Vogliamo ringraziare tutti i colleghi che hanno fornito aiuto, consiglio e contribuisce in modo fondamentale a vari passi di questo progetto. La produzione di questo articolo video è stato sostenuto con i fondi IMIDIA ( <a href="http://www.imidia.org" target="_blank">http://www.imidia.org</a> ), il Ministero tedesco dell&#39;Istruzione e della ricerca (BMBF) al Centro tedesco per la ricerca del diabete (DZD, <a href="http://www.dzd-ev.de" target="_blank">http://www.dzd -ev.de</a> ) e l&#39;Ospedale universitario Carl Gustav Carus presso l&#39;Università di Tecnologia di Dresda. Il lavoro ha portato a questa pubblicazione ha ricevuto il sostegno della iniziativa medicinali innovativi impresa comune ai sensi della convenzione di sovvenzione n ° 155005 (IMIDIA), le risorse di cui sono composte di partecipazione finanziaria del Settimo programma quadro dell&#39;Unione europea (FP7/2007-2013) e dall&#39;EFPIA società &#39;contributo di natura.

1. Congelamento di tessuto pancreatico umano <ol><li> Rimuovere il tessuto adiposo, vasi sanguigni, nervi e tessuto parenchimale non con un bisturi e pinzette, e tagliare il tessuto pancreatico in pezzi (~ 0,5-1 cubetti di cm). </li><li> Posizionare un pezzo tessuto pancreatico nel centro di un Cryomold, e coprire completamente con freddo Tissue-Tek Compound ottobre Mettere il Cryomold in un vaso il cui fondo è ricoperto di pre-raffreddato 2-metilbutano e snap-congelamento in azoto liquido. Conservare il campione congelato a -80 ° C. </li></ol> 2. Preparazione di sezioni congelate <ol><li> Indossare guanti durante tutte le misure per evitare la denaturazione di RNA. </li><li> Pulire il coltello criostato, portacampione e pennello con il freddo di etanolo 100% e RNase Away. Posizionare il tessuto congelato all&#39;interno del criostato. </li><li> Attendere che il, coltello e pennello tessuto raggiungere -20 ° C. Etichettare il SuperFrost Plus diapositive e pre-raffreddare dentro la camera di criostato in attesaper il tessuto di raggiungere -20 ° C. </li><li> Applicare il Tissue-Tek Compound PTOM porta campione e posizionare il tessuto congelato in alto. </li><li> Una volta che il Tissue-Tek OCT è congelato tagliare il cryoblock e rimuovere ogni eccesso di Tissue-Tek Compound ottobre con una lama di rasoio. </li><li> Tagliare un totale di 40 consecutivi 10 fette micron dal blocco di tessuto pancreatico. Inoltre si consiglia di tagliare una prima fetta e mezzo per essere salvati per i disegni panoramica della sezione del pancreas che può facilitare l&#39;individuazione di isole all&#39;interno delle fette durante il processo di microdissezione. </li><li> Trasferire la sezione della lama al centro di un vetrino SuperFrost Plus toccando il retro del vetrino con il dito (coperta da un guanto) per riscaldare solo la regione in cui la sezione deve aderire. </li><li> Asciugare il 1-2 min sezioni all&#39;interno del criostato a -20 ° C, dopo di che li metti in una scatola scorrevole posta in ghiaccio secco. Conservare le sezionia -80 ° C. </li><li> Se necessario, pulire la lama del coltello con tovaglioli di carta e fredda di etanolo al 100%. </li></ol> 3. Disidratazione delle sezioni congelate <ol><li> Preparare i reagenti: versare 30 ml preparata etanolo 70%, 30 ml di acqua trattata con DEPC, 2x30 ml di etanolo 100% e 30 ml di xilene in provette da 50 ml Cellstar (Falcons); freddo e conservare tutti i reagenti (tranne xilene) su ghiaccio. </li><li> Pulire lo spazio di lavoro sotto il cofano con il 100% di etanolo e