Name: human pancreatic islet isolation: part ii: purification and culture of human islets
Uploaded: 2009-05-26T00:00:00
Description: Watch this Scientific Journal Video about Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets at JoVE.com

胰岛细胞资源中心美国国立卫生研究院授予（05-003 RFA - RR），克里斯托弗基金会，Efroymson基金会，和泰乐基金会成立在芝加哥伊利诺伊大学以及支持。

1。胰岛的分离纯化<ol><li>要启动胰岛的纯化过程，加载的COBE袋，所有的管子钳，除了绿管，并设置速度到1500超到0。 </li><li>在引擎盖成立泵和梯度烧杯和收集管连接到绿管。 </li><li>现在可以开始加载聚蔗糖1.100：推“旋转”，然后添加110毫升聚蔗糖1.100前烧杯，并启动水泵，以负载的COBE袋。由于聚蔗糖加载，按超，停泵，松开泵的扬程，并设置超出速度100。 </li><li>当聚蔗糖达到烧杯中，所有?...

<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>Nano, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+isolation+for+allotransplantation:+variables+associated+with+successful+islet+yield+and+graft+function.">Islet isolation for allotransplantation: variables associated with successful islet yield and graft function.</a> Diabetologia. 48, 906-912 (2005).</li><li>Barbaro, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+human+pancreatic+islet+purification+with+the+refined+UIC-UB+density+gradient.">Improved human pancreatic islet purification with the refined UIC-UB density gradient.</a> Transplantation. 84, 1200-1203 (2007).</li><li>Lakey, J. R., Rajotte, R. V., Warnock, G. L., Kneteman, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+pancreas+preservation+prior+to+islet+isolation.+Cold+ischemic+tolerance.">Human pancreas preservation prior to islet isolation. Cold ischemic tolerance.</a> Transplantation. 59, 689-694 (1995).</li><li>Fridell, 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=Comparison+of+histidine-tryptophan-ketoglutarate+solution+and+University+of+Wisconsin+solution+for+organ+preservation+in+clinical+pancreas+transplantation.">Comparison of histidine-tryptophan-ketoglutarate solution and University of Wisconsin solution for organ preservation in clinical pancreas transplantation.</a> Transplantation. 77, 1304-1306 (2004).</li><li>Potdar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+experience+using+histidine-tryptophan-ketoglutarate+solution+in+clinical+pancreas+transplantation.">Initial experience using histidine-tryptophan-ketoglutarate solution in clinical pancreas transplantation.</a> Clin Transplant. 18, 661-665 (2004).</li><li>Salehi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+islet+isolation+outcomes+from+pancreata+preserved+with+Histidine-Tryptophan+Ketoglutarate+versus+University+of+Wisconsin+solution.">Human islet isolation outcomes from pancreata preserved with Histidine-Tryptophan Ketoglutarate versus University of Wisconsin solution.</a> Transplantation. 82, 983-985 (2006).</li><li>Ricordi, C., Lacy, P. E., Scharp, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+islet+isolation+from+human+pancreas.">Automated islet isolation from human pancreas.</a> Diabetes. 38 Suppl 1, 140-142 (1989).</li><li>Ricordi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+isolation+assessment+in+man+and+large+animals.">Islet isolation assessment in man and large animals.</a> Acta Diabetol Lat. 27, 185-195 (1990).</li></ol>

尽管在人胰岛分离技术方面取得了长足的进步，胰岛产量仍充满变数和不可预知的。净化消化胰腺组织移植成功的酶消化后的恢复足够岛大规模的关键。 UIC的净化方法的建议，因为优越的高纯度的人类胰岛复苏，通过使用这种方法来证明。此外，可装载高达50毫升的消化组织进行净化处理运行一个单一的COBE，从而最大限度地减少缺血相关的人类胰岛的损伤，单独使用的总时间缩短。

