Wir wollen alle unsere Kollegen, die Hilfe, Rat und kritischen Input auf verschiedenen Stufen des Projektes bedanken. Die Produktion dieses Video Artikel wurde aus Mitteln IMIDIA (unterstützt <a href="http://www.imidia.org" target="_blank">http://www.imidia.org</a> ), das Bundesministerium für Bildung und Forschung (BMBF) mit dem Deutschen Zentrum für Diabetesforschung (DZD, <a href="http://www.dzd-ev.de" target="_blank">http://www.dzd -ev.de</a> ) und das Universitätsklinikum Carl Gustav Carus an der Technischen Universität Dresden. Die Arbeiten, die zu dieser Veröffentlichung wird unterstützt von der Innovative Medicines Initiative Joint Undertaking erhalten unter Finanzhilfevereinbarung n ° 155005 (IMIDIA), Ressourcen, von denen die finanzielle Beteiligung Siebten Rahmenprogramms der Europäischen Union (FP7/2007-2013) und EFPIA bestehen Unternehmen in Form von Sachleistungen.

Ein. Freezing of Human Pankreasgewebe <ol><li> Entfernen Fettgewebe, Blutgefäße, Nerven und nicht Parenchymgewebe mit einem Skalpell und Pinzette, und schneiden Sie das Pankreasgewebe in Stücke (~ 0,5-1 cm große Würfel schneiden). </li><li> Legen Sie eine Pankreasgewebe Stück in der Mitte eines Cryomold, und decken Sie es komplett mit kaltem Tissue-Tek OCT Compound. Legen Sie die Cryomold in ein Gefäß, dessen Boden mit vorgekühlten 2-Methylbutan und Snap-freeze in flüssigem Stickstoff bedeckt. Bewahren Sie die gefrorene Probe bei -80 ° C. </li></ol> 2. Herstellung von Gefrierschnitten <ol><li> Tragen Sie Handschuhe bei allen Schritten zur Denaturierung der RNA zu vermeiden. </li><li> Reinigen Sie den Kryostaten Messer, Probenhalter und Pinsel mit kaltem 100% Ethanol und RNase Away. Legen Sie die gefrorenen Gewebe im Inneren des Kryostaten. </li><li> Warten Sie, bis das Gewebe, Messer und Pinsel zu erreichen -20 ° C. Beschriften Sie die SuperFrost Plus Objektträger und pre-cool sie innerhalb der Kryostatkammer während der Wartezeitfür das Gewebe zu erreichen -20 ° C. </li><li> Übernehmen Sie die Tissue-Tek OCT Compound auf dem Probenhalter und legen Sie die gefrorenen Gewebe an der Spitze. </li><li> Sobald das Tissue-Tek OCT Compound eingefroren trimmen cryoblock und entfernen Sie über Tissue-Tek OCT Compound mit einer Rasierklinge. </li><li> Schneiden Sie insgesamt 40 aufeinanderfolgende 10 um Scheiben aus dem Pankreasgewebe Block. Zusätzlich empfehlen wir das Schneiden eines ersten und mittleren Scheibe für Übersichtszeichnungen des Pankreas Abschnitt, der die Identifizierung von Inseln innerhalb der Scheiben erleichtern kann während der Mikrodissektion eingespart werden. </li><li> Übertragen Sie die Abschnitte aus der Messerklinge in die Mitte eines SuperFrost Plus Objektträger berühren die Rückseite der Folie mit dem Finger (abgedeckt mit einem Handschuh) zum Aufwärmen nur die Region, in die der Abschnitt einzuhalten haben. </li><li> Trocknen Sie die Abschnitte 1-2 min innerhalb des Kryostaten bei -20 ° C, nach denen Sie steckte sie in eine Folie Box auf Trockeneis gelegt. Bewahren Sie die Abschnittebei -80 ° C. </li><li> Falls notwendig, reinigen Sie das Messer mit Papiertüchern und kalt 100% Ethanol. </li></ol> 3. Dehydratisierung von Gefrierschnitte <ol><li> Bereiten