Name: effective isolation of functional islets from neonatal mouse pancreas
Uploaded: 2017-01-06T11:30:00.000+00:00
Duration: 7 min 4 s
Description: Watch this Scientific Journal Video about Effective Isolation of Functional Islets from Neonatal Mouse Pancreas at JoVE.com

This work was supported by grant from NIDDK (DK065949 for GG). We thank Dr. Brenda M. Jarvis and Jeff Duryea Jr. for reading the manuscript.

utilização de animais segue os procedimentos especificados no protocolo M / 11/181 aprovado pelo Institutional Animal Care e do Comitê Use Vanderbilt para Gu. ratinhos CD1 ou CBA / Bl6 foram adquiridos de fornecedores comerciais e cruzou no biotério Vanderbilt obter camundongos neonatais. 1. Preparação de Ratos, Soluções de Stock, and Equipment <ol><li> Para a cruz mouse: configurar uma cruz mouse e registrar as datas entupimento para auxiliar o planejamento experimental. ratinhos CD1 normalmente dão à luz por volta do dia 19 após o acasalamento. Use CD1 ou CBA / BL6 cruza para obter CD1 ou CBA / Bl6 neonatos, respectivamente. Intercruzamentos entre as duas linhas de utilizar fêmeas CD1 e machos CBA / Bl6. </li><li> Para o estoque de colagenase: pesar 200 mg de colagenase tipo IV com uma balança de alta precisão. Transferir para um tubo de centrífuga de 50 mL. Dissolve-se em 40 ml de HBSS com Ca2 + / Mg2 + (1,26 e 0,5 mM, respectivamente) para produzir uma solução stock de 5 mg / mL. <ol><li> Permitir que a colagenasedissolver para ~ 30 min com agitação suave. Alíquotas e manter a solução congelada em -20 ° C como estoque, que permanece activo durante pelo menos um ano. </li></ol></li><li> Autoclave para esterilizar vários pares de pinças, tesouras e dicas P20 para dissecção e picking ilhéu. </li><li> Para o estoque de glucose 1 H: pesar 9 g de glucose em um tubo de centrífuga de 50 ml, adicionar 50 ml de meio RPMI 1640 para dissolver a glicose. Tenha em 4 ° C até à sua utilização, mas não mais do que 6 meses. </li></ol> 2. As soluções de trabalho NOTA: O dia de isolamento de ilhotas, preparar os seguintes reagentes. <ol><li> Para a solução IV trabalho colagenase tipo: descongelar e diluir estoque colagenase no gelo. Dispensar 0,15 mL estoque em cada tubo de microcentrífuga de 1,7 mL. Adicionar 1,35 mL de HBSS com Ca2 + / Mg2 +. Inverta o tubo 5 vezes. Deixar em gelo durante 15 min. <ol><li> Girar em alta velocidade por 3 min numa microcentrífuga. Transferir o sobrenadante para um novo tubo. Deixar em gelo até à sua utilização. Rejeitar o tubo de detritos insolúveis. </li></ol></li><li> Para completo de mídia RPMI 1640: adicionar 2,5 mL 1 glucose M, 50 mL de calor FBS inactivado, e 5 mL 100x estoque Pen-strep em 500 ml RPMI 1640. Deixar em gelo até à sua utilização. </li></ol> 3. Isolamento pâncreas e Digestão <ol><li> Eutanásia neonatos com isoflurano, seguido por decapitação de acordo com protocolos aprovados. </li><li> Coloque o mouse com a barriga virada para cima. Spray com 70% de etanol para esterilização. </li><li> Levantar a pele de barriga para cima, com uma pinça e cortar as camadas da pele e músculo longitudinalmente ao longo da linha média com um par de tesouras, a partir da área genital para a caixa torácica. Isto abre a cavidade abdominal para expor todos os órgãos internos. </li><li> Transferir todos os órgãos internos, de 100 cm de placa com um par de pinças. Localize o pâncreas, que é um órgão-dispersa com uma porção dorsal que se agarra ao estômago e baço e uma porção ventral que se agarra ao duodeno 11. </li&gt; <li> Adicionar HBSS no prato para submergir os órgãos. Em solução, os tecidos pancreáticos não se agarram ao duodeno, estômago, ou baço. É facilmente reconhecível sob um escopo de dissecação causa de sua cor branca típica. Use um par de pinças para descascar os tecidos pancreáticos longe do tecido circundante e transferir para um novo prato 60 mm, com HBSS. </li><li> Corte cada pâncreas com uma tesoura em pedaços menores que 5 mm em qualquer dimensões. Transferir para um tubo de microcentrífuga de 1,5 mL. Até cinco pancreata (menores de P7) pode ser digerida em um tubo. Para ratos P8-P16, use um tubo por pâncreas. </li><li> Adicionar 0.5 - Solução de trabalho de 1 ml a cada tubo colagenase (0,2 ml para cada pâncreas P1-P7 Utilizar 0,5 mL por cada pâncreas P8-P16.). Deixar o tubo numa incubadora a 37 ° C durante até 15 min. Inverta o tubo duas vezes a cada min. </li><li> Depois de ~ 5 min, toque no tubo para monitorar o estado de digestão do tecido pancreático. Prosseguir o processo de digestão até que a maior parte do pâncreastecido aparece como grupos de células fragmentadas, com diâmetro &lt;2 mm. O ligado aparece turva, o que é devido a autólise celular acinar normal. </li></ol> 4. Lysate de lavar roupa <ol><li> Girar o lisado a 500 xg durante 10 s numa