<p class="jove_content">UCSF Diabetes Center. NIH UCSF DERC Islet Core. JDRF. LIfeScan Inc. Johnson &amp; Johnson Company.</p>

<p class="jove_title">Preparation of Islets for Transplant (Tx)</p>
<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 syr...

<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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro-expanded+antigen-specific+regulatory+T+cells+suppress+autoimmune+diabetes.">In vitro-expanded antigen-specific regulatory T cells suppress autoimmune diabetes.</a> <em style='font-style: italic'>J Exp Med</em>. <b>199</b>, 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inability+to+induce+tolerance+through+direct+antigen+presentation.">Inability to induce tolerance through direct antigen presentation.</a> <em style='font-style: italic'>Am J Transplant</em>. <b>2</b>, 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+transplant+rejection+following+treatment+with+anti-B7-2+and+anti-B7-1+antibodies.+Transplantation.">Inhibition of transplant rejection following treatment with anti-B7-2 and anti-B7-1 antibodies. Transplantation.</a> <em style='font-style: italic'>Transplantation</em>. <b>60</b>, 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+survival+of+xenogeneic+pancreatic+islet+grafts+induced+by+CTLA4lg.">Long-term survival of xenogeneic pancreatic islet grafts induced by CTLA4lg.</a> <em style='font-style: italic'>Science</em>. <b>257</b>, 789-792 (1992).</li></ol>

<p class="jove_content">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.</p>

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		<div>
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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Surgical gloves</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>11-394-95(sz)</td>
<td>&nbsp;</td>
<tr>
<td>Surgical scrub sponges</td>
<td>&nbsp;</td>
<td>Moore medical</td>
<td>42940AK</td>
<td>&nbsp;</td>
<tr>
<td>Forceps</td>
<td>&nbsp;</td>
<td>Miltex</td>
<td>6-114 and 6-26</td>
<td>2 with teeth, 2 straight</td>
</tr>
<tr>
<td>Dissecting scissors n=2</td>
<td>&nbsp;</td>
<td>Miltex</td>
<td>5-290</td>
<td>&nbsp;</td>
<tr>
<td>Oster razor (sz. 40 blade)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>01-305-10B</td>
<td>&nbsp;</td>
<tr>
<td>4x4 Sterile gauze pads</td>
<td>&nbsp;</td>
<td>Moore Medical</td>
<td>08252AK</td>
<td>&nbsp;</td>
<tr>
<td>Povidone Iodine pads</td>
<td>&nbsp;</td>
<td>Moore Medical</td>
<td>08486AK</td>
<td>&nbsp;</td>
<tr>
<td>Alcohol pads</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>14-819-2</td>
<td>&nbsp;</td>
<tr>
<td>Heating pad</td>
<td>&nbsp;</td>
<td>Moore Medical</td>
<td>42508AK</td>
<td>&nbsp;</td>
<tr>
<td>23G or 25G needles</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>1" long</td>
</tr>
<tr>
<td>Cidex solution</td>
<td>&nbsp;</td>
<td>Moore Medical</td>
<td>07535AK</td>
<td>for cleaning/santizing instruments</td>
</tr>
<tr>
<td>PE50 polyethylene tubing</td>
<td>&nbsp;</td>
<td>BD</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>&nbsp;</td>
<td>Moore</td>
<td>35625AK</td>
<td>&nbsp;</td>
<tr>
<td>Silicone tubing</td>
<td>&nbsp;</td>
<td>Spectrum Chromat.</td>
<td>123732</td>
<td>5/32&#x201D;OD x 1/32&#x201D; ID</td>
</tr>
<tr>
<td>Instrument sterilizing container</td>
<td>&nbsp;</td>
<td>Moore Medical</td>
<td>39074AK</td>
<td>&nbsp;</td>
<tr>
<td>Straight Pipet Tips</td>
<td>&nbsp;</td>
<td>TipOne</td>
<td>1111-0810</td>
<td>sterile</td>
</tr>
<tr>
<td>Glass syringe w/screw-drive</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>1001</td>
<td>&nbsp;</td>
<tr>
<td>Cauterizing tool</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>RS230</td>
<td>&nbsp;</td>
<tr>
<td>Needle Holder</td>
<td>&nbsp;</td>
<td>Moore Medical</td>
<td>41-067</td>
<td>&nbsp;</td>
<tr>
<td>5-0 silk</td>
<td>&nbsp;</td>
<td>Look</td>
<td>754B</td>
<td>for suture (6-C) </td>
</tr>
<tr>
<td>Cotton tipped swab</td>
<td>&nbsp;</td>
<td>Moore Medical</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>9mm autoclip stapler</td>
<td>&nbsp;</td>
<td>BD</td>
<td>7630</td>
<td>&nbsp;</td>
<tr>
<td>9mm staples</td>
<td>&nbsp;</td>
<td>BD</td>
<td>7631</td>
<td>&nbsp;</td>
<tr>
<td>9mm staple remover</td>
<td>&nbsp;</td>
<td>BD</td>
<td>7637</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
			<div>
					</div>
		

transplantation of pancreatic islets into the kidney capsule of diabetic mice

<p class="jove_content">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.</p>

<p class="jove_content">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.</p>
<p class="jove_content">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.</p>

Watch this Scientific Journal Video about Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice at JoVE.com

Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice

<p class="jove_content">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.</p>

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 ...

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

Research

JoVE Journal

Biology

26.9K visualizações.  University of California, San Francisco - UCSF. Nosso protocolo foi desenvolvido para fornecer precisão e facilidade ilhotas ou células sob a cápsula renal de camundongos. Células estão concentradas em pelotas na tubulação final utilizada para o transplante de células sob a cápsula renal. A facilidade desta técnica reduz o estresse nas células e mouse.