RNaseZAP. </li><li> Processo due criosezioni come segue: fissare in ghiaccio freddo etanolo al 70% per 30 secondi, lavare con 5-6 rapidi cali in ghiaccio freddo DEPC-acqua trattata, disidratare due volte per 1 minuto in etanolo ghiacciato al 100%, incubare per 4 min in xilene a temperatura ambiente (25 ° C), e asciugare all&#39;aria per 3-5 min. Ripetere questa operazione con un altro paio di criosezioni. </li><li> Inserire due criosezioni secchi back to back in una provetta da 50 ml Cellstar avvolto con un foglio di alluminio per proteggerli dalla luce e dall&#39;umidità. </li></ol> &lt;p class = &quot;jove_title&quot;&gt; 4. Microdissezione laser di beta-cellule con PALM MicroBeam, Zeiss <ol><li> Pulire il microscopio con RNaseZAP e montare il tappo adesivo in RoboMover. </li><li> Impostare le seguenti impostazioni: set di filtri 09 (eccitazione: 450-490 nm, emissione&gt; 515 nm), AutoLPC modo, l&#39;energia laser del 50%, 65% fuoco del laser, laser 100% della velocità, distanza di 5 micron tra gli scatti AutoLPC, 6 distanza pm alla linea, altezza di lavoro: -10.100. </li><li> Inserire una diapositiva nello stadio. </li><li> Individuare le cellule beta autofluorescenti sotto visualizzazione microscopica (5x o 10x). </li><li> Passare alla lente 40x, selezionare le cellule beta con lo strumento &quot;mano libera&quot; di selezione e microdissect utilizzando il laser. </li><li> Cattura il tessuto di quattro criosezioni disidratati in una AdhesiveCap. Commento 1: il tempo per analizzare le cellule beta di un cryosection disidratato non deve superare i 10-20 minuti, per evitare la reidratazione delle sezioni di tessuto che porterebbe aRNA degrado. Commento 2: verificare la riuscita del processo di microdissezione mediante ispezione visiva dopo aver spostato il palco per il checkpoint. </li></ol> 5. Lisi di microdissezione tessuto cellulare Beta Enriched <ol><li> Rimuovere il AdhesiveCap dal RoboMover. </li><li> Pipettare 10 microlitri di tampone di estrazione (XB, PicoPure RNA Isolation Kit, Arcturus) nel coperchio del AdhesiveCap e incubare capovolta a 42 ° C per 30 min. </li><li> Centrifugare a 10.000 xg e mettere il lisato in ghiaccio secco. Conservarlo a -80 ° C fino a quando l&#39;estrazione dell&#39;RNA viene eseguita. </li><li> Ripetere i passaggi da 3.) A 5.) Con tutte le altre sezioni. </li></ol> 6. Estrazione RNA <ol><li> Unire tutti e dieci i lisati 10 pl </li><li> Procedere con l&#39;isolamento di RNA, lavorando a temperatura ambiente secondo il protocollo del RNA Isolation Kit PicoPure, Arturo, con un rapporto di 1:1 tampone di estrazione (XB) al 70% di etanolo (incluso il trattamento DNasi). </li></ol> 7. RNA Analisi <ol><li> Utilizzare 1 RNA pl per l&#39;analisi di qualità e quantità utilizzando un Agilent 2100 Bioanalyser (picoChip). Valutazione quantitativa viene effettuata in riferimento ad un RNA standard, caricati in parallelo sul chip. </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>

Nessun conflitto di interessi dichiarati.

Descriviamo un metodo affidabile per la microdissezione laser (LMD) di isole umane da un campione del pancreas chirurgico. A condizione che un microscopio LMD è disponibile, questa procedura potrebbe essere attuata in qualsiasi istituto di ricerca di eseguire pancreatectomies parziali, aumentando in tal modo l&#39;accesso al materiale umano da entrambe le isole non-diabetici e di tipo 2 soggetti diabetici. Questo è particolarmente rilevante data la scarsità di pancreas offerto per l&#39;isolamento delle isole. Aspett...