microcentrifuga. Retirar a camada sobrenadante com um Pipetman P1,000. O sobrenadante deve aparecer turva mas desprovido de agregados de células. Não utilizar aspiração, que pode aspirar rapidamente afastado os fragmentos de tecido na parte inferior devido à presença de uma amostra de DNA pegajoso. </li><li> Adicione 1 mL RPMI 1640 mídia completo. Toque suavemente o tubo para voltar a suspender o lisado fragmentado. Inverta cima e para baixo 10 vezes. Repita o passo 4.1. </li><li> Repita o passo 4.2 mais duas vezes ou até que o sobrenadante claro. Re-suspender pancreï¿½ticos lavadas em 1 mL completos RPMI 1640. </li></ol> 5. Isolamento Islet NOTA: Para um pequeno número de pancreata, mão-picking direto como a seguir (5.1) pode ser usado para o isolador do ilhéução. Para grandes números de pâncreas (&gt; 6), é preferido o método descrito no ponto 5.2. <ol><li> mão-picking direto: <ol><li> Transferir o lisado do passo 4.3 para um prato de 60 mm. Adicionar 5 ml completos RPMI 1640. Traga sob um microscópio de dissecação. </li><li> Configurar a iluminação de campo brilhante no microscópio. Ajustar a intensidade da luz de ~ 50% da potência máxima. Sob esta condição, ilhotas aparecer como um pouco cachos cor de rosa, enquanto as células acinares aglomerados de forma irregular como escuras (Figura 1A). A ampliação de ~ 50X (combinando o poder da objetiva e da parte do olho) é recomendado para oferecer a melhor maneira de ilhotas visualização e a abertura ponta da pipeta, simultaneamente, para a colheita. </li><li> Pegar ilhotas com uma ponta de pipeta P20, evitando aglomerados acinares. Fracções dispensar ilhotas enriquecido a um novo prato de 60 milímetros com meio RPMI completo (Figura 1B). </li><li> Repita o processo de mão-picking mais duas vezes. Deixar ilhotas puro em uma nova 60prato mm com 4 ml de meio RPMI completo (Figura 1C). </li></ol></li><li> centrifugação em gradiente: <ol><li> Preparar um tubo de centrífuga de 15 ml que é pré-carregado com 2 mL de polissacarose e diatrizoato de sódio, a uma densidade de 1,077 g / mL. Deixe o tubo em gelo até à sua utilização. </li><li> Transferir a suspensão do passo 4.3 sobre o topo da solução de diatrizoato de sódio e polissacarose com uma pipeta de Pasteur. Não perturbe a camada inferior. Os tecidos de até 10 recém-nascidos ou 2 P17 pancreata pode ser colocada em cada tubo de 15 ml. </li><li> Girar o tubo de a ~ 600 x g num rotor oscilante durante 15 min a 4 ° C. Use as configurações de freio-off. </li><li> Após a centrifugação, transferir a mídia e camadas interfase (note que em condições de operação ideais, as ilhotas será localizado na interfase entre o polissacarose e cultura da mídia após a centrifugação) para um novo tubo de 15 mL. Adicionar 10 ml de mídia, misture delicadamente mas completamente. </li><li> Spin down a fração enriquecida com ilhéua 300 xg durante 5 min. Remova o meio de lavagem. Repetir a lavagem mais uma vez com meio RPMI completo. Pelotas Ressuspender em 4 mL de mídia, que contém a maioria dos ilhéus e dutos (Figura 1D). </li><li> Mão-pegar as ilhotas, conforme descrito na secção 5.1. </li></ol></li></ol> 6. Os ensaios GSIS em isolados Ilhotas <ol><li> Incubar os ilhéus a partir do passo 5.1.4 em meio RPMI completo, durante 2 h num incubador de cultura de tecido C 37 °. </li><li> Usar uma pipeta para remover RPMI sob um microscópio de dissecação. Adiciona-se 3 mL de solução de KRB com glucose 2,8 mM e a placa 3. Agitar 10 vezes para lavar as ilhotas. Remover soluções KRB. <ol><li> Repetir o processo de lavagem uma vez. Incubar as ilhotas com 3 mL de solução de KRB a 37 ° C durante 1 hora numa incubadora. </li></ol></li><li> Aliquota de 1 mL de KRB a cada poço de uma placa de 12 poços. Pré-aqueça o estoque KRB e placa a 37 ° C em uma incubadora. </li><li> Lavar as ilhotas duas vezes com KRB pré-aquecido como em 6.2. transfer 10 ilhotas para cada poço das placas pré-aquecido. Incubar numa incubadora a 37 ° C. </li><li> Após 45 minutos, retirar-se 50 ul de sobrenadante. Este contém a solução de insulina segregada sob basais de glucose. </li><li> Adicionar glucose 32 mL de 500 mM. Agitar a placa 20 vezes para misturar a solução. Incubar numa incubadora a 37 ° C. Agitar a placa 5 vezes a cada 5 min. </li><li> Após 45 minutos, retirar-se 50 uL de sobrenadante. Esta é a insulina segregada sob alto teor de glucose. </li><li> Transfira todas as ilhotas a um tubo de 1,5 mL. Congelar em -20 ° C durante 30 min. Descongelar à temperatura ambiente durante 5 min. Repita o processo de congelamento e descongelamento. </li><li> Adicionar 0,5 mL de etanol a 70% com HCl 20 mM. Deixe durante a noite. Esta é a insulina nas ilhotas. </li><li> Use de ELISA convencional para ensaiar os níveis de insulina, descrito anteriormente 3. </li></ol>

