Supportato da un finanziamento della University of Illinois a Chicago, nonché delle cellule delle isole Resource Center NIH di sovvenzione (RFA-RR 05-003), la Fondazione Cristoforo, la Fondazione Efroymson, e la Fondazione Tellabs.

1. Programma di setup <ol><li> Isolamento isole umane è una procedura momento delicato che coinvolge il lavoro di squadra e, quindi, quattro individui sono necessarie per condurre questa procedura. Il team è guidato dal chirurgo, che analizza e cannulates il pancreas. </li><li> L&#39;isolamento delle isole inizia il minuto la squadra entra nel UIC di isolamento delle isole. I membri del team in modo che tutta la strumentazione necessaria, come la centrifuga, viene controllata e acceso. Inoltre assicuratevi di disinfettare tutte le cappe e superfici. </li><li> Due persone si impostare la tabella incannulazione, granitore, circuito digestione, unità di perfusione, macchina purificazione COBE, e tavoli operatori. </li><li> Contemporaneamente, due altri membri del team si preparano i mezzi di comunicazione che è necessario durante l&#39;isolamento. </li><li> Avanti, tubi batteriologia (bottiglie, tubi, la forma e la borsa), tubi di campionamento (pre-riempite con lavaggio), e tubi di valutazione e piatti sono preparati. </li><li> Preparate il tubo di purificazione impostata come segue: Il 1 ° tubo vuoto e segnato a 150 ml, tubi 2-12 riempito con 200 ml di M199 mezzi di comunicazione integrata con albumina umana (soluzione di lavaggio) e segnato a 230 ml, e infine un tubo da 50 ml riempito con 50 ml di soluzione di lavaggio. Una volta che questi passaggi di preparazione sono completi siamo pronti per iniziare la preparazione e la perfusione del pancreas. </li></ol> 2. Pancreas preparazione e perfusione <ol><li> Per iniziare la preparazione e la perfusione del pancreas, un operatore rimuove l&#39;organo dalla confezione utilizzando una tecnica sterile. Il chirurgo trasferirà il pancreas al tavolo di decontaminazione. </li><li> Il chirurgo riempire una siringa da 60 ml con la soluzione originale preservazione del pancreas e consegnarla ad un operatore, che lo utilizzano per riempire preparato &quot;Procurement&quot; bottiglie di batteriologia. </li><li> Dopo un controllo visivo per stabilire se l&#39;organo è adatto per l&#39;isolamento delle isole (Se il pancreas viene danneggiato durante il prelievo o anatomicamente anomali, che non possono essere utilizzati per l&#39;isolamento delle isole), il chirurgo dovrà chiedere a un operatore per preparare collagenasi. Ricorda che tale ri-sospensione del tempo varia tra i marchi di enzimi diversi e molto. </li><li> Quando il chirurgo è pronto, portare le soluzioni di decontaminazione (Betadine, Fungizone / Cefazolina e HBSS) alla tabella di decontaminazione e versarli in appositi contenitori. Chirurgo esegue decontaminazione pancreas lavaggio per un minuto il pancreas in ciascuna delle soluzioni di decontaminazione. Il pancreas viene quindi posto in un vasetto sterile in cui sarà pesato. Dopo che il peso è entrato nel batch record, il pancreas viene posizionato in padella con un po cannulazione soluzione di conservazione. </li><li> Il chirurgo esporre e parzialmente incidere il condotto principale e cannulate ogni metà del pancreas con una cannula. La cannula viene garantito da sutura. Per