Soutenu par un financement de l&#39;Université de l&#39;Illinois à Chicago ainsi que des cellules des îlots Resource Center NIH (RFA-05-003 RR), la Fondation Christopher, la Fondation Efroymson, et la Fondation Tellabs.

1. Facilité de configuration <ol><li> L&#39;isolement des îlots humains est une procédure qui implique le temps sensible équipe, et donc, quatre personnes sont nécessaires pour mener à bien cette procédure. L&#39;équipe est dirigée par le chirurgien, qui dissèque et cannulates le pancréas. </li><li> L&#39;isolement des îlots commence à la minute de l&#39;équipe entre dans l&#39;installation des îlots UIC isolement. Membres de l&#39;équipe d&#39;assurer que tous les instruments nécessaires, comme la centrifugeuse, est vérifié et mis sous tension. Ils s&#39;assurent aussi de désinfecter toutes les hottes et les surfaces. </li><li> Deux personnes seront mis en place la table de canulation, machine à neige fondante, circuit de la digestion, l&#39;unité de perfusion, la machine de purification COBE, et les tables chirurgicales. </li><li> Simultanément, deux autres membres d&#39;équipe va préparer le support qui est nécessaire lors de l&#39;isolement. </li><li> Ensuite, les tubes de bactériologie (bouteilles, tubes, la forme et le sac), des tubes de prélèvement (pré-rempli avec de lavage), et les tubes et les plaques d&#39;évaluation sont préparés. </li><li> Préparer le tube de purification fixés comme suit: Le 1er tube vide et marqué à 150 ml, tubes remplis 2-12 avec 200 ml d&#39;M199 milieux supplémentés avec de l&#39;albumine humaine (solution de lavage) et marqué à 230 ml, et enfin un tube conique de 50 ml remplie avec 50 ml de solution de lavage. Une fois ces étapes de préparation sont complets, nous sommes prêts à commencer la préparation et la perfusion du pancréas. </li></ol> 2. La préparation du pancréas et de la perfusion <ol><li> Pour commencer la préparation et la perfusion du pancréas, un opérateur enlève l&#39;organe de l&#39;emballage en utilisant une technique stérile. Le chirurgien va transférer le pancréas à la table de décontamination. </li><li> Le chirurgien va remplir une seringue de 60 ml avec la solution de préservation du pancréas d&#39;origine et le remettre à un opérateur qui va l&#39;utiliser pour remplir des bouteilles préparées &quot;achats&quot; bactériologie. </li><li> Après une inspection visuelle pour déterminer si l&#39;organe est approprié pour l&#39;isolement des îlots (Si le pancréas est endommagé au cours d&#39;achat ou anatomique anormale, il ne peut être utilisé pour l&#39;isolement des îlots), le chirurgien va demander à un opérateur pour préparer la collagénase. Rappelez-vous que ce temps de remise en suspension varie entre marques différentes enzymes et des lots. </li><li> Lorsque le chirurgien est prêt, apporter les solutions de décontamination (Betadine, Fungizone / céfazoline et HBSS) à la table de décontamination et les verser dans les contenants appropriés. Chirurgien effectue la décontamination du pancréas à laver pendant une minute le pancréas dans chacune des solutions de décontamination. Le pancréas est ensuite placé dans un petit bocal stérile dans lequel il sera pesé. Après le poids est entré dans le dossier de lot, le pancréas est placé dans la poêle avec quelques canulation solution de conservation. </li><li> Le chirurgien va exposer et partiellement inciser le conduit principal et cathétériser chaque moitié du pancréas