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	<li>Matsumoto, 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=Improvement+of+pancreatic+islet+cell+isolation+for+transplantation.">Improvement of pancreatic islet cell isolation for transplantation.</a> Proc (Bayl Univ Med Cent). 20 (4), 357-362 (2007).</li><li>Zmuda, E. J., Powell, C. A., Hai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+murine+islet+isolation+and+subcapsular+kidney+transplantation.">A method for murine islet isolation and subcapsular kidney transplantation.</a> J Vis Exp. 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D., 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=Nonenzymic+in+vitro+isolation+of+perinatal+islets+of+Langerhans.">Nonenzymic in vitro isolation of perinatal islets of Langerhans.</a> In Vitro. 19 (8), 611-620 (1983).</li><li>Dolen&#353;ek, J., Rupnik, M. A., Sto&#382;er, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+similarities+and+differences+between+the+human+and+the+mouse+pancreas.">Structural similarities and differences between the human and the mouse pancreas.</a> Islets. 7 (1), e102445 (2015).</li><li>White, J., White, D. 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The authors declare no competing financial interests for the described work.

Aqui, nós fornecemos um protocolo passo-a-passo de isolamento de ilhotas de pâncreas que são demasiado pequenos para perfusão convencional. Espera-se, para se obter ilhotas prontos para todos os estudos baseados em purificação de ilhotas, tais como beta-célula, a análise da expressão do gene, a maturação das células beta dos ilhéus pancreáticos, proliferação, as respostas de stress celular, sobrevivência celular, metabolismo, e manutenção GSIS funcional, etc. Este será, para o melhor do noss...