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Centrifuge</td>
<td>equipment</td>
<td>Beckman Coulter</td>
<td>424803</td>
<td>&nbsp;</td>
<tr>
<td>Cobe 2991 Cell Processor</td>
<td>equipment</td>
<td>Gambro. BCT</td>
<td>2556</td>
<td>islet purification</td>
</tr>
<tr>
<td>MZ6 Inverted Microscopy TLBD4.1</td>
<td>equipment</td>
<td>Leica Diagnostic Instrument Co</td>
<td>4103</td>
<td>&nbsp;</td>
<tr>
<td>Cell Culture Dish with 2 mm Grid</td>
<td>equipment</td>
<td>Nunc Brand Products</td>
<td>174926</td>
<td>islet counting</td>
</tr>
<tr>
<td>Digital Sight DS L1</td>
<td>equipment</td>
<td>Nikon</td>
<td>217267</td>
<td>&nbsp;</td>
<tr>
<td>COBE bag</td>
<td>disposable equipment</td>
<td>O.R. Solution</td>
<td>66707003</td>
<td>&nbsp;</td>
<tr>
<td>T175 culture flask</td>
<td>disposable equipment</td>
<td>Sarstedt</td>
<td>83.1812.500</td>
<td>&nbsp;</td>
<tr>
<td>University of Wisconcin (UW) solution</td>
<td>reagent</td>
<td>DURAMED</td>
<td>1000-46-06</td>
<td>&nbsp;</td>
<tr>
<td>Hank's Buffered Salt Solution (HBSS)</td>
<td>reagent</td>
<td>Mediatech Inc</td>
<td>99-597-CM</td>
<td>&nbsp;</td>
<tr>
<td>Ficoll 1.100</td>
<td>reagent</td>
<td>Bichrom AG</td>
<td>L6155</td>
<td>&nbsp;</td>
<tr>
<td>M199 media (wash solution)</td>
<td>reagent</td>
<td>Mediatech Inc</td>
<td>99-784-CM</td>
<td>&nbsp;</td>
<tr>
<td>Final wash/Culture Medium</td>
<td>reagent</td>
<td>Mediatech Inc</td>
<td>99-785-CV</td>
<td>&nbsp;</td>
<tr>
<td>Dithizone</td>
<td>reagent</td>
<td>Sigma </td>
<td>D5130</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

human pancreatic islet isolation: part ii: purification and culture of human islets

1型糖尿病的管理负担，既要对个人和社会，每年耗资超过100亿美元。尽管血糖监测和新的胰岛素制剂的广泛使用，许多人仍然发展毁灭性的继发性并发症。胰岛移植可以恢复接近正常的糖尿病患者血糖控制<sup&gt; 1</sup&gt;胰岛素强化治疗相关的严重的低血糖发作的危险，没有。胰岛移植成功的，重要的是提供足够的胰岛质量。但是，捐助者的特征，器官获取和保存的影响隔离的结果<sup&gt; 2</sup&gt;。在美国伊利诺伊大学芝加哥分校（UIC），我们开发了一种改进的净化梯度成功分离协议<sup&gt; 3</sup&gt;。该计划开始于2004年1月和2008年11月超过300隔离。 “胰腺被送往在冷保存液（UW液，威斯康星州或HTK的大学，组氨酸，色氨酸酮戊二酸）<sup&gt; 4-7</sup&gt;在UIC的细胞分离胰岛隔离的实验室。使用UIC的方法，这是最初所Ricordi描述的方法修改后的版本中分离出胰岛<em&gt;等</em&gt;<sup&gt; 8</sup&gt;。正如在第一部分所述：消化和胰腺组织收集，人体胰腺被修剪，空心，灌注，消化。组织收集和UW液中孵育至少30分钟后，被装在细胞分离机（COBE 2991，COBE，翠湖，二氧化碳）净化<sup&gt; 3</sup&gt;。经过净化，胰岛产量（表现为胰岛等值，IEQ），组织的体积，和纯度测定按标准方法<sup&gt; 9</sup&gt;。隔离胰岛培养媒体CMRL -​​ 1066（Mediatech，赫恩登，VA），辅以1.5％的人血白蛋白，0.1％的胰岛素转硒（简称ITS），1毫升，5毫升1M HEPES环丙沙星，和14.5毫升7.5％，在T175烧瓶钠碳酸氢盐在37 ° C过夜培养胰岛移植前或用于研究。

Watch this Scientific Journal Video about Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets at JoVE.com

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets

实现高​​品质和适当数量的人类胰岛是胰岛移植成功的突出的先决条件之一。在这个视频中，我们描述一步一步人类胰岛隔离（第二部分：人类胰岛的纯化和文化）的程序，采用改良的自动方法。

人类胰岛的分离：第二部分：人类胰岛的分离纯化及文化

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