avec une canule. La canule sera garanti par une suture. Pour perfuser le pancréas, d&#39;abord de le déplacer à l&#39;unité de la perfusion et versez la solution enzymatique dans le bassin. Temps de perfusion totale est d&#39;environ 10 minutes, à 80 mmHg pendant 5 minutes, puis à 180 mm Hg pendant 5 minutes. Pancréas distendus après la perfusion. </li><li> Déplacer le pancréas perfusé à la casserole canulation, où le tissu adipeux est coupé par le pancréas attentivement. Ensuite, couper le pancréas en 10-12 morceaux et transfert à Ricordi Chambre pour la digestion. </li></ol> 3. La digestion du pancréas <ol><li> Pour commencer étape de digestion, le transfert du pancréas à la chambre vide Ricordi, ajouter un flacon de 2,5 ml de Pulmozyme, mis à l&#39;écran (éviter les gros morceau de bloc de tissu dans la chambre), à ​​proximité de la chambre, et remplissez le système. </li><li> Commencer à pomper la solution pendant 5 minutes. Secouez doucement la chambre pour mieux répartir la solution chaude partout. Après cinq minutes, secouez doucement la chambre pour la phase de digestion ensemble. Maintenir la température à 37 ˚ C. </li><li> Rocher de la chambre doucement jusqu&#39;à ce que la température atteint 37 °, puis secouez la chambre. Prendre un échantillon toutes les deux minutes et de détecter si îlots libres se trouvent sous la portée. Enregistrer les informations d&#39;évaluation des îlots dans le tableau de digestion du dossier de lot. Continuer à surveiller le pourcentage d&#39;îlots jusqu&#39;à il ya 50% d&#39;entre eux flottent librement et isolées dans la solution. </li></ol> 4. Collection de tissus <ol><li> Une fois que 50% des îlots se trouvent libres dans les échantillons, la digestion est arrêtée en ajoutant une solution froide de dilution RPMI dans le circuit. Tissulaire est recueilli dans 500 ml tubes coniques pré-rempli avec 30 ml d&#39;albumine humaine. Une fois les tubes sont filées, aspiration le surnageant et laver le culot avec la solution de lavage à froid. Faites tourner les tubes coniques à 1100 RPM (tours par minute) pendant 1 minute, 4 ˚ C. </li><li> Combiner tous les tubes de prélèvement suspendu avec du tissu pancréatique digère en une seule conique de 500 ml. Lorsqu&#39;un tube est plein de tissus lavés, spin et répétez le processus de lavage jusqu&#39;à ce que tous les tissus ont été lavés plusieurs fois. Spin ce tube et le transfert des tissus dans un seul tube conique de 250 ml. Porter le volume à 250 ml avecM199 solution de lavage complété avec HA et se préparer pour l&#39;échantillonnage. </li><li> Prenez un échantillon de 1 ml avec une pipette et ajouter à un tube de 15 ml conique pré-rempli avec 9 ml de solution de lavage. Puis mélanger doucement en inversant le tube de 15 ml conique et de cela, l&#39;échantillon à 1 ml directement dans un plat grille de comptage. Compter les cellules, et calculer le nombre de cellules en utilisant un facteur de dilution de 2500 (10x250). </li><li> Spin le tube conique de 250 ml et ajouter 150 ml d&#39;UW (Université du Wisconsin) Solution qui sert d&#39;ordinaire pour la préservation des organes. Lorsque l&#39;UW est ajouté, mélanger la suspension cellulaire à fond à l&#39;aide de la pipette. Enregistrer et communiquer à l&#39;opérateur de purification de l&#39;heure de début dans l&#39;UW. Laisser le tube (s) sur la glace et agiter de temps en temps. Maintenant, les îlots peuvent être purifié davantage. </li></ol>