Isolating pure pancreatic islets is essential for assaying glucose stimulated insulin secretion (GSIS) of beta cells and for islet transplantation from cadaveric donors 1-3. It is also necessary to establish endocrine gene expression in islet cells 4, 5. For this purpose, detailed protocols have been established to allow for isolation of pancreatic islets from large pancreata (6 and references therein). These methods are based on enzymatic perfusion to dissociate acinar from islet tissues, coupled with gradient separation and hand picking. Thus, islet isolation from large pancreas can be performed readily in most laboratories. On the other hand, no detailed step-by-step protocol exists to allow for the isolation of islets from pancreata that are too small to perfuse.
Studying gene expression and function of neonatal islets is important. Neonate islets have different properties from adults in insulin secretion and proliferation capability 7, 8. However, isolating islets from newly born animals, especially mice is challenging due to the small size of the newly born pancreas. The size prevents the usual perfusion process when collagenase is injected though the pancreatic duct. Indeed, several papers have presented studies along these lines, with enzyme or non-enzyme aided isolation procedures 7, 9, 10. However, detailed description of the islet isolation process with visual aid is lacking 7, 9, making it a challenge for most researchers to perform similar studies. 
We have explored several different conditions that yield high quality islets from neonatal mice. Here we present a protocol that is expected to help researchers learn the key details in the islet isolation process. This protocol is applicable to mouse pancreas up to two weeks of age, after which perfusion can be performed for routine islet isolation. Islets can be directly used for insulin secretion and gene expression assays.

<table><tbody><tr><td>Collagenase Type IV</td><td>Sigma Aldrich, St Louis, MO</td><td>C5138</td><td></td></tr><tr><td>1x HBSS with Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>Mediatech/Cellgro, Manassas, VA</td><td>MT21020CV</td><td>If HBSS wihout Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt; is obtained, CaCl&lt;sub&gt;2&lt;/sub&gt; and MgSO&lt;sub&gt;4&lt;/sub&gt; can be added to 1.26 and 0.5 mM, respectively.&amp;nbsp;</td></tr><tr><td>RPMI 1640 w/o glucose</td><td>Thermo Fisher Scientific/Life Technologies, Waltham, MA</td><td>11879-020</td><td>Glucose needs to be added to specific levels to not interfere with subsequent islet usage.&amp;nbsp;</td></tr><tr><td>Glucose</td><td>Sigma Aldrich, St Louis, MO</td><td>G-7021&amp;nbsp;</td><td></td></tr><tr><td>polysucrose and sodium diatrizoate solution</td><td>Sigma Aldrich, St Louis, MO</td><td>Histopaque-10771</td><td></td></tr><tr><td>Stereoscope</td><td>Carl-Zeiss, Oberkochen, Germany</td><td>Stemi2000</td><td></td></tr><tr><td>Stereoscope</td><td>Leica, Wetzlar, Germany</td><td>Leica M165</td><td></td></tr><tr><td>Microcentrifuge</td><td>Eppendorf, Hauppauge, NY</td><td>Centrifuge 5417C</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf, Hauppauge, NY</td><td>Centrifuge 5810R</td><td></td></tr><tr><td>15-mL centrfuge tubes</td><td>VWR, Radnor, PA</td><td>89039-666</td><td></td></tr><tr><td>50-mL centrfuge tubes</td><td>VWR, Radnor, PA</td><td>89039-658</td><td></td></tr><tr><td>Precision balance</td><td>VWR, Radnor, PA</td><td>VWR-225AC</td><td></td></tr><tr><td>Microfuge tubes</td><td>VWR, Radnor, PA</td><td>87003-294</td><td></td></tr><tr><td>Pipetman P1000</td><td>Fisher Scientific,&amp;nbsp; Waltham, MA</td><td>F123602</td><td></td></tr><tr><td>Pipetman P20</td><td>Fisher Scientific,&amp;nbsp; Waltham, MA</td><td>F123600</td><td></td></tr><tr><td>100 x 15 mm dish</td><td>VWR, Radnor, PA</td><td>25384-088</td><td></td></tr><tr><td>60 x 15 mm dish</td><td>VWR, Radnor, PA</td><td>25384-168</td><td></td></tr><tr><td>12-well plates</td><td>VWR, Radnor, PA</td><td>665-180</td><td></td></tr><tr><td>Scissor</td><td>Fine Scientific Tools, Foster City, CA</td><td>14080-11</td><td></td></tr><tr><td>Tweezers</td><td>Fine Scientific Tools, Foster City, CA</td><td>5708-5</td><td></td></tr><tr><td>CD1 mice</td><td>Charles River Laboratories, Wilminton, MA&amp;nbsp;</td><td>CD-1</td><td></td></tr><tr><td>C57BL/6J</td><td>The Jackson laboratory, Farmington, CT&amp;nbsp;</td><td>C57BL/6J</td><td></td></tr><tr><td>B6CBAF1/J</td><td>The Jackson laboratory, Farmington, CT&amp;nbsp;</td><td>B6CBAF1/J</td><td></td></tr></tbody></table>

