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

Supported by founding from the University of Illinois at Chicago as well as Islet Cell Resource Center NIH grant (RFA-RR 05-003), the Christopher Foundation, the Efroymson Foundation, and the Tellabs Foundation.

1. Purification of islets
<ol>
<li>To start the islet purification procedure, load the COBE bag, clamp all tubes except the green tube, and set the speed to 1,500 and the super out to 0.</li>
<li>In the hood set up the pump and gradient beakers and connect the collection tubing to the green tube.</li>
<li>Now the loading of the ficoll 1.100 can begin: push &ldquo;spin&rdquo; then add 110 ml of Ficoll 1.100 to the front beaker, and start the pump in order to load the COBE bag. As the Ficoll is loaded, press super out, ...

<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>

In spite of significant advancements in techniques of human islet isolation, islet yield remain highly variable and unpredictable. Purification of digested pancreatic tissue is crucial to recover a sufficient isle mass after successful enzymatic digestion for transplantation. The UIC purification method is recommended because a superior recovery of highly pure human islets was demonstrated by using this method. Moreover, up to 50 ml of digested tissue can be loaded in a single Cobe run for purification, thus minimizing t...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristics, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004 and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. As described in Part I: Digestion and Collection of Pancreatic Tissue, human pancreas was trimmed, cannulated, perfused, and digested. After collection and at least 30 minutes of incubation in UW solution, the tissue was loaded in the cell separator (COBE 2991, Cobe, Lakewood, CO) for purification 3. Following purification, islet yield (expressed as islet equivalents, IEQ), tissue volume, and purity was determined according to standard methods 9. Isolated islets were cultured in CMRL-1066 media (Mediatech, Herndon, VA), supplemented with 1.5% human albumin, 0.1% insulin-transferrin-selenium (ITS), 1 ml of Ciprofloxacin, 5 ml o f 1M HEPES, and 14.5 ml of 7.5% Sodium Bicarbonate in T175 flasks at 37&deg;C overnight culture before islets were transplanted or used for research.

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

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part II: purification and culture of human islets) using a modified automated method.

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

Research

JoVE Journal

Biology

Murine Pancreatic Islet Isolation

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part II: Carbohydrates

Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part II: Carbohydrates

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st ...

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

En metode for mus bukspyttkjertelens Isolering og Intracellulær cAMP Bestemmelse

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

Isolasjon og kultur av mus Primær acinøse Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Effektiv produksjon og rensing av rekombinant mus Kindlin-3 fra insektceller for biofysiske studier

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Ex vivo Culture of Mouse Embryonic Hud og Live-avbildning av Melanoblast Migration

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...