<ol>
	<li>Shapiro, A. M., et al. <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., et al. <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., et al. <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., et al. <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., et al. <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., et al. <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, 140-142 (1989).</li><li>Hanley, S. C., Paraskevas, S., Rosenberg, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donor+and+isolation+variables+predicting+human+islet+isolation+success.">Donor and isolation variables predicting human islet isolation success.</a> Transplantation. 85, 950-955 (2008).</li><li>Toso, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+affecting+human+islet+of+Langerhans+isolation+yields.">Factors affecting human islet of Langerhans isolation yields.</a> Transplant Proc. 34, 826-827 (2002).</li></ol>

Il existe une variété de facteurs influant sur le résultat 9,10 isolement. Parmi eux, la digestion du tissu pancréatique joue un rôle de premier plan. Basé sur notre propre expérience d&#39;isolement d&#39;îlots humains, nous avons résumé ci-après les points critiques, qui pourraient potentiellement influencer le rendement de l&#39;îlot et de qualité. <ol><li> Le temps de digestion est différente lorsque les différents types d&#39;enzymes sont utilisés. </li><li> Dès que 40-50% des îlots complètem...

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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>Ice Slush Machine</td>
<td>equipment</td>
<td>Taylor Company</td>
<td>K4036546</td>
<td>make sterile ice</td>
</tr>
<tr>
<td>Centrifuge</td>
<td>equipment</td>
<td>Beckman Coulter</td>
<td>424803</td>
<td>&nbsp;</td>
<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 Water Bath EX7</td>
<td>equipment</td>
<td>NesLab</td>
<td>105361058</td>
<td>&nbsp;</td>
<tr>
<td>Thermometer</td>
<td>equipment</td>
<td>IBI:IR</td>
<td>60444</td>
<td>&nbsp;</td>
<tr>
<td>MonoBloc Balance AB 104-S</td>
<td>equipment</td>
<td>Metler Toledo</td>
<td>1119430864</td>
<td>measure pancreas weight</td>
</tr>
<tr>
<td>MasterFlex II Speed Controller</td>
<td>equipment</td>
<td>Cole Parmer Instrument Co</td>
<td>J00006445</td>
<td>adjust flow speed during degestion</td>
</tr>
<tr>
<td>Digital Sight DS L1</td>
<td>equipment</td>
<td>Nikon</td>
<td>217267</td>
<td>&nbsp;</td>
<tr>
<td>Perfusion Unit</td>
<td>equipment</td>
<td>Custom Made/Swiss</td>
<td>n/a</td>
<td>&nbsp;</td>
<tr>
<td>Cooler for perfusion unit</td>
<td>equipment</td>
<td>Fisher Scientific</td>
<td>3013P</td>
<td>&nbsp;</td>
<tr>
<td>Ricordi Chamber</td>
<td>equipment</td>
<td>University of Miami</td>
<td>n/a</td>
<td>&nbsp;</td>
<tr>
<td>RPMI dilution solution</td>
<td>reagent</td>
<td>Mediatech Inc</td>
<td>99783024</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>25% Human Albumin</td>
<td>reagent</td>
<td>GRIFOLS</td>
<td>NDC 61953-0002-2</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>Collagenase NB1 GMP grade</td>
<td>reagent</td>
<td>SERVA Electrophoresis</td>
<td>N0002937</td>
<td>&nbsp;</td>
<tr>
<td>Neutral Protease NB1 GMP grade</td>
<td>reagent</td>
<td>SERVA Electrophoresis</td>
<td>N0002936</td>
<td>&nbsp;</td>
<tr>
<td>Betadine</td>
<td>reagent</td>
<td>Cardinal Health</td>
<td>29906-004</td>
<td>&nbsp;</td>
<tr>
<td>Cefazolin</td>
<td>reagent</td>
<td>West-Ward Pharmaceutical</td>
<td>NDC 0143-9924-90</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 i: digestion and collection of pancreatic tissue

Gestion diabète de type 1 est lourde, tant pour l&#39;individu et la société, ce qui coûte plus de 100 milliards de dollars annuellement. Malgré l&#39;utilisation répandue de la surveillance du glucose et des préparations d&#39;insuline nouvelles, de nombreuses personnes encore développer des complications secondaires dévastateurs. Transplantation d&#39;îlots pancréatiques peut restaurer à proximité de contrôle de la glycémie normale chez les patients diabétiques<sup&gt; 1</sup&gt;, Sans courir le risque d&#39;épisodes hypoglycémiques graves qui sont associées à une insulinothérapie intensive. Fournir la masse des îlots suffisant est important pour la transplantation d&#39;îlots de succès. Toutefois, caractéristique des donateurs, le prélèvement d&#39;organes et de la préservation affecter le résultat d&#39;isolement<sup&gt; 2</sup&gt;. À l&#39;Université de l&#39;Illinois à Chicago (UIC), nous avons développé un protocole d&#39;isolement de succès avec un gradient de purification améliorée<sup&gt; 3</sup&gt;. Le programme a démarré en Janvier 2004, et plus de 300 isolements ont été effectuées jusqu&#39;à Novembre 2008. Le pancréas ont été envoyés dans les solutions de conservation par le froid (UW, Université du Wisconsin ou de HTK, histidine-tryptophane cétoglutarate)<sup&gt; 4-7</sup&gt; À l&#39;isolement des cellules en laboratoire à l&#39;UIC pour l&#39;isolement des îlots. Les îlots pancréatiques ont été isolés en utilisant la méthode de l&#39;UIC qui est une version modifiée de la méthode initialement décrite par Ricordi<em&gt; Et al</em&gt;<sup&gt; 8</sup&gt;. Brièvement, après le nettoyage du pancréas du tissu environnant, il a été perfusé avec une solution enzymatique (collagénase Serva + Neutre protéase ou Sigma enzymes V). Le pancréas distendu a ensuite été transféré à la chambre de digestion Ricordi, connecté à une modification, le système de circulation tube fermé et chauffé à 37 ° C. Pendant la digestion, la chambre a été secoué doucement. Les échantillons ont été prises en permanence pour surveiller la progression de digestion. Une fois les îlots libres ont été détectées sous le microscope, la digestion a été arrêtée par rinçage à froid (4 ° C) RPMI dilution solution (Mediatech, Herndon, VA) dans le système de circulation afin de diluer l&#39;enzyme. Après avoir été recueilli et lavé au M199 milieux supplémentés avec l&#39;albumine humaine, le tissu a été échantillonnée pour une pré-purification compter et incubées avec la solution UW avant purification. Processus de purification seront décrites dans la Partie II: Purification et culture d&#39;îlots humains.

