Apoiada por um financiamento da Universidade de Illinois em Chicago, assim como Cell Resource Islet Centro NIH subvenção (RFA-RR 05-003), a Fundação Christopher, a Fundação Efroymson, ea Fundação Tellabs.

1. Facilidade de instalação <ol><li> Isolamento de ilhotas humana é um procedimento sensível ao tempo que envolve o trabalho em equipe e, portanto, quatro pessoas são necessários para realizar este procedimento. A equipe é liderada pelo cirurgião, que disseca e cannulates pâncreas. </li><li> O isolamento das ilhotas começa o minuto a equipe entra na instalação de isolamento de ilhotas UIC. Membros da equipe assegurar que toda a instrumentação necessária, como a centrífuga, é verificada e ligado. Também certifique-se de higienizar todos os capuzes e superfícies. </li><li> Duas pessoas irá definir a tabela de canulação, máquina de lama, circuito digestão, unidade de perfusão, máquina de purificação COBE, e mesas cirúrgicas. </li><li> Simultaneamente, dois outros membros da equipe irá preparar os meios de comunicação que é necessária durante o isolamento. </li><li> Em seguida, os tubos de bacteriologia (garrafas, tubos, forma e saco), tubos de amostragem (pré-carregada com lavagem), e tubos e placas de avaliação estão preparados. </li><li> Prepare o tubo de purificação definido da seguinte forma: O primeiro tubo vazio e marcou a 150 ml, tubos de 2-12 preenchido com 200 ml de M199 meio suplementado com humanos (solução de lavagem) e albumina marcados em 230 ml e, finalmente, um tubo de 50 ml cônico preenchido com 50 ml de solução de lavagem. Uma vez que estas etapas de preparação estão completas estamos prontos para começar a preparar e irrigando o pâncreas. </li></ol> 2. Preparação pâncreas e perfusão <ol><li> Para começar a preparação e perfusão do pâncreas, um operador remove o órgão a partir de embalagens utilizando técnica estéril. O cirurgião fará a transferência do pâncreas para a mesa de descontaminação. </li><li> O cirurgião vai encher uma seringa de 60 ml com a solução original pâncreas preservação e entregá-lo a um operador, que irá usá-lo para preencher preparado &quot;Procurement&quot; garrafas de bacteriologia. </li><li> Depois de uma inspeção visual para determinar se o órgão é adequado para o isolamento das ilhotas (Se o pâncreas for danificado durante a colheita ou anatomicamente anormal, não pode ser utilizado para o isolamento das ilhotas), o cirurgião vai pedir um operador para preparar colagenase. Lembre-se que esse tempo re-suspensão varia entre as marcas e lotes diferentes de enzimas. </li><li> Quando o cirurgião está pronto, traga as soluções de descontaminação (Betadine, Fungizone / Cefazolina e HBSS) para a tabela de descontaminação e despeje-os em recipientes apropriados. Cirurgião realiza a descontaminação de lavagem pâncreas por um minuto do pâncreas em cada um a solução de descontaminação. O pâncreas é então colocada em um pequeno frasco esterilizado no qual ele vai ser pesado. Depois que o peso é inserido no registro do lote, o pâncreas é colocado no pan canulação com algumas solução de preservação. </li><li> O cirurgião irá expor e parcialmente inciso o duto principal e canular