Sie die Reagenzien: pour 30 ml frisch zubereitet 70% Ethanol, 30 ml DEPC-behandeltem Wasser, 2x30 ml 100% Ethanol und 30 ml Xylol in 50 ml CELLSTAR Röhren (Falcons); Kälte und halten alle Reagenzien (außer Xylol) auf Eis. </li><li> Reinigen Sie den Arbeitsbereich unter der Haube mit 100% Ethanol und RNaseZAP. </li><li> Prozess zwei Kryoschnitten wie folgt: in eiskaltem 70% Ethanol fix für 30 sec, waschen mit 5-6 schnelle Dips in eiskaltem DEPC-behandeltem Wasser, zweimal austrocknen für 1 min in eiskaltem 100% Ethanol inkubieren 4 min in Xylol bei Raumtemperatur (25 ° C), und an der Luft trocknen für 3-5 min. Wiederholen Sie diesen Schritt mit einem anderen Paar von Kryoschnitten. </li><li> Legen Sie zwei getrocknete Kryoschnitten zurück in eine 50 ml CELLSTAR Rohr mit Aluminiumfolie eingewickelt, um sie vor Licht und Feuchtigkeit zu schützen sichern. </li></ol> &lt;p class = &quot;jove_title&quot;&gt; 4. Laser Mikrodissektion der Beta-Zellen mit PALM MicroBeam, Zeiss <ol><li> Reinigen Sie das Mikroskop mit RNaseZAP und montieren Sie die Adhesive Cap im RoboMover. </li><li> Legen Sie die folgenden Einstellungen: Filter-Set 09 (Anregung: 450-490 nm, Emission&gt; 515 nm), AutoLPC-Modus, 50% Laserenergie, 65% Laserfokus, 100% Laser-Geschwindigkeit, 5 mm Abstand zwischen AutoLPC Schüsse, 6 um Abstand die Linie, Arbeitshöhe: -10.100. </li><li> Legen Sie ein Abgleiten in die Bühne. </li><li> Suchen Sie die autofluoreszierenden Beta-Zellen unter dem Mikroskop Visualisierung (5x oder 10x Objektiv). </li><li> Wechseln Sie zu dem 40x Objektiv, wählen Sie die Beta-Zellen mit dem &quot;Freehand&quot; Auswahl-Werkzeug und microdissect sie mit dem Laser. </li><li> Nehmen Sie das Gewebe von vier dehydrierte Kryoschnitten in einem AdhesiveCap. Kommentar 1: Die Zeit, um die Beta-Zellen ein dehydrierte Kryoschnitt sezieren sollte nicht länger als 10-20 min Rehydratation der Gewebeschnitte, die dazu führen würde, zu vermeidenRNA-Abbau. Bemerkung 2: überprüfen Sie die erfolgreiche Mikrodissektionsverfahren durch visuelle Inspektion nach dem Umzug von der Bühne zum Checkpoint. </li></ol> 5. Lyse von mikrodissezierten Beta-Zellen Enriched Tissue <ol><li> Entfernen Sie die AdhesiveCap aus dem RoboMover. </li><li> Pipet 10 ul Extraktionspuffer (XB, PicoPure RNA Isolation Kit, Arcturus) in den Deckel des AdhesiveCap und inkubieren sie auf den Kopf bei 42 ° C für 30 min. </li><li> Spin-Down bei 10.000 xg und legte das Lysat auf Trockeneis. Speichern sie bei -80 ° C bis zur RNA-Extraktion durchgeführt wird. </li><li> Wiederholen Sie die Schritte 3.) Bis 5.) Mit allen anderen Abschnitten. </li></ol> 6. RNA-Extraktion <ol><li> Kombinieren Sie alle zehn 10 ul Lysaten </li><li> Verfahren mit der RNA-Isolierung durch Arbeiten bei Raumtemperatur gemäß dem Protokoll des PicoPure RNA Isolation Kit, Arcturus, unter Verwendung eines 1:1-Verhältnis Extraktionspuffer (XB) zu 70% Ethanol (einschließlich DNase Behandlung). </li></ol> 7. RNA-Analyse <ol><li> Verwenden Sie 1 ul RNA für die Analyse von Qualität und Quantität mit einem Agilent 2100 Bioanalyzer (picoChip). Quantitative Auswertung ist in Bezug auf eine Standard-RNA parallel auf dem Chip geladen getan. </li></ol>

<ol>
	<li>Bonner, R. F., Emmert-Buck, 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=Laser+capture+microdissection:+molecular+analysis+of+tissue.">Laser capture microdissection: molecular analysis of tissue.</a> Science. 278, 1481-1483 (1997).</li><li>Suarez-Quian, C. A., Goldstein, S. 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=Laser+capture+microdissection+of+single+cells+from+complex+tissues.">Laser capture microdissection of single cells from complex tissues.</a> Biotechniques. 26 (2), 328-3235 (1999).</li><li>Espina, V., Wulfkuhle, J. 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=Laser-capture+microdissection.">Laser-capture microdissection.</a> Nature Protocols. 1, 586-603 (2006).</li><li>Mikulowska-Mennis, A., Taylor, T. 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=High-quality+RNA+from+cells+isolated+by+laser+capture+microdissection.">High-quality RNA from cells isolated by laser capture microdissection.</a> Biotechniques. 33 (1), 176-179 (2002).</li><li>B&#246;tticher, G., Sturm, D., Ehehalt, F., Knoch, K. P., Kersting, S., Gr&#252;tzmann, 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=Isolation+of+Human+Islets+from+Partially+Pancreatectomized+Patients.">Isolation of Human Islets from Partially Pancreatectomized Patients.</a> J. Vis. Exp. (53), e2962 (2011).</li><li>Marselli, L., Sgroi, D. 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=Laser+capture+microdissection+of+human+pancreatic+beta-cells+and+RNA+preparation+for+gene+expression+profiling.">Laser capture microdissection of human pancreatic beta-cells and RNA preparation for gene expression profiling.</a> Methods in Molecular Biology. 560, 87-98 (2009).</li><li>Marselli, L., Thorne, J., 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=Gene+Expression+of+Purified+{beta}-Cell+Tissue+Obtained+from+Human+Pancreas+with+Laser+Capture+Microdissection.">Gene Expression of Purified {beta}-Cell Tissue Obtained from Human Pancreas with Laser Capture Microdissection.</a> Journal of Clinical Endocrinology &amp; Metabolism. 93, 1046-1053 (2008).</li><li>Bottino, R., Balamurugan, A. N., 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=Response+of+human+islets+to+isolation+stress+and+the+effect+of+antioxidant+treatment.">Response of human islets to isolation stress and the effect of antioxidant treatment.</a> Diabetes. 53 (10), 2559-2568 (2004).</li><li>Abdelli, S., Ansite, J., 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=Intracellular+stress+signaling+pathways+activated+during+human+islet+preparation+and+following+acute+cytokine+exposure.">Intracellular stress signaling pathways activated during human islet preparation and following acute cytokine exposure.</a> Diabetes. 53 (11), 2815-2823 (2004).</li><li>Negi, S., Jetha, 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=Analysis+of+beta-cell+gene+expression+reveals+inflammatory+signaling+and+evidence+of+dedifferentiation+following+human+islet+isolation+and+culture.">Analysis of beta-cell gene expression reveals inflammatory signaling and evidence of dedifferentiation following human islet isolation and culture.</a> PLoS One. 7 (1), e30415 (2012).</li></ol>

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Wir beschreiben einen zuverlässigen Ansatz für die Laser-Mikrodissektion (LMD) der menschlichen Inselzellen von chirurgischen Pankreas Probe. Sofern ein LMD Mikroskop zur Verfügung steht, könnte dieses Verfahren an irgendeiner Forschungseinrichtung Ausführung partieller Pankreatektomien umgesetzt werden, wodurch sich Zugang zu menschlichen Inseln Material aus beiden Nicht-Diabetiker und Typ 2 Diabetikern. Dies ist besonders relevant in Anbetracht der Mangel an Bauchspeicheldrüsen für Inselisolierung angeboten. Vo...

<table border="1"><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> 2-Methylbutan (Isopentan) </td><td> ROTH </td><td> 3927,1 </td><td></td></tr><tr><td> AdhesiveCap (opak, 500 ul) </td><td> ZEISS </td><td> 415190-9201-000 </td><td></td></tr><tr><td> CELLSTAR Röhrchen (50 ml) </td><td> Greiner Bio-One </td><td> 210 261 </td><td> mit Unterstützung skirt </td></tr><tr><td> CELLSTAR Röhrchen (50 ml) </td><td> Greiner Bio-One </td><td> 227,261 </td><td></td></tr><tr><td> Diethylpyrocarbonat (DEPC) </td><td> SIGMA </td><td> D 5758 </td><td></td></tr><tr><td> Trockeneis </td><td></td><td></td><td></td></tr><tr><td> Ethanol absolut </td><td> VWR </td><td> 20821.310 </td><td></td></tr><tr><td> Flüssiger Stickstoff </td><td></td><td></td><td></td></tr><tr><td> Paint brush </td><td></td><td></td><td></td></tr><tr><td> PALM MicroBeam </td><td> ZEISS </td><td></td><td></td></tr><tr><td> Peel-a-Way Einbettformen (abgeschnitten), 12 x 12 mm </td><td> ProSciTech </td><td> RR12 </td><td> Top internen 22x22 mm, Tiefe 21 mm </td></tr><tr><td> Arcturus PicoPure Gefrorene RNA Isolation Kit </td><td> Applied Biosystems </td><td> KIT 0204 </td><td></td></tr><tr><td> Kunststoff-Klemme </td><td></td><td></td><td></td></tr><tr><td> Rasierer </td><td></td><td></td><td></td></tr><tr><td> RNase-Free DNase Set </td><td> Qiagen </td><td> 79254 </td><td></td></tr><tr><td> RNaseZAP </td><td> SIGMA </td><td> R 2020 </td><td></td></tr><tr><td> Scalpel /Chirurgische Klingenanordnung </td><td> Techno Cut </td><td> 2800111 </td><td></td></tr><tr><td> SuperFrost Plus Haftung Objektträger </td><td> Thermo Scientific </td><td> J1800AMNZ </td><td> 25x75x1.0 mm </td></tr><tr><td> Tissue-Tek OCT Compound </td><td> Sakura </td><td> 4583 oder 0807000022 </td><td></td></tr><tr><td> Pinzette </td><td> BRAUN </td><td> BD168R </td><td></td></tr><tr><td> Xylol </td><td> VWR </td><td> 28975.291 </td><td></td></tr></table>

improved protocol for laser microdissection of human pancreatic islets from surgical specimens

Lasermikrodissektion (LMD) ist eine Technik, die die Rückgewinnung ausgewählter Zellen und Geweben von winzigen Mengen von Parenchym 1,2 ermöglicht. Die präparierten Zellen können für eine Vielzahl von Untersuchungen, wie transkriptomischen oder Proteom-Studien, DNA Beurteilung oder Chromosomenanalyse 2,3 verwendet werden. Eine besonders anspruchsvolle Anwendung von LMD Transkriptomanalyse ist, die aufgrund der Labilität der RNA 4, besonders ausgeprägt, wenn die von Geweben, die reich von RNasen, wie der Bauchspeicheldrüse sind seziert kann. Ein Protokoll, das Mikrodissektion schnelle Identifikation und Erfassung von Zielzellen ermöglicht ist in dieser Einstellung, um das Gewebe Handhabung zu verkürzen und damit zu RNA-Konservierungsmedien zu gewährleisten. Hier beschreiben wir ein Protokoll zum Erfassen menschlichen Pankreas-Beta-Zellen aus chirurgischen Proben für transkriptomischen Studien 5 verwendet werden. Kleine Stücke der Bauchspeicheldrüse von etwa 0,5-1 cm 3 wurden von den gesunden erscheinenden Rande der resezierten Bauchspeicheldrüse Proben, in Tissue-Tek OCT Compound eingebettet, sofort gekühlt 2-Methylbutan zerteilt gefroren und bei -80 ° C bis zum Schneiden. Vierzig Serienschnitten von 10 um Dicke wurden auf einem Kryostaten bei -20 ° C eine Einstellung geschnitten, individuell auf Glasobjektträgern im Kryostat für 1-2 Minuten getrocknet, und bei -80 ° C überführt Unmittelbar vor der Lasermikrodissektion Verfahrens wurden die Schnitte fixiert in eiskaltem, frisch zubereitete 70% Ethanol für 30 sec. gewaschen, um 5-6 Dips in eiskaltem DEPC-behandeltem Wasser und entwässert durch zwei einminütigen Inkubationen in eiskaltem 100% Ethanol gefolgt von Xylol (welches zum Gewebe Dehydratisierung verwendet) für 4 min; Gewebeschnitte wurden dann luftgetrocknet anschließend für 3-5 min. Wichtig ist, dass alle Schritte, mit Ausnahme der Inkubation in Xylol, wurden mit eiskaltem Reagenzien - eine Änderung über einen zuvor beschriebenen Protokoll 6. Utilization eiskaltem Reagenzien führte zu einem deutlichen Anstieg der intrinsischen Autofluoreszenz der Beta-Zellen, und erleichtert ihre Anerkennung. Für Mikrodissektion wurden vier Abschnitte dehydratisiert jeweils erneut beiden wurden in einer Folie umwickelte 50 ml Röhrchen gegeben, um das Gewebe vor Feuchtigkeit schützen und Bleichen; die verbleibenden zwei wurden sofort mikrodissezierten. Dieses Verfahren wurde unter Verwendung eines PALM MicroBeam Instrument (Zeiss) unter Verwendung der Auto Laser Pressure Catapulting (AutoLPC) Modus. Der Abschluss der Beta-Zell-/ Insel Dissektion von vier Kryoschnitten nicht länger als 40-60 min benötigt. Zellen wurden in ein AdhesiveCap gesammelt und lysiert mit 10 ul Lysepuffer. Jede einzelne RNA Probe für Transkriptom-Analyse wurde durch die Kombination von 10-Zellen mikrodissezierten Proben, gefolgt von RNA Extraktion mit dem Pico Pure RNA Isolation Kit (Arcturus) erhalten. Dieses Protokoll verbessert die intrinsische Autofluoreszenz der menschlichen Beta-Zellen, um so ihren schnellen und präzisen Erkennung und collection. Weitere Verbesserung dieses Verfahrens konnte ermöglichen die Dissektion der phänotypisch unterschiedlichen Beta-Zellen, mit möglichen Folgen für ein besseres Verständnis der Veränderungen mit Typ 2 Diabetes.

Wie in 1 gezeigt, führte die modifizierte Dehydratisieren Protokoll zu einer Verbesserung der Betazellen Autofluoreszenz im Vergleich zum vorherigen veröffentlichten Protokoll 6. Aufbringen der beschriebenen Protokolls wurde jedes der 39 Proben verwendet chirurgischen Pankreas bis 40 serielle Kryoschnitten für einen Durchschnitt von 3 31&#39;544&#39;704 um Gewebe / Probe Pankreas (Bereich: 8&#39;742&#39;390 - 81&#39;522&#39;153 um 3) zu erzeugen wie in Tabelle...

Watch this Scientific Journal Video about Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens at JoVE.com

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Lasermikrodissektion ist eine Technik, die die Gewinnung von ausgewählten Zellen aus winzigen Mengen von Parenchym ermöglicht. Hier beschreiben wir ein Protokoll zum Erfassen menschlicher Pankreasinseln aus chirurgischen Proben für transkriptomischen Studien verwendet werden. Unser Protokoll verbessert die intrinsische Autofluoreszenz der menschlichen Beta-Zellen und erleichtert so ihre Sammlung.

Verbessertes Protokoll für Laser Mikrodissektion Of Human Pankreasinseln Von chirurgischen Proben

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

improved-protocol-for-laser-microdissection-human-pancreatic-islets

Research

JoVE Journal

Medicine

14.3K Aufrufe.  Paul Langerhans Institute Dresden. Lasermikrodissektion ist eine Technik, die die Gewinnung von ausgewählten Zellen aus winzigen Mengen von Parenchym ermöglicht. Hier beschreiben wir ein Protokoll zum Erfassen menschlicher Pankreasinseln aus chirurgischen Proben für transkriptomischen Studien verwendet werden. Unser Protokoll verbessert die intrinsische Autofluoreszenz der menschlichen Beta-Zellen und erleichtert so ihre Sammlung.

Serie: Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens

The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined by current drug efficacy tests and the efficacy on patients. Thus, novel and reliable methods for evaluating drug efficacy are warranted in pre-clinical phase. In vitro culture of tumor tissues, including cell lines, has substantial phenotypic, genetic, and epigenetic alterations of cancer cells caused by artificial environment of cell culture, which may not reflect the biology of original tumors in situ. Xenograft models with the immunodeficient mice also have limitations, i.e., the lack of immune system and interspecies genetic and epigenetic discrepancies in microenvironment. Here, we demonstrate a novel method using the surgical specimens of malignant glioma as undissociated tumor blocks to evaluate treatment effects. To validate this method, data with the current first-line chemotherapeutic agent, temozolomide (TMZ), are described.


We used the freshly-removed surgical specimen of malignant glioma for our experiments. We performed intratumoral injection of TMZ or other drug candidates, followed by incubation and analysis on surgical specimens. Here, we sought to establish a tumor tissue explant method as a platform to determine the efficacy of novel anti-cancer therapies so that we may be able to overcome, at least, some of the current limitations and fill the existing gap between the current experimental data and the efficacy on an actual patient's tumor. This method may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment.

Here, we established a method for drug efficacy testing with surgical specimens of brain tumors, termed &#x201C;tumor explant method&#x201D;. With this method, we can evaluate drug efficacy without breaking the microenvironment of solid tumors. To validate reliability of this method, we describe representative data with our glioma specimen treated with the current first-line chemotherapeutic agent, temozolomide.

Verfahren zur Novel Anti-Cancer Drug Development mit Tumor Explantate von chirurgischen Proben


The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined ...

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 ...

Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

Sammlung Protokoll zur menschlichen Pankreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

Staining Protocols for Human Pancreatic Islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. 
Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region.
The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&amp;E and IHC stains. 
The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.

This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&amp;E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.

Färbeprotokollen for Human Pankreasinseln

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2, immunological responses 3,4, hemodynamics 5,6, and small-for-size syndrome 7. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8 since the first model was described by Lee et al. 9 in 1973, the elimination of the hepatic arterial reconstruction 10 and the introduction of the cuff anastomosis technique by Kamada et al. 11 were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al., the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12 and the liver integrity 8,13,14.
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.

Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

Chirurgische Verfahren für eine Ratte Modell Teilweise Lebertransplantation mit arteriellen Rekonstruktion

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl

Retinal detachment (RD) describes a separation of the neurosensory retina from the retinal pigmented epithelium (RPE). The RPE is essential for normal function of the light sensitive neurons, the photoreceptors. Detachment of the retina from the RPE creates a physical gap that is filled with extracellular fluid. RD initiates cellular and molecular adverse events that affect both the neurosensory retina and the RPE since the physiological exchange of ions and metabolites is severely perturbed. The consequence for vision is related to the duration of the detachment since a rapid reapposition of the two tissues results in the restoration of vision 1. The treatment of RD is exclusively surgical. Removal of vitreous gel (vitrectomy) is followed by the removal non essential part of the retina around the detached area to favor retinal detachment. The removed retinal specimens are res nullius (nothing) and consequently normally discarded. To recover RNA from these surgical specimens, we developed the procedure jouRNAl that allows RNA conservation during the transfer from the surgical block to the laboratory. We also standardized a protocol to purify RNA by cesium chloride ultracentrifugation to assure that the purified RNAs are suitable for global gene expression analysis. The quality of the RNA was validated both by RT-PCR and microarray analysis. Analysis of the data shows a simultaneous involvement of inflammation and photoreceptor degeneration during RD.

We used retinal samples from retinectomy for a transcriptomic analysis of retinal detachment. We developed a procedure that allows RNA conservation between the surgical blocks and the laboratory. We standardized a protocol to purify RNA by cesium chloride ultracentrifugation to assure that the purified RNAs are suitable for microarray analysis.

Transkriptom-Analyse der menschlichen Retina chirurgischen Proben mit Journal

Retinal detachment (RD) describes a separation of the neurosensory retina  from the retinal pigmented epithelium (RPE). The RPE is essential for normal  ...

Optimization of High Grade Glioma Cell Culture from Surgical Specimens for Use in  Clinically Relevant Animal Models and 3D Immunochemistry

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell cultures that preserve the genomic profile of the parental tumors, for use in patient specific in vitro and in vivo models, has the potential to revolutionize the preclinical development of new treatments for glioblastoma tailored to the molecular characteristics of each tumor.
Starting with fresh high grade astrocytoma tumors dissociated into single cells, we use the neurosphere assay as an enrichment method for cells presenting cancer stem cell phenotype, including expression of neural stem cell markers, long term self-renewal in vitro, and the ability to form orthotopic xenograft tumors. This method has been previously proposed, and is now in use by several investigators. Based on our experience of dissociating and culturing 125 glioblastoma specimens, we arrived at the detailed protocol we present here, suitable for routine neurosphere culturing of high grade astrocytomas and large scale expansion of tumorigenic cells for preclinical studies. We report on the efficiency of successful long term cultures using this protocol&#160;and suggest affordable alternatives for culturing dissociated glioblastoma cells that fail to grow as neurospheres. We also describe in detail a protocol for preserving the neurospheres 3D architecture for immunohistochemistry. Cell cultures enriched in CSCs, capable of generating orthotopic xenograft models that preserve the molecular signatures and heterogeneity of GBMs, are becoming increasingly popular for the study of the biology of GBMs and for the improved design of preclinical testing of potential therapies.

Protocol for propagation of dissociated high grade glioma surgical specimens in serum-free neurosphere medium to select for cells with cancer stem cell phenotype. For specimens that fail to grow as neurospheres, an alternative protocol is suggested. A paraffin embedding technique for maintaining the 3D neurosphere architecture for immunocytochemistry is described.

Optimierung der hochwertigen Gliom-Zellkultur von chirurgischen Proben für den Einsatz in klinisch relevanten Tiermodellen und 3D-Immunchemie

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell ...

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell culture systems arising from human endometrial samples are no exception. Applications range from normal cyclic physiological processes to endometrial pathologies such as gynecological cancers, infectious diseases, and reproductive deficiencies. Here, we provide two methods for establishing primary endometrial stromal cells from surgically resected endometrial hysterectomy specimens. The first method is referred to as &#8220;the scraping method&#8221; and incorporates mechanical scraping using surgical or razor blades whereas the second method is termed &#8220;the trypsin method.&#8221; This latter method uses the enzymatic activity of trypsin to promote the separation of cells and primary cell outgrowth. We illustrate step-by-step methodology through digital images and microscopy. We also provide examples for validating endometrial stromal cell lines via quantitative real time polymerase chain reactions (qPCR) and immunofluorescence (IF).

Establishing primary endometrial stromal cell culture systems from hysterectomy specimens is a valuable biological technique and a crucial step prior to pursuing a vast array of research aims. Here, we describe two methods used to establish stromal cultures from surgically resected endometrial tissues of human patients.&#160;

Zwei Methoden zur Festlegung von primären humanen Endometrium Stromazellen aus Hysterektomie Proben

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell ...

Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types. Healthy prostate is comprised of fibromuscular stroma surrounding discrete epithelial-lined secretory lumens and a very small population of immune and neuroendocrine cells. In contrast, areas of prostate cancer have increased dysplastic luminal epithelium with greatly reduced or absent stromal population. Given the profound difference between stromal and epithelial cell types, it is imperative to separate the cell types for any type of downstream molecular analysis. Despite this knowledge, the bulk of gene expression studies compare benign prostate to cancer without micro-dissection, leading to stromal bias in the benign samples. Laser-capture micro-dissection (LCM) is an effective method to physically separate different cell types from a specimen section. The goal of this protocol is to show that RNA can be successfully isolated from LCM-collected human prostatic epithelium and used for downstream gene expression studies such as RT-qPCR and RNAseq.

The goal of this protocol is to use laser-capture micro-dissection as an effective method to isolate pure populations of cell types from heterogeneous prostate tissues for downstream RNA analysis.

Laser-Mikrodissektion für Menschen Prostataepithel für die RNA-Analyse

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types.  Healthy prostate is comprised of ...

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

As a functional status of microcirculation, microvascular vasomotion is important for the delivery of oxygen and nutrients and the removal of carbon dioxide and waste products. The impairment of microvascular vasomotion might be a crucial step in the development of microcirculation-related diseases. In addition, the highly vascularized pancreatic islet is adapted to support endocrine function. In this respect, it seems possible to infer that the functional status of pancreatic islet microvascular vasomotion might affect pancreatic islet function. Analyzing the pathological changes of the functional status of pancreatic islet microvascular vasomotion may be a feasible strategy to determine contributions that pancreatic islet microcirculation makes to related diseases, such as diabetes mellitus, pancreatitis, etc. Therefore, this protocol describes using a laser Doppler blood flow monitor to determine the functional status of pancreatic islet microvascular vasomotion, and to establish parameters (including average blood perfusion, amplitude, frequency, and relative velocity of pancreatic islet microvascular vasomotion) for evaluation of the microcirculatory functional status. In a streptozotocin-induced diabetic mouse model, we observed an impaired functional status of pancreatic islet microvascular vasomotion. In conclusion, this approach for assessing pancreatic islet microvascular vasomotion in vivo may reveal mechanisms relating to pancreatic islet diseases.

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

Pancreatic islet microvascular vasomotion regulates islet blood distribution and maintains the physiological function of islet &#946; cells. This protocol describes using a laser Doppler monitor to determine the functional status of pancreatic islet microvascular vasomotion in vivo and to assess the contributions of pancreatic islet microcirculation to pancreatic-related diseases.

Laser-Doppler: Ein Instrument zur Messung der Pankreas Islet mikrovaskuläre Vasomotion In Vivo

As a functional status of microcirculation, microvascular vasomotion is important for the delivery of oxygen and nutrients and the removal of carbon ...