<table border="1"><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> 2-metilbutano (isopentano) </td><td> ROTH </td><td> 3.927,1 </td><td></td></tr><tr><td> AdhesiveCap (opaco, 500 pl) </td><td> ZEISS </td><td> 415190-9201-000 </td><td></td></tr><tr><td> Cellstar Tubes (50 ml) </td><td> Greiner Bio-One </td><td> 210 261 </td><td> con gonna supporto </td></tr><tr><td> Cellstar Tubes (50 ml) </td><td> Greiner Bio-One </td><td> 227,261 </td><td></td></tr><tr><td> Dietile pirocarbonato (DEPC) </td><td> SIGMA </td><td> D 5758 </td><td></td></tr><tr><td> Ghiaccio secco </td><td></td><td></td><td></td></tr><tr><td> Etanolo assoluto </td><td> VWR </td><td> 20821.310 </td><td></td></tr><tr><td> Azoto liquido </td><td></td><td></td><td></td></tr><tr><td> Pennello </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 embedding stampi (troncato), 12 x 12 mm </td><td> ProSciTech </td><td> RR12 </td><td> Top mm interno 22x22, profondità 21 millimetri </td></tr><tr><td> Arcturus PicoPure congelato RNA Isolation Kit </td><td> Applied Biosystems </td><td> KIT 0204 </td><td></td></tr><tr><td> Morsetto di plastica </td><td></td><td></td><td></td></tr><tr><td> Rasoio </td><td></td><td></td><td></td></tr><tr><td> RNase-Free DNasi 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 /lama chirurgica </td><td> Techno Taglio </td><td> 2800111 </td><td></td></tr><tr><td> SuperFrost Plus adesione microscopio diapositive </td><td> Thermo Scientific </td><td> J1800AMNZ </td><td> 25x75x1.0 millimetri </td></tr><tr><td> Tissue-Tek ottobre Compound </td><td> Sakura </td><td> 4583 o 0807000022 </td><td></td></tr><tr><td> Pinzetta </td><td> BRAUN </td><td> BD168R </td><td></td></tr><tr><td> Xilene </td><td> VWR </td><td> 28975.291 </td><td></td></tr></table>

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

Microdissezione laser (LMD) è una tecnica che consente il recupero di cellule e tessuti selezionati da piccole quantità di parenchima 1,2. Le cellule sezionato possono essere utilizzati per una varietà di indagini, come gli studi di proteomica, trascrittomica o di valutazione o analisi del DNA cromosomico 2,3. Un&#39;applicazione particolarmente impegnativa LMD è l&#39;analisi del trascrittoma, che, a causa della instabilità del RNA 4, può essere particolarmente importante quando le cellule vengono sezionati da tessuti che sono ricchi di RNasi, come il pancreas. Un protocollo microdissezione che consente una rapida identificazione e raccolta delle cellule bersaglio è essenziale in questa impostazione per abbreviare il tempo di movimentazione del tessuto e, di conseguenza, per garantire la conservazione dell&#39;RNA. Qui si descrive un protocollo per l&#39;acquisizione di cellule beta pancreatiche da campioni chirurgici di essere utilizzati per lo studio trascrittomica 5. Piccoli pezzi di pancreas di circa 0,5-1 cm 3 sono stati tagliati dai margini sani figuranti di campioni resecati pancreas, incorporati in Tissue-Tek Compound ottobre, immediatamente congelati in refrigerata 2-metilbutano, e conservati a -80 ° C fino al sezionamento. Quaranta sezioni seriali di spessore 10 um sono state tagliate su un criostato in un ambiente -20 ° C, trasferite individualmente i vetrini, essiccate all&#39;interno del criostato per 1-2 min, e conservati a -80 ° C. Immediatamente prima della procedura microdissezione laser, le sezioni sono state fissate in ghiaccio freddo, preparata etanolo al 70% per 30 sec, lavato da 5-6 cali di ghiaccio freddo acqua trattata con DEPC, e disidratato per un minuto due incubazioni in ghiaccio freddo 100% etanolo seguita da xilene (che viene usato per la disidratazione del tessuto) per 4 min, sezioni di tessuto sono state poi essiccate dopo per 3-5 min. È importante sottolineare che tutti i passi, eccetto l&#39;incubazione in xilene, sono stati eseguiti utilizzando ghiacciata reagenti - una modifica su un protocollo precedentemente descritto 6. Utilization di reagenti ghiaccio freddo ha determinato un aumento marcato della autofluorescenza intrinseca delle cellule beta, e facilitato il loro riconoscimento. Per microdissezione, quattro sezioni erano disidratati ogni volta: due sono stati posti in una pellicola protettiva tubo da 50 ml, per