cada metade do pâncreas com uma cânula. A cânula será fixada por sutura. Para perfundir o pâncreas, primeiro movê-lo para a unidade de perfusão e despeje a solução enzimática para a bacia. Tempo de perfusão total é de cerca de 10 minutos, a 80 mmHg por 5 minutos, então a 180 mmHg por 5 minutos. Pâncreas distendido após a perfusão. </li><li> Mover o pâncreas perfundido para o pan canulação, onde o tecido adiposo é cortado a partir do pâncreas com cuidado. Em seguida, corte do pâncreas em 10-12 pedaços e transferência para Ricordi Câmara para a digestão. </li></ol> 3. Digestão do pâncreas <ol><li> Para começar a etapa de digestão, o pâncreas a transferência da Câmara Ricordi vazia, adicione um frasco de 2,5 ml de Pulmozyme, colocar na tela (evitar grande parte do bloco de tecido da câmara), perto da câmara, e encher o sistema. </li><li> Começar a bombear a solução por 5 minutos. Suavemente de rock da câmara para uniformemente dispersar solução quente por toda parte. Após cinco minutos, agite a câmara para a fase de digestão todo. Manter a temperatura a 37 ˚ C. </li><li> Rock na câmara suavemente até a temperatura atinge 37 °, em seguida, agitar a câmara. Tomar uma amostra a cada dois minutos e detectar se ilhotas livres são encontrados sob o escopo. Registrar as informações de avaliação de ilhotas na tabela a digestão do registro do lote. Continuar a acompanhar o percentual de ilhotas até que haja 50% deles flutuando livres e isolados na solução. </li></ol> 4. Coleta de tecido <ol><li> Uma vez que 50% das ilhotas são encontrados livres nas amostras, a digestão é interrompida pela adição de solução de diluição RPMI frio no circuito. Tecido é recolhido em tubos de 500 ml cônico pré-cheias com 30 ml de Albumina Humana. Uma vez que os tubos são giradas, aspiração de fora o sobrenadante e lavar o pellet com solução de lavagem a frio. Girar os tubos cônicos a 1100 RPM (rotações por minuto) por 1 minuto, 4 ˚ C. </li><li> Combine todos os tubos com exigibilidade suspensa tecido pancreático digerido em uma única de 500 ml. Quando um tubo está cheio de tecido lavado, spin e repita o processo de lavagem até que todo o tecido foi lavado várias vezes. Girar o tubo e transfira o tecido em um único tubo de 250 ml. Traga o volume até 250 ml comM199 solução de lavagem suplementado com HA e se preparar para a amostragem. </li><li> Tomar uma amostra de 1 ml com uma pipeta e adicionar a um tubo cônico de 15 ml pré-cheia com 9 ml de solução de lavagem. Em seguida, misture suavemente invertendo o tubo cônico de 15 ml e deste, amostra para fora 1ml diretamente em um prato grade de contagem. Contar as células, e calcular o número de células usando um fator de diluição de 2500 (10x250). </li><li> Girar o tubo de 250 ml e adicionar 150 ml de UW (University of Wisconsin) Solução que costumo usar para preservação de órgãos. Quando UW é adicionado, misture a suspensão de células, cuidadosamente, com a pipeta. Registrar e comunicar ao operador de purificação a hora de início na UW. Deixe o tubo (s) no gelo e agite ocasionalmente. Agora as ilhotas podem ser purificado ainda mais. </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>