Watch this Scientific Journal Video about Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue at JoVE.com

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

Atteindre de haute qualité et la quantité appropriée d&#39;îlots humains est l&#39;une des conditions préalables importantes pour la transplantation d&#39;îlots de succès. Dans cette vidéo, nous décrire étape par étape les procédures pour l&#39;isolement des îlots pancréatiques humains (partie I: la digestion et la collecte des tissus pancréatiques) en utilisant une méthode modifiée automatisés.

Isolement îlots pancréatiques humaines: Partie I: La digestion et la perception des tissus pancréatiques

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

human-pancreatic-islet-isolation-part-i-digestion-collection

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Biology

19.2K vues.  University of Illinois, Chicago. Atteindre de haute qualité et la quantité appropriée d'îlots humains est l'une des conditions préalables importantes pour la transplantation d'îlots de succès. Dans cette vidéo, nous décrire étape par étape les procédures pour l'isolement des îlots pancréatiques humains (partie I: la digestion et la collecte des tissus pancréatiques) en utilisant une méthode modifiée automatisés.

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

Murine Pancreatic Islet Isolation

Isolement murin îlots pancréatiques

Recherche

Biologie

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.

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.

Isolement îlots pancréatiques humaines: Partie II: Purification et culture d&#39;îlots humains

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 and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on
ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human
xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,
a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

Isolement des cellules souches de l&#39;homme xénogreffes de cancer du pancréas

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

Computer-assisted Large-scale Visualization and Quantification of Pancreatic Islet Mass, Size Distribution and Architecture

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), and delta-cells (somatostatin) that are embedded in the exocrine tissues and comprise 1-2% of the entire pancreas. There is a close correlation between body and pancreas weight. Total beta-cell mass also increases proportionately to compensate for the demand for insulin in the body. What escapes this proportionate expansion is the size distribution of islets. Large animals such as humans share similar islet size distributions with mice, suggesting that this micro-organ has a certain size limit to be functional. The inability of large animal pancreata to generate proportionately larger islets is compensated for by an increase in the number of islets and by an increase in the proportion of larger islets in their overall islet size distribution. Furthermore, islets exhibit a striking plasticity in cellular composition and architecture among different species and also within the same species under various pathophysiological conditions. In the present study, we describe novel approaches for the analysis of biological image data in order to facilitate the automation of analytic processes, which allow for the analysis of large and heterogeneous data collections in the study of such dynamic biological processes and complex structures. Such studies have been hampered due to technical difficulties of unbiased sampling and generating large-scale data sets to precisely capture the complexity of biological processes of islet biology. Here we show methods to collect unbiased "representative" data within the limited availability of samples (or to minimize the sample collection) and the standard experimental settings, and to precisely analyze the complex three-dimensional structure of the islet. Computer-assisted automation allows for the collection and analysis of large-scale data sets and also assures unbiased interpretation of the data. Furthermore, the precise quantification of islet size distribution and spatial coordinates (i.e. X, Y, Z-positions) not only leads to an accurate visualization of pancreatic islet structure and composition, but also allows us to identify patterns during development and adaptation to altering conditions through mathematical modeling. The methods developed in this study are applicable to studies of many other systems and organisms as well.

Novel computer-assisted methods of large-scale procurement and analysis of immunohistochemically stained pancreatic specimens are described: (1) Virtual Slice capture of the entire section; (2) Mass analysis of large-scale data; (3) Reconstruction of 2D Virtual Slices; (4) 3D islet mapping; and (5) Mathematical analysis.

Assistée par ordinateur à grande échelle de visualisation et quantification de la messe des îlots pancréatiques, distribution de taille et d&#39;architecture

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), ...

