival+of+xenogeneic+pancreatic+islet+grafts+induced+by+CTLA4lg%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Long-term survival of xenogeneic pancreatic islet grafts induced by CTLA4lg.</a> Science. 257, 789-792 (1992).</li>
</ol>

This protocol provides a practical and efficient option for cleanly and easily delivering islets or cells under the kidney capsule of diabetic or normal mice. The technique of concentrating and pelleting the islets or cells into the final delivery tubing (PE50) used to transplant the cells under the kidney capsule offers an easy and effective method of transplanting cells while reducing any undue stress to the cells or to the mouse.

<table><tbody><tr><td>Surgical gloves</td><td>Fisher Scientific</td><td>11-394-95(sz)</td><td></td></tr><tr><td>Surgical scrub sponges</td><td>Moore Medical</td><td>42940AK</td><td></td></tr><tr><td>Forceps</td><td>Miltex Inc.</td><td>6-114 and 6-26</td><td>2 with teeth, 2 straight</td></tr><tr><td>Dissecting scissors n=2</td><td>Miltex Inc.</td><td>5-290</td><td></td></tr><tr><td>Oster razor (sz. 40 blade)</td><td>Fisher Scientific</td><td>01-305-10B</td><td></td></tr><tr><td>4x4 Sterile gauze pads</td><td>Moore Medical</td><td>08252AK</td><td></td></tr><tr><td>Povidone Iodine pads</td><td>Moore Medical</td><td>08486AK</td><td></td></tr><tr><td>Alcohol pads</td><td>Fisher Scientific</td><td>14-819-2</td><td></td></tr><tr><td>Heating pad</td><td>Moore Medical</td><td>42508AK</td><td></td></tr><tr><td>23G or 25G needles</td><td></td><td></td><td>1&quot; long</td></tr><tr><td>Cidex solution</td><td>Moore Medical</td><td>07535AK</td><td>for cleaning/santizing instruments</td></tr><tr><td>PE50 polyethylene tubing</td><td>BD Biosciences</td><td>427411</td><td>PE50, 0.965mm O.D. x 0.58mm I.D.</td></tr><tr><td>Cidex + 28 day soln</td><td>Moore Medical</td><td>35625AK</td><td></td></tr><tr><td>Silicone tubing</td><td>Spectrum Chromat.</td><td>123732</td><td>5/32&amp;#8221;OD x 1/32&amp;#8221; ID</td></tr><tr><td>Instrument sterilizing container</td><td>Moore Medical</td><td>39074AK</td><td></td></tr><tr><td>Straight Pipet Tips</td><td>USA Scientific, Inc.</td><td>1111-0810</td><td>sterile</td></tr><tr><td>Glass syringe w/screw-drive</td><td>Hamilton Co</td><td>1001</td><td></td></tr><tr><td>Cauterizing tool</td><td>Roboz Surgical Instruments Co.</td><td>RS230</td><td></td></tr><tr><td>Needle Holder</td><td>Moore Medical</td><td>41-067</td><td></td></tr><tr><td>5-0 silk</td><td>LOOK Surgical</td><td>754B</td><td>for suture (6-C) </td></tr><tr><td>Cotton tipped swab</td><td>Moore Medical</td><td></td><td></td></tr><tr><td>9mm autoclip stapler</td><td>BD Biosciences</td><td>7630</td><td></td></tr><tr><td>9mm staples</td><td>BD Biosciences</td><td>7631</td><td></td></tr><tr><td>9mm staple remover</td><td>BD Biosciences</td><td>7637</td><td></td></tr></tbody></table>

transplantation of pancreatic islets into the kidney capsule of diabetic mice

Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice. We found that it was easier to concentrate the islets or cells into pellets in the final delivery tubing (PE50) used to transplant the cells under the kidney capsule. This technique provides both speed and ease while reducing any undue stress to the cells or to the mouse.