profumato il pancreas, prima spostare l&#39;unità perfusione e versare la soluzione enzimatica nel bacino. Perfusione tempo totale è di circa 10 minuti, a 80 mmHg per 5 minuti, poi a 180 mmHg per 5 minuti. Pancreas disteso dopo la perfusione. </li><li> Spostare il pancreas perfuso alla padella incannulazione, dove viene tagliato il tessuto adiposo dal pancreas con attenzione. Poi tagliare il pancreas in 10-12 pezzi e trasferimento a Ricordi Camera per la digestione. </li></ol> 3. Pancreas digestione <ol><li> Per iniziare la fase di digestione, il pancreas trasferimento alla Camera vuota Ricordi, aggiungere una fiala 2,5 ml di Pulmozyme, mettere sullo schermo (evitare grossa fetta del blocco tessuto della camera), chiudere la camera, e riempire il sistema. </li><li> Iniziare a pompare la soluzione per 5 minuti. Agitare delicatamente la camera a disperdersi uniformemente soluzione calda per tutto. Dopo cinque minuti, agitare delicatamente la camera per la fase di digestione intero. Mantenere la temperatura a 37 ˚ C. </li><li> Rock the camera delicatamente fino a quando la temperatura raggiunge i 37 °, poi scuotere la camera. Prelevare un campione ogni due minuti e rilevare se isolotti liberi si trovano nel campo d&#39;applicazione. Registrare le informazioni isolotto di valutazione nella tabella digestione dei batch record. Continuerà a monitorare la percentuale di isolotti finché non ci sono il 50% di loro flottante libero e isolato nella soluzione. </li></ol> 4. Raccolta dei tessuti <ol><li> Una volta che il 50% delle isole si trovano liberi nei campioni, la digestione viene interrotta con l&#39;aggiunta di freddo soluzione di diluizione RPMI nel circuito. Tessuto viene raccolto in 500 ml provette coniche pre-riempite con 30 ml di albumina umana. Una volta che i tubi sono filata, aspirazione fuori dal surnatante e lavare il pellet con la soluzione di lavaggio a freddo. Gira i tubi conici a 1100 RPM (rotazioni al minuto) per 1 minuto, 4 ˚ C. </li><li> Combina tutti i tubi di raccolta sospesa con tessuto pancreatico digeriti in un singolo da 500 ml. Quando un tubo è pieno di tessuto lavato, girare e ripetere il processo di lavaggio fino a tutto il tessuto è stato lavato più volte. Spin questo tubo e trasferire il tessuto in un unico tubetto da 250 ml. Portare il volume fino a 250 ml conM199 soluzione di lavaggio integrato con HA e prepararsi per il campionamento. </li><li> Prendete un campione di 1 ml con una pipetta e aggiungere in una provetta da 15 ml conica pre-riempita con 9 ml di soluzione di lavaggio. Quindi mescolare delicatamente invertendo il tubo da 15 ml e da questa, campione fuori 1ml direttamente in un piatto della griglia di conteggio. Contare le celle, e calcolare il numero di cellulare utilizzando un fattore di diluizione del 2500 (10x250). </li><li> Spin il tubetto da 250 ml conica e aggiungere 150 ml di UW (Università del Wisconsin), soluzione che di solito uso per la conservazione degli organi. Quando UW si aggiunge, mescolare la sospensione cellulare accuratamente con la pipetta. Registrare e comunicare all&#39;operatore purificazione l&#39;ora di inizio di UW. Lasciare il tubo (s) su ghiaccio e agitare di tanto in tanto. Ora le isole possono essere ulteriormente purificato. </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>

Ci sono una varietà di fattori che influenzano il risultato dell&#39;isolamento 9,10. Tra questi, la digestione del tessuto pancreatico gioca un ruolo di primo piano. Sulla base della nostra esperienza umana isolamento delle isole, abbiamo riassunto i seguenti punti critici, che potrebbe potenzialmente influenzare il rendimento delle isole e la qualità. <ol><li> Il tempo di digestione è diversa quando i diversi tipi di enzimi sono utilizzati. </li><li> Non appena il 40-50% delle isole completamente separate dal t...

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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

Gestione del diabete di tipo 1 è onerosa, sia per l&#39;individuo e la società, che costano oltre 100 miliardi di dollari all&#39;anno. Nonostante l&#39;uso diffuso di monitoraggio della glicemia e insulina formulazioni nuove, molte persone ancora sviluppare devastanti complicanze secondarie. Trapianto di isole pancreatiche in grado di ripristinare nei pressi di controllo normale del glucosio in pazienti diabetici<sup&gt; 1</sup&gt;, Senza il rischio di gravi episodi di ipoglicemia che sono associati con la terapia insulinica intensiva. Fornire sufficiente massa isolotto è importante per il trapianto di isole pancreatiche di successo. Tuttavia, caratteristica dei donatori, reperimento di organi e la conservazione influenzare l&#39;esito isolamento<sup&gt; 2</sup&gt;. Presso l&#39;Università dell&#39;Illinois a Chicago (UIC), abbiamo sviluppato un protocollo di successo isolamento con pendenza purificazione migliorato<sup&gt; 3</sup&gt;. Il programma è iniziato nel gennaio 2004, e più di 300 isolamenti sono stati effettuati fino a novembre 2008. Il pancreas sono stati inviati nelle soluzioni di conservazione a freddo (UW, University of Wisconsin o HTK, Istidina, Triptofano chetoglutarato)<sup&gt; 4-7</sup&gt; Al Laboratorio cella d&#39;isolamento a UIC per l&#39;isolamento delle isole. Isole pancreatiche sono stati isolati con il metodo UIC, che è una versione modificata del metodo originariamente descritto da Ricordi<em&gt; Et al</em&gt;<sup&gt; 8</sup&gt;. In breve, dopo aver pulito il pancreas dal tessuto circostante, è stato perfuso con soluzione enzimatica (Serva Collagenasi + Neutro Sigma enzima proteasi o V). Il pancreas è stato dilatato poi trasferito nella camera di digestione Ricordi, collegato ad una modifica, sistema chiuso tubi circolazione, e riscaldato a 37 ° C. Durante la digestione, la camera era scosso dolcemente. I campioni sono stati presi costantemente per monitorare il processo di digestione. Una volta libero isolette sono state rilevate al microscopio, la digestione è stato fermato da lavaggio a freddo (4 ° C) RPMI soluzione di diluizione (Mediatech, Herndon, VA) nel sistema di circolazione per diluire l&#39;enzima. Dopo essere raccolte e lavate in M199 mezzi di comunicazione integrato con albumina umana, il tessuto è stato campionato per la pre-purificazione contare e incubati con soluzione UW prima purificazione. Processo di purificazione saranno descritti nella Parte II: La purificazione e la cultura di isole umane.

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

Il raggiungimento di alta qualità e la quantità appropriata di isole umane è uno dei presupposti importanti per il trapianto di isole pancreatiche di successo. In questo video, si descrive passo dopo passo le procedure per l&#39;isolamento delle isole pancreatiche umane (I parte: la digestione e la raccolta di tessuto pancreatico) utilizzando un metodo modificato automatizzato.

Isolamento delle isole pancreatiche umane: Parte I: Digestione e Collezione di tessuto pancreatico

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

Research

JoVE Journal

Biology

19.2K Views.  University of Illinois, Chicago. Il raggiungimento di alta qualità e la quantità appropriata di isole umane è uno dei presupposti importanti per il trapianto di isole pancreatiche di successo. In questo video, si descrive passo dopo passo le procedure per l'isolamento delle isole pancreatiche umane (I parte: la digestione e la raccolta di tessuto pancreatico) utilizzando un metodo modificato automatizzato.

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

Murine Pancreatic Islet Isolation

Murino pancreatica isolamento Islet

Ricerca

Biologia

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.

Isolamento del pancreas umano Islet: Parte II: La purificazione e la cultura di isole umane

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.

Isolamento di cellule staminali da xenotrapianti di cancro pancreatico umano

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.

Assistita da computer su larga scala di visualizzazione e quantificazione della Messa di isole pancreatiche, distribuzione delle dimensioni e architettura

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.

Un metodo per l&#39;isolamento del mouse isole pancreatiche e intracellulare cAMP Determinazione

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.

Isolamento e Cultura del mouse principale acinose pancreatiche Cellule

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 semplice protocollo ad alta efficienza per l'isolamento delle isolozioni pancreatiche dai topi

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.

Isole pancreatiche incorporamento per sezioni di paraffina

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.

Imaging calcio Dynamics in sottopopolazioni di cellule di islet pancreatica del topo

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

Differenziazione delle cellule staminali pluripotenti umane in cellule β pancreatiche

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.

Autore in primo piano: Progressi e sfide nella differenziazione delle cellule β dalle cellule staminali pluripotenti

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.