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.

Tiernutzung nach den festgelegten Verfahren in Protokoll M / 11/181 genehmigt von der Vanderbilt Institutional Animal Care und Use Committee für Gu. CD1 oder CBA / Bl6 Mäuse wurden von kommerziellen Anbietern gekauft und in der Vanderbilt Tieranlage gekreuzt neugeborenen Mäusen erhalten. 1. Herstellung von Mäusen, Stock-Lösungen und Geräte <ol><li> Für die Maus Kreuz: eine Maus Kreuz und notieren Sie die Aufstecken Termine einrichten experimentelle Planung zu unterstützen. CD1-Mäuse geben in der Regel Geburt um Tag 19 nach der Paarung. Verwenden Sie CD1 oder CBA / Bl6 kreuzt CD1 oder CBA / Bl6 Neugeborenen bzw. zu erhalten. Kreuzungen zwischen den beiden Linien nutzen CD1 Weibchen und CBA / Bl6 Männer. </li><li> Für die Kollagenase Lager: 200 mg Kollagenase Typ IV mit einem hohen Präzisionswaage wiegen. Transfer in ein 50 ml Zentrifugenröhrchen. Man löst in 40 ml HBSS mit Ca 2+ / Mg 2+ (1,26 und 0,5 mM jeweils) , um eine 5 mg / ml Stammlösung herzustellen. <ol><li> Lassen Sie Kollagenasefür ~ 30 min unter leichtem Schütteln lösen. Aliquotieren und die Lösung in -20 ° C als Vorrat eingefroren halten, die für mindestens ein Jahr lang aktiv bleibt. </li></ol></li><li> Autoklaven auf mehrere Paare von Pinzetten, Scheren und P20-Tipps für die Präparation und Inselchen Picking sterilisieren. </li><li> Für die 1 M Glucose Lager: wiegen 9 g Glucose in einem 50 ml Zentrifugenröhrchen, 50 ml RPMI 1640-Medium, das Glukose zu lösen. Halten Sie in 4 ° C bis zur Verwendung, aber nicht mehr als 6 Monate. </li></ol> 2. Arbeitslösungen HINWEIS: Der Tag der Inselisolierung, bereiten Sie die folgenden Reagenzien. <ol><li> Für die Kollagenase Typ IV Arbeitslösung: Tauwetter und Kollagenase Lager auf Eis verdünnt. Dispense 0,15 ml Brühe in jedem 1,7 ml Mikrozentrifugenröhrchen. In 1,35 ml HBSS mit Ca 2+ / Mg 2+. Drehen Sie das Röhrchen 5 mal. Lassen Sie auf Eis für 15 min. <ol><li> Spin bei hoher Geschwindigkeit für 3 Minuten in einer Mikro. Überstand in ein neues Röhrchen. Lassen Sie auf Eis bis zum Gebrauch. Entsorgen Sie das Rohr mit unlöslichen Ablagerungen. </li></ol></li><li> Zur vollständigen RPMI 1640 Medium: 2,5 ml 1 M Glucose, 50 ml hitzeinaktiviertem FBS und 5 ml 100x Pen-Strep Lager in 500 ml RPMI 1640 Medien hinzuzufügen. Lassen Sie auf Eis bis zum Gebrauch. </li></ol> 3. Pancreas Isolierung und Verdauung <ol><li> Euthanize Neugeborenen mit Isofluran, gefolgt von Enthauptung nach anerkannten Protokollen. </li><li> Legen Sie die Maus mit dem Bauch nach oben zeigt. Spray mit 70% Ethanol für die Sterilisation. </li><li> Heben Sie die Bauchhaut mit einer Pinzette und schneiden Sie die Haut und Muskelschichten in Längsrichtung entlang der Mittellinie mit einer Schere aus dem Genitalbereich des Brustkorbs. Dies öffnet die Bauchhöhle alle inneren Organe zu belichten. </li><li> Übertragen Sie alle inneren Organe zu einer 100-cm-Schale mit einer Pinzette. Suchen Sie die Bauchspeicheldrüse, die ein Streu Orgel mit einem Rückenteil ist , das in den Magen und Milz und eine ventrale Teil klammert , die 11 bis Duodenum klammert. </li&gt; <li> In HBSS in die Schüssel, die