Loading: Settled, hand picked, islets or pelleted cells are carefully aspirated off the bottom of a 1.5 mL microcentrifuge tube using a p200 pipetteman and a straight, thin-wall pipette tip. A length of PE50 tubing is attached to the pipette tip using a small silicone adapter tubing. Cells are allowed to settle, in the tip, and then are transferred to the PE50 tubing by slowly dialing the pipetteman. Once the cells are near the end of the PE50 tubing, a kink is made and the silicone adaptor tubing is placed over the kink. The PE50 tubing is transferred to a 15 mL conical containing a cut 5 mL pipet, and the PE50 tubing is taped over the side of the 5 mL pipet to prevent curling during centrifuging. Cells are allowed to reach 1,000 rpm and stopped.
Transplantation: Recipient mice are anesthetized, shaved, and cleaned. A small incision is made on the left flank of the mouse and the kidney is exposed. The kidney, fat, and tissue are kept moist with normal saline swab. The distal end of the PE50 is attached to a Hamilton screw drive syringe, containing a pipette tip, using the silicone adaptor tubing. A small nick is made on the right flank side of the kidney, not too large nor too deep. The beveled end of the PE50 tubing, nearest the cells, is carefully placed under the capsule, the tubing is moved around gently to make space while swabbing normal saline; a dry capsule can tear easily. A small air bubble is delivered under the capsule by slowly dialing the syringe screw drive. Islets are then slowly delivered behind the air bubble. Once the islets have been delivered kidney homeostasis is maintained and the knick is cauterized with low heat. The kidney is placed back into the cavity and the peritoneum and skin are sutured and stapled. Mice are immediately treated with Flunixin and Buprenorphine s.q. and placed in a cage on a heating pad.

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of mice. Cells are concentrated into pellets in the final tubing used for transplanting the cells under the kidney capsule. The ease of this technique reduces stress to the cells and the mouse.

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice.  We found that it was easier ...

Hi, My name is Greg Zod. I'm at UCSF Diabetes Center at the Bluestone Laboratory, and today I'm gonna show you how to transplant mirroring eyelets under the kidney capsule of diabetic mice. This procedure involves taking eyelets that have been purified from either syngeneic or allogeneic strain of mice, taking those eyelets, purifying them, and transplanting em under the kidney capsule of diabetic mice.Mice that could be naturally diabetic like the NOD or mice that are made diabetic artificially with the injection Of strep pitocin. Now we're Ready to handpick the eyelets. I'm going to handpick the eyelets and count them while I'm sucking them up with a pasture with a pipette.I'm gonna leave behind any a in our tissue, and you can see just by the, the, the way the lights refracted in the tissue, what is an eyelet and what's not an eyelet. And then I count the eyelets. I'll put them in a micro centri tube and, and we'll collect them for transplant.The eyelets have a more dense, almost reddish look to them, and the ace in R is lighter and fluffier. And it also has like a whitish look. So we, you're going to get some a in r tissue while you do the eyelets, but as long as you grab a maj

transplantation-pancreatic-islets-into-kidney-capsule-diabetic

Research

JoVE Journal

Biology

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

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

This video describes the protocol for grafting human pancreatic islets into the anterior chamber of the mouse’s eye to monitor revascularization of human islet grafts in vivo. It also helps to understand the contribution of recipient versus donor cells in promoting the encapsulation and vascularization of the graft.

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

This protocol demonstrates murine islet isolation and seeding onto a decellularized scaffold. Scaffold-supported islets were transplanted into the epididymal fat pad of streptozotocin (STZ)-induced diabetic mice. Islets survived at the transplantation site and reversed the hyperglycemic condition.

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

Among abdominal solid organ transplantation, pancreatic grafts are prone to develop severe ischemia reperfusion injury-associated graft damage, leading eventually to early graft loss. This protocol describes a model of murine pancreas transplantation using a non-suture cuff technique, ideally suited for analyzing these early, deleterious damages.

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

A method for the omental transplantation of islets in a mouse is introduced. The isolated islets are mixed with hydrogel and the mixture is placed into the omental pouch of a diabetic mouse. Then, the blood glucose is monitored, and immuno-histochemical analysis is performed.

A Method for Islet Transplantation to the Omentum in Mouse

Pancreatic islet transplantation is a way to achieve normoglycemia in type 1 diabetes. This article focuses on the transplantation technique through the intraportal route to maintain normal glucose levels in diabetic mice.

Intraportal Transplantation of Pancreatic Islets in Mouse Model

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

The goal of this protocol is to continuously monitor the dynamics of the human pancreatic islet engraftment process and the contributing host versus donor cells. This is accomplished by transplanting human islets into the anterior chamber of the eye (ACE) of an NOD.(Cg)-Gt(ROSA)26Sortm4-Rag2-/-mouse recipient followed by repeated 2-photon imaging.

Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the Anterior Eye Chamber

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

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

We provide a minimally invasive protocol for the intramuscular transplantation of human stem cell-derived pancreatic stem cell islets into immunodeficient mice for in vivo implantation and downstream analysis.

Intramuscular Transplantation of Human Pluripotent Stem Cell-derived Pancreatic Endocrine Cells in Mice

Learn islet transplantation under the kidney capsule in diabetic mice using purified syngeneic or allogeneic islets. This method minimizes stress on cells and recipients to improve transplantation outcomes.

Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice

Explore More Videos