effective isolation of functional islets from neonatal mouse pancreas

protocolos de isolamento de ilhotas-à base de perfusão de grande pâncreas de mamíferos estão bem estabelecidos. Tais protocolos estão prontamente realizado em muitos laboratórios, devido ao grande tamanho do ducto pancreático que permite a injecção pronto colagenase e subsequente perfusão tecidual. Em contraste, o isolamento de ilhotas do pâncreas pequena, como a de ratinhos neonatais, é um desafio porque perfusão não é facilmente realizável na pequena pâncreas. Aqui nós descrevemos um procedimento simples detalhado que recupera um número substancial de ilhotas de ratos recém-nascidos com assistência visual. pancreata inteiro recentemente dissecados foram digeridos com 0,5 mg / ml de colagenase IV dissolvido em Hanks &#39;Solução Salina Equilibrada (SSHE) a 37 ° C, em tubos de microcentrífuga. Os tubos foram aproveitado regularmente para ajudar a dispersão do tecido. Quando a maior parte do tecido foi disperso em pequenos aglomerados de cerca de 1 mM, lisados ​​foram lavados três a quatro vezes com meio de cultura com 10% de soro fetal de bovino (FBS). aglomerados de ilhotas,desprovidas de tecidos acinares reconhecíveis, pode então ser recuperado sob dissecando estereoscópio. Este método recupera 20 - 80 pequenas e ilhotas de grande porte por pâncreas de rato recém-nascido. Estas ilhotas são adequadas para ensaios a jusante mais possíveis, incluindo a secreção de insulina, a expressão do gene, e a cultura. Um exemplo do ensaio de secreção de insulina é apresentado para validar o processo de isolamento. O fundo genético e grau de digestão são os maiores fatores determinantes do rendimento. solução de colagenase recentemente feito com alta atividade é preferível, uma vez que auxilia no isolamento endócrino-exócrina. A presença de catiões [cálcio (Ca2 +) e magnésio (Mg2 +)] em todas as soluções e soro fetal de bovino na lavagem / meios de recolha são necessárias para um bom rendimento de ilhotas com integridade adequada. Um escopo de dissecação com bom contraste e ampliação também vai ajudar.

Em condições óptimas, o método apresentado pode rendimento de 20 - 80 de cada pequena ilhotas do pâncreas do rato. Este número depende do fundo genético, idade de ratinhos, e o tamanho das ilhotas para ser recuperado. Entre os comumente usados, CD1 fora de raça C57BL / 6J de raça pura produzem menos ilhotas com tamanho menor do que híbridos entre CD1 e C57BL / 6 ou B6CBAF1 comercial / mice J fazer. Mão directa colheita geralmente deu um menor número de ilhotas, provavelmente ...

Watch this Scientific Journal Video about Effective Isolation of Functional Islets from Neonatal Mouse Pancreas at JoVE.com

Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

Nós descrevemos um protocolo aqui para isolar ilhotas intactas de ratos recém-nascidos. Pâncreas foi parcialmente digerido com colagenase, seguido por lavagem e colheita manual. 20 - 80 agrupamentos de ilhéus podem ser obtidos por pâncreas de ratinhos recém-nascidos, que são adequados para vários estudos da ilhota.

Isolamento eficaz de ilhotas funcionais a partir de ratinho neonato Pâncreas

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories ...

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10.0K visualizações.  Vanderbilt University Medical School.  Nós descrevemos um protocolo aqui para isolar ilhotas intactas de ratos recém-nascidos. Pâncreas foi parcialmente digerido com colagenase, seguido por lavagem e colheita manual. 20 - 80 agrupamentos de ilhéus podem ser obtidos por pâncreas de ratinhos recém-nascidos, que são adequados para vários estudos da ilhota. 

Vídeo: Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

Um método para isolamento de ilhotas murino e transplante renal subcapsular

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

Medicina

Isolation of Human Islets from Partially Pancreatectomized Patients

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 diabetic islets from organ donors2. Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area.
To extend the applicability of our protocol to include the occasional case in which a large amount (&gt;15g) of human pancreatic tissue is available , we used a Ricordi chamber (50 ml) to digest the tissue. During digestion, we manually shook the Ricordi chamber3 at an intensity that varied by specimen according to its level of tissue fibrosis. A discontinous Ficoll gradient was then used to separate the islets from acinar tissue. We noted that the tissue pellet should be small enough to be homogenously resuspended in Ficoll medium with a density of 1.125 g/ml. After isolation, we cultured the islets under stress free conditions (no shaking or rotation) with 5% CO2 at 37 &deg;C for at least 48 h in order to facilitate their functional recovery. Widespread application of our protocol and its future improvement could enable the timely harvesting of large quantities of human islets from diabetic and clinically matched non-diabetic subjects, greatly advancing type 2 diabetes research.

The supply of type 2 diabetic islets for research is insufficient. Here we share our protocol for isolating islets from patients undergoing partial pancreatectomy. This approach represents a unique venue for obtaining islets from type 2 diabetic and clinically matched non-diabetic subjects in adequate numbers for basic and clinical studies.

Isolamento de Ilhotas Humanos de pacientes parcialmente pancreatectomizado

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 ...

Scaffold-supported Transplantation of Islets in the Epididymal Fat Pad of Diabetic Mice

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy may incur acute whole blood reactions and result in poor islet engraftment. Here, we report a robust protocol for the transplantation of islets at the extrahepatic transplantation site-the epididymal fat pad (EFP)-in a diabetic mouse model. A protocol to isolate and purify islets at high yields from C57BL/6J mice is described, as well as a transplantation method performed by seeding islets onto a decellularized scaffold (DCS) and implanting them at the EFP site in syngeneic C57BL/6J mice rendered diabetic by streptozotocin. The DCS graft containing 500 islets reversed the hyperglycemic condition within 10 days, while the free islets without DCS required at least 30 days. The normoglycemia was maintained for up to 3 months until the graft was explanted. In conclusion, DCS enhanced the engraftment of islets into the extrahepatic site of the EFP, which could easily be retrieved and might provide a reproducible and useful platform for investigating the scaffold materials, as well as other transplantation parameters required for a successful islet engraftment.

This protocol demonstrates murine islet isolation and seeding onto a decellularized scaffold. Scaffold-supported islets were transplanted into the epididymal fat pad of streptozotocin (STZ)-induced diabetic mice. Islets survived at the transplantation site and reversed the hyperglycemic condition.

Transplante de islotes apoiado em andaimes na almofada de gordura epidídmica de ratos diabéticos

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy ...

Bioengenharia

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

Lepr<sup&gt; Db</sup&gt; Mouse Modelo de Diabetes Tipo 2: Isolamento de ilhotas pancreáticas e células vivas 2-Photon Imagens de ilhotas intactas

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic &#946; cells which will lead to the development of type 2 diabetes mellitus (T2DM). Although the mechanisms for the chemical-induced &#946; cell damage were unclear and likely to be complex, one recurring finding is that these chemicals induce oxidative stress leading to the generation of excessive reactive oxygen species (ROS) which induce damage to the &#946; cell. To identify potential diabetogenic environmental chemicals, we isolated pancreatic islet cells from C57BL/6 mice and cultured islet cells in 96-well cell culture plates; then, the islet cells were dosed with chemicals and the ROS generation was detected by 2',7'-dichlorofluorescein (DCFH-DA) fluorescent dye. Using this method, we found that bisphenol A (BPA), Benzo[a]pyrene (BaP), and polychlorinated biphenyls (PCBs), could induce high levels of ROS, suggesting that they may potentially induce damage in islet cells. This method should be useful for screening diabetogenic xenobiotics. In addition, the cultured islet cells may also be adapted for in vitro analysis of chemical-induced toxicity in pancreatic cells.

Here we present a protocol to isolate mouse pancreatic islet cells for screening the ROS inductions by the xenobiotics in order to identify the potential diabetogenic xenobiotic chemicals.

Um rastreio de baseada em célula de murino ilhotas pancreáticas para produtos químicos ambientais Diabetogenic

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic &#946; cells which will lead to ...

Imunologia e Infecção

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet &#946;-cell replication and to develop methods for stimulating &#946;-cell regeneration. Herein a high-content screening method to identify and assess the &#946;-cell replication-promoting activity of small molecules is presented.

A-alta conteúdo<em&gt; In Vitro</em&gt; Replicação plataforma de descoberta de células-β das ilhotas pancreáticas

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines to the study of isolated rodent islets. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular signaling pathways and secretory responses. Whereas the method of islet isolation using tissue dissociating enzyme (TDE) preparations has been well established in many laboratories1-4, variations in the consistency of islet yield and quality from any given rodent strain limit the extent and feasibility of primary islet studies. These variations often occur as a result of the crude partially purified TDEs used in the islet isolation procedure; TDEs frequently exhibit lot-to-lot variations in activity and often require adjustments to the dose of enzyme used. A small number of reports have used purified TDEs for rodent cell isolations5, 6, but the practice is not widespread despite the routine use and advantages of purified TDEs for human islet isolations. In collaboration with VitaCyte, LLC (Indianapolis, IN), we developed a modified mouse islet isolation protocol based on that described by Gotoh7, 8, in which the TDEs are perfused directly into the pancreatic duct of mice, followed by crude tissue fractionation through a Histopaque gradient9, and isolation of purified islets. A significant difference in our protocol is the use of purified collagenase (CIzyme MA) and neutral protease (CIzyme BP) combination. The collagenase was characterized by the use of a6 fluorescence collagen degrading activity (CDA) assay that utilized fluorescently labeled soluble calf skin fibrils as substrate6. This substrate is more predictive of the kinetics of collagen degradation in the tissue matrix because it relies on native collagen as the substrate. The protease was characterized with a sensitive fluorescent kinetic assay10. Utilizing these improved assays along with more traditional biochemical analysis enable the TDE to be manufactured more consistently, leading to improved performance consistency between lots. The protocol described in here was optimized for maximal islet yield and optimal islet morphology using C57BL/6 mice. During the development of this protocol, several combinations of collagenase and neutral proteases were evaluated at different concentrations, and the final ratio of collagenase:neutral protease of 35:10 represents enzyme performance comparable to Sigma Type XI. Because significant variability in average islet yields from different strains of rats and mice have been reported, additional modifications of the TDE composition should be made to improve the yield and quality of islets recovered from different species and strains.

A detailed description of mouse islet isolation is described using the technique of in situ pancreatic ductal cannulation and perfusion of a combination of purified collagenase and neutral protease.

Rato Ilhéu de Langerhans isolamento usando uma combinação de colagenase purificada e Protease Neutra

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines ...

Biologia

Intraportal Transplantation of Pancreatic Islets in Mouse Model

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation site in experimental islet transplantation is the kidney capsule. However, as less is known about the interaction of pancreatic islets with blood constituents, it also makes sense to utilize the portal vein approach in experimental islet transplantation.
This protocol demonstrates an intraportal islet transplantation technique in NMRI nude mice. Streptozotocin (180 mg/kg) is injected intraperitoneally to induce hyperglycemia in recipient mice. They are considered as diabetic at a non-fasting blood glucose level greater than 20 mmol/L. One day prior to transplantation, mouse pancreatic islets are isolated from the donor pancreas by collagenase digestion; a minimum of 350 islets are utilized per diabetic recipient. Depending upon the islet isolation yield, two or more donor mice are utilized per recipient. After overnight culture at 37 &#176;C, islets are administered into the recipient liver via the portal vein. After surgery, the mice are protected in red Makrolon houses and observed until are awake. This protocol maintains glycemic control for 120 days in syngeneic mice and 15 days in allogeneic mice.

Pancreatic islet transplantation is a way to achieve normoglycemia in type 1 diabetes. This article focuses on the transplantation technique through the intraportal route to maintain normal glucose levels in diabetic mice.

Intraportal transplante de ilhotas pancreáticas no modelo de Mouse

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation ...

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 ...

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.

Isolamento eficaz de ilhotas funcionais a partir de ratinho neonato Pâncreas

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