Organe zu versenken. In Lösung klammern die Pankreasgewebe nicht mehr auf den Zwölffingerdarm, Magen oder Milz. Es ist ohne weiteres erkennbar unter einem Binokular wegen seiner typischen weißen Farbe. Verwenden einer Pinzette die Pankreasgewebe weg vom umgebenden Gewebe und an einem neuen 60-mm-Schale mit HBSS zu schälen. </li><li> Schneiden Sie jede Bauchspeicheldrüse mit einer Schere in Stücke kleiner als 5 mm über beliebigen Abmessungen. Transfer zu einem 1,5 ml Mikrozentrifugenröhrchen. Bis zu fünf Bauchspeicheldrüsen (jünger als P7) kann in einem Röhrchen verdaut werden. Für P8-P16 Mäuse, verwenden Sie eine Röhre pro Bauchspeicheldrüse. </li><li> In 0,5-1 ml Kollagenase Arbeitslösung in jedes Röhrchen (0,2 ml pro P1-P7 Bauchspeicheldrüse Verwenden Sie 0,5 ml für jede P8-P16 Bauchspeicheldrüse.). Lassen Sie das Rohr in einem 37 ° C Inkubator für bis zu 15 min. Drehen Sie das Rohr zwei Mal pro min. </li><li> Nach ca. 5 min, tippen Sie auf das Rohr die Verdauung Status des Pankreasgewebe zu überwachen. Weiter den Verdauungsprozess, bis die meisten der PankreasGewebe erscheint als fragmentierten Zellcluster, mit einem Durchmesser von &lt;2 mm. Das Lysat wird trüb erscheinen, die den normalen Azinuszelltumoren Autolyse zurückzuführen ist. </li></ol> 4. Lysate Waschen <ol><li> Drehen Sie das Lysat bei 500 × g für 10 s in einer Mikro. Entfernen Sie die überstehende Schicht mit einem P1,000 Pipetman. Der Überstand sollte erscheinen trübe, aber ohne Zellhaufen. verwenden aspiration nicht, die die Gewebefragmente an der Unterseite durch die Anwesenheit von etwas klebrig DNA schnell abzusaugen weg kann. </li><li> 1 ml RPMI 1640 Komplettmedium. Tippen Sie auf das Rohr sanft die fragmentierte Lysat wieder zu suspendieren. Kehren nach oben und unten 10-mal. Wiederholen Sie Schritt 4.1. </li><li> Wiederholen Sie Schritt 4.2 zwei weitere Male oder bis der Überstand klar erscheint. Re-suspend gewaschenen Pankreas Fragmente in 1 ml komplettem RPMI 1640-Medium. </li></ol> 5. Islet Isolation HINWEIS: Bei einer kleinen Anzahl von Bauchspeicheldrüsen, direkte Handlese, wie unten (5.1) kann für die Insel isol verwendet werdenation. Für große Anzahlen von Bauchspeicheldrüsen (&gt; 6), wobei das Verfahren in 5.2 skizziert ist bevorzugt. <ol><li> Direkt Handlese: <ol><li> Übertragen Sie Lysat aus Schritt 4.3 zu einer 60-mm-Schale. In 5 ml komplettem RPMI 1640-Medium. Bringen Sie unter einem Binokular. </li><li> Stellen Sie die Hellfeldbeleuchtung auf dem Mikroskop auf. Stellen Sie die Lichtintensität auf ~ 50% der maximalen Leistung. Unter dieser Bedingung erscheinen Inseln als leicht rosa Cluster, während Acinuszellen als dunkle unregelmäßig geformte Cluster (Abbildung 1A). Eine Vergrößerung von ~ 50X (die Leistung des Objektivs zu kombinieren und das Augenteil) wird empfohlen, die beste Art und Weise zu visualisieren Inseln und die Pipettenspitze Öffnung gleichzeitig für die Kommissionierung zu bieten. </li><li> Pick-up-Inseln mit einem P20-Pipettenspitze während acinar Cluster zu vermeiden. Dispense Inselchen angereicherten Fraktionen auf eine neue 60 - mm - Schale mit RPMI Komplettmedium (1B). </li><li> Wiederholen Sie die Hand-Picking-Prozess noch zweimal. Lassen Sie rein Inseln in eine neue 60mm - Schale mit 4 ml komplettem RPMI Medium (1C). </li></ol></li><li> Gradientenzentrifugation: <ol><li> Vorbereiten eines 15 ml Zentrifugenröhrchen, die mit 2 ml Polysaccharose und Natriumdiatrizoat in einer Dichte von 1,077 g / ml vorgespannt ist. Lassen Sie das Röhrchen auf Eis bis zum Gebrauch. </li><li> Übertragung der Suspension aus Schritt 4.3 auf der Oberseite des Polysaccharose und Natriumdiatrizoat-Lösung mit einer Pasteur-Pipette. Sie nicht die untere Schicht stören. Gewebe von bis zu 10 neu geboren oder 2 P17 Bauchspeicheldrüsen können in jede 15 ml-Röhrchen geladen werden. </li><li> Drehe das Röhrchen bei ~ 600 xg in einem Schwenkrotor für 15 min bei 4 ° C. Verwenden Bremse-Aus-Einstellungen. </li><li> Nach Zentrifugation übertragen die Medien und Zwischenphasenschichten (man beachte, dass unter idealen Betriebsbedingungen werden die Inseln in der Zwischenphase zwischen Polysaccharose und Kulturmedien nach dem Zentrifugieren befinden) in ein neues 15 ml-Röhrchen. In 10 ml Medien mischen vorsichtig , aber gründlich. </li><li> Spin down die Insel angereicherte Fraktionbei 300 xg für 5 min. Entfernen Sie die Waschmedien. Wiederholen Sie die Wäsche noch einmal mit komplettem RPMI-Medien. Resuspendieren Pellets in bis 4 ml Medien, die meist kleinen Inseln und Kanälen (Abbildung 1D) enthält. </li><li> Hand holen die kleinen Inseln, wie in Abschnitt 5.1 beschrieben. </li></ol></li></ol> 6. GSIS Assays in isolierten Inseln <ol><li> Inkubieren der Inseln aus Schritt 5.1.4 in kompletten RPMI-Medium für 2 h in einem 37 ° C Gewebekultur-Inkubator. </li><li> Verwenden Sie eine Pipette RPMI unter einem Binokular zu entfernen. In 3 ml KRB - Lösung mit 2,8 mM Glucose zu der Platte 3. Swirl 10-mal die Inseln zu waschen. Entfernen KRB-Lösungen. <ol><li> Wiederholen Sie einmal, um den Waschvorgang. Inkubieren der Inselchen mit 3 ml KRB-Lösung bei 37 ° C für 1 h in einem Inkubator. </li></ol></li><li> Aliquot 1 ml KRB in jede Vertiefung einer 12-Well-Platte. Pre-warm die KRB Lager und Platte auf 37 ° C in einem Inkubator. </li><li> Waschen Sie die kleinen Inseln zweimal mit vorgewärmten KRB wie in 6.2. Transfer 10 Inselchen in jede Vertiefung der vorgewärmten Platten. Inkubieren in einem 37 ° C Inkubator. </li><li> Nach 45 Minuten wurden 50 ul Überstand zurücktreten. Diese enthält die Insulinlösung unter basalen Glukose ausgeschieden. </li><li> In 32 &amp; mgr; l 500 mM Glucose. Schwenken Sie die Platte 20-mal die Lösung zu mischen. Inkubieren in einem 37 ° C Inkubator. Schwenken Sie die Platte 5 mal einmal alle 5 min. </li><li> Nach 45 min zurückziehen 50 &amp; mgr; l Überstand. Dies ist das Insulin unter hohem Glucose sekretiert. </li><li> Übertragen Sie alle Inseln in ein 1,5 ml-Röhrchen. Einfrieren in -20 ° C für 30 min. Tauen Sie für 5 Minuten bei Raumtemperatur. Wiederholen Sie das Einfrieren und Auftauen Prozess. </li><li> In 0,5 ml 70% Ethanol mit 20 mM HCl. Lassen Sie über Nacht. Dies ist das Insulin links in Inselchen. </li><li> Verwenden herkömmlicher ELISA auf den Insulinspiegel Assay, zuvor beschriebenen 3. </li></ol>

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The authors declare no competing financial interests for the described work.

Hier bieten wir eine Schritt-für-Schritt-Protokoll auf Inselisolierung von Bauchspeicheldrüsen, die für konventionelle Perfusion zu klein sind. Es wird erwartet , Inselchen bereit für alle islet-basierten Untersuchungen wie beta-Zellreinigung, Genexpressionsanalyse, islet beta-Zell - Reifung, Proliferation, Zellstressreaktionen, Zellüberleben, Metabolismus und funktionelle GSIS Wartung, usw. zu ergeben. Dies wird nach bestem Wissen unseren Erkenntnissen die erste detaillierte visuelle Protokoll, das neue F...

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

Perfusions-basierte Inselisolierung Protokolle von großen Säugetieren Bauchspeicheldrüsen sind gut etabliert. Solche Protokolle sind in vielen Laboratorien aufgrund der großen Größe der Pankreasgang, der ermöglicht bereit Kollagenase Injektion und anschließende Gewebeperfusion leicht durchgeführt. Im Gegensatz dazu Inselisolierung von kleinen Bauchspeicheldrüsen, wie die von neugeborenen Mäusen, ist schwierig, weil die Perfusion nicht leicht erreichbar in dem kleinen pancreata ist. Hier haben wir ein detailliertes einfaches Verfahren beschreiben, die von neu geborenen Mäusen, die mit visuellen Unterstützung einer beträchtlichen Zahl von Inseln erholt. Frisch sezierte ganze Bauchspeicheldrüsen wurden mit 0,5 mg / ml Kollagenase IV in Hanks &#39;Balanced Salt Solution (HBSS) bei 37 ° C, in Mikrozentrifugenröhrchen gelöst verdaut. Die Röhrchen wurden regelmäßig zu unterstützen Gewebe Zerstreuung angezapft. Wenn der größte Teil des Gewebes zu kleinen Gruppen etwa 1 mm dispergiert, drei- bis viermal mit Nährmedien Lysate wurden mit 10% fötalem Rinderserum (FBS), gewaschen. Islet Cluster,ohne erkennbare acinar Gewebe kann dann unter Sezieren Stereoskop zurückgewonnen werden. Dieses Verfahren gewinnt 20 - 80 kleine bis große Inseln pro Bauchspeicheldrüse von Neugeborenen Maus. Diese Inseln sind für die meisten denkbaren Downstream-Assays, einschließlich der Insulinsekretion, Genexpression und Kultur. Ein Beispiel für die Insulinsekretion Assay dargestellt, um den Isolationsprozess zu validieren. Der genetische Hintergrund und der Grad der Verdauung sind die größten Faktoren, die Ausbeute zu bestimmen. Frisch mit hoher Aktivität hergestellt Kollagenase-Lösung wird bevorzugt, da es in endokrin exokrinen Isolation hilft. Die Anwesenheit von Kationen [Calcium (Ca 2+) und Magnesium (Mg 2+)] in allen Lösungen und fötales Rinderserum in der Wasch- / Kommissionierung Medien sind notwendig für eine gute Ausbeute an kleinen Inseln mit der richtigen Integrität. Ein Binokular mit gutem Kontrast und Vergrößerung wird auch dazu beitragen.

Unter optimalen Bedingungen kann das vorgestellte Verfahren ergeben 20 - 80 Inseln aus jeder kleinen Maus Bauchspeicheldrüse. Diese Zahl hängt von dem genetischen Hintergrund, dem Alter der Mäuse, und die Größe der Inseln zurückgewonnen werden. Unter den üblicherweise verwendeten, CD1 aus gezüchtet und C57BL / 6J reinrassige Mäuse produzieren weniger Inselchen mit geringerer Größe als Hybrid zwischen CD1 und C57BL / 6 oder kommerziellen B6CBAF1 / J-Mäuse tun. Direkt Hand Komm...

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

Wir beschreiben ein Protokoll hier für intakte Inseln von neugeborenen Mäusen zu isolieren. Bauchspeicheldrüsen wurden teilweise mit Kollagenase, gefolgt von Waschen und Hand picking verdaut. 20-80 islet Cluster können pro Bauchspeicheldrüse von neugeborenen Mäusen erhalten werden, die für mehrere islet Studien geeignet sind.

Effektive Isolation von funktionellen Inselchen von Neonatal Maus Pancreas

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

effective-isolation-of-functional-islets-from-neonatal-mouse-pancreas

Research

JoVE Journal

Developmental Biology

10.0K Aufrufe.  Vanderbilt University Medical School.  Wir beschreiben ein Protokoll hier für intakte Inseln von neugeborenen Mäusen zu isolieren. Bauchspeicheldrüsen wurden teilweise mit Kollagenase, gefolgt von Waschen und Hand picking verdaut. 20-80 islet Cluster können pro Bauchspeicheldrüse von neugeborenen Mäusen erhalten werden, die für mehrere islet Studien geeignet sind. 

Serie: 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.

Eine Methode zur Murine Islet Isolation und subkapsulären Nierentransplantation

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

Forschung

Medizin

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.

Isolierung humaner Inselzellen aus teilweise Pankreatektomierten

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.

Gerüstgestützte Transplantation von Inseln in der epididymalen Fettauflage von diabetischen Mäusen

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

Biotechnik

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; Mausmodell des Typ-2-Diabetes: Pancreatic Islet Isolation und Live-cell 2-Photonen-Imaging von intakten Inseln

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.

Ein murinen Pancreatic Islet Cell-basierte Screening für diabetogene Umweltchemikalien

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 ...

Immunologie und Infektion

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.

Ein hoher Gehalt<em&gt; In Vitro</em&gt; Pancreatic Islet β-Zell-Replikation-Discovery-Plattform

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.

Maus Islet der Langerhans Isolation durch eine Kombination von gereinigtem Collagenase und 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 ...

Biologie

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 Transplantation der Bauchspeicheldrüse Inselchen im Mausmodell

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.

Eine Methode zur Maus-Inselzellen der Bauchspeicheldrüse Isolation und intrazellulären cAMP-Bestimmung

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.

Effektive Isolation von funktionellen Inselchen von Neonatal Maus Pancreas

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