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

Une méthode pour la souris Isolement îlots pancréatiques et AMPc intracellulaire Détermination

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

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 week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

Isolement et culture de cellules primaires de souris acineuses du pancréas

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 ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

Un protocole simple à haut rendement pour l'isolement des îlettes pancréatiques des souris

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

Pancreatic Islet Embedding for Paraffin Sections

Experiments using isolated pancreatic islets are important for diabetes research, but islets are expensive and of limited abundance. Islets contain a mixed cell population in a structured architecture that impacts function, and human islets are widely variable in cell type composition. Current frequently used methods to study cultured islets include molecular studies performed on whole islets, lumping disparate islet cell types together, or microscopy or molecular studies on dispersed islet cells, disrupting islet architecture. For in vivo islet studies, paraffin-embedded pancreas sectioning is a powerful technique to assess cell-specific outcomes in the native pancreatic environment. Studying post-culture islets by paraffin sectioning would offer several advantages: detection of multiple outcomes on the same islets (potentially even the exact-same islets, using serial sections), cell-type-specific measurements, and maintaining native islet cell-cell and cell-substratum interactions both during experimental exposure and for analysis. However, existing techniques for embedding isolated islets post-culture are inefficient, time consuming, prone to loss of material, and generally produce sections with inadequate islet numbers to be useful for quantifying outcomes. Clinical pathology laboratory cell block preparation facilities are inaccessible and impractical for basic research laboratories. We have developed an improved, simplified bench-top method that generates sections with robust yield and distribution of islets. Fixed islets are resuspended in warm histological agarose gel and pipetted into a flat disc on a standard glass slide, such that the islets are distributed in a plane. After standard dehydration and embedding, multiple (10+) 4 - 5 &#181;m sections can be cut from the same islet block. Using this method, histological&#160;and immunofluorescent analyses can be performed on mouse, rat, and human islets. This is an effective, inexpensive, time-saving approach to assess cell-type-specific, intact-architecture outcomes from cultured islets.

Ex vivo pancreatic islet studies are important for diabetes research. Existing techniques to study cultured islets in their native 3-dimensional architecture are time consuming, inefficient, and infrequently used. This work describes a new, simple, and efficient method for generating high-quality paraffin sections of whole cultured islets.

Îlots pancréatiques, enrobage pour les Sections de paraffine

Experiments using isolated pancreatic islets are important for diabetes research, but islets are expensive and of limited abundance. Islets contain a ...

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from &#946;-cells), glucagon (&#945;-cells) and somatostatin (&#948;-cells). &#946;-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, &#945;-cells and &#948;-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest.
Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of &#946;-cells is minimized.
Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase.
Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

Imagerie de la dynamique du calcium dans les sous-populations de cellules d'islet pancréatique de souris

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells ...

Différenciation des cellules souches pluripotentes humaines en cellules β pancréatiques

Optimized Protocol for Generating Functional Pancreatic Insulin-secreting Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) can differentiate into any kind of cell, making them an excellent alternative source of human pancreatic &#946;-cells. hPSCs can either be embryonic stem cells (hESCs) derived from the blastocyst or induced pluripotent cells (hiPSCs) generated directly from somatic cells using a reprogramming process. Here a video-based protocol is presented to outline the optimal culture and passage conditions for hPSCs, prior to their differentiation and subsequent generation of insulin-producing pancreatic cells. This methodology follows the six-stage process for &#946;-cell directed differentiation, wherein hPSCs differentiate into definitive endoderm (DE), primitive gut tube, posterior foregut fate, pancreatic progenitors, pancreatic endocrine progenitors, and ultimately pancreatic &#946;-cells. It is noteworthy that this differentiation methodology takes a period of 27 days to generate human pancreatic &#946;-cells. The potential of insulin secretion was evaluated through two experiments, which included immunostaining and glucose-stimulated insulin secretion.

Pleins feux sur l’auteur : Avancées et défis dans la différenciation des cellules β à partir de cellules souches pluripotentes

This article presents a protocol for directed differentiation and functional analysis of &#946;-cell like cells. We describe optimal culture conditions and passages for human pluripotent stem cells before generating insulin-producing pancreatic cells. The six-stage differentiation progresses from definitive endoderm formation to functional &#946;-cell like cells secreting insulin in response to glucose.

Protocole optimisé pour la génération de cellules pancréatiques fonctionnelles sécrétant de l’insuline à partir de cellules souches pluripotentes humaines