Há uma variedade de fatores que afetam o resultado de isolamento 9,10. Entre eles, a digestão do tecido pancreático desempenha um papel proeminente. Baseado em nossa experiência humana de isolamento de ilhotas própria, resumimos seguintes pontos críticos, o que poderia influenciar o rendimento de ilhotas e qualidade. <ol><li> O tempo de digestão é diferente quando diferentes tipos de enzimas são utilizadas. </li><li> Assim que 40-50% das ilhotas completamente separado do tecido acinar, a digestão deve ser ...

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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

Gestão da diabetes tipo 1 é onerosa, tanto para o indivíduo ea sociedade, custando mais de 100 bilhões de dólares anualmente. Apesar do uso generalizado de monitoramento de glicose e insulina novas formulações, muitas pessoas ainda desenvolvem devastadoras complicações secundárias. Transplante de ilhotas pancreáticas pode restaurar controle quase normais de glicose em pacientes diabéticos<sup&gt; 1</sup&gt;, Sem o risco de graves episódios de hipoglicemia que estão associados com a terapia insulínica intensiva. Fornecimento de massa de ilhotas suficiente é importante para o transplante de ilhotas bem sucedido. No entanto, características dos doadores, a colheita de órgãos e preservação afetar o resultado de isolamento<sup&gt; 2</sup&gt;. Na Universidade de Illinois em Chicago (UIC), temos desenvolvido um protocolo de isolamento bem sucedido com um gradiente de purificação melhorou<sup&gt; 3</sup&gt;. O programa foi iniciado em janeiro de 2004, e mais de 300 isolamentos foram realizados até novembro de 2008. O pancreata foram enviados em soluções frias de conservação (UW, University of Wisconsin ou HTK, histidina-triptofano Ketoglutarate)<sup&gt; 07/04</sup&gt; Para o Laboratório de Isolamento Celular na UIC para isolamento de ilhotas. Ilhotas pancreáticas foram isolados utilizando o método de UIC, que é uma versão modificada do método originalmente descrito por Ricordi<em&gt; Et al</em&gt;<sup&gt; 8</sup&gt;. Resumidamente, após a limpeza do pâncreas do tecido circundante, foi perfundido com solução enzimática (Serva Colagenase + Neutro Protease ou enzima Sigma V). O pâncreas distendido foi então transferido para a câmara de digestão Ricordi, conectado a um sistema modificado de tubulação, fechada a circulação, e aquecido a 37 ° C. Durante a digestão, a câmara foi agitada suavemente. As amostras foram tomadas de forma contínua para monitorar o progresso digestão. Uma vez livre ilhotas foram detectadas ao microscópio, a digestão foi parado por lavagem a frio (4 ° C) RPMI solução de diluição (Mediatech, Herndon, VA) para o sistema de circulação para diluir a enzima. Depois de ser coletados e lavados em M199 meio suplementado com albumina humana, o tecido foi amostrado para a purificação de pré-contagem e incubadas com solução de UW antes de purificação. Processo de purificação serão descritos na Parte II: Purificação e Cultura de Ilhotas Humanos.

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

Alcançar alta qualidade e quantidade adequada de ilhotas humanas é um dos pré-requisitos importantes para o transplante de ilhotas bem sucedido. Neste vídeo, descrevemos passo a passo os procedimentos para o isolamento de ilhotas pancreáticas humanas (parte I: a digestão e coleta de tecido pancreático), utilizando um método modificado automatizado.

Isolamento Islet humana Pancreatic: Parte I: Digestão e Coleta de tecido pancreático

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 visualizações.  University of Illinois, Chicago. Alcançar alta qualidade e quantidade adequada de ilhotas humanas é um dos pré-requisitos importantes para o transplante de ilhotas bem sucedido. Neste vídeo, descrevemos passo a passo os procedimentos para o isolamento de ilhotas pancreáticas humanas (parte I: a digestão e coleta de tecido pancreático), utilizando um método modificado automatizado.

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

Murine Pancreatic Islet Isolation

Isolamento Islet murino de pâncreas

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 Islet humana Pancreatic: Parte II: Purificação e Cultura de Ilhotas Humanos

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 de células-tronco de xenoenxertos Humano do Câncer de pâncreas

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.

Assistida por computador visualização de grande escala e Quantificação da Missa das ilhotas pancreáticas, distribuição de tamanho e Arquitetura

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.

Um Método para Mouse ilhotas pancreáticas Isolamento e intracelular cAMP Determinação

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 do mouse primárias células pancreáticas acinares

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.

Um protocolo de alta eficiência simples para isolamento de Islet pancreático de camundongos

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.

Ilhota pancreatic incorporação para cortes de parafina

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.

Dinâmica de cálcio de imagem em subpopulações de células de ilhotas pancreáticas do rato

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

Diferenciando células-tronco pluripotentes humanas em células β pancreáticas

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.

Autor em destaque: Avanços e desafios na diferenciação de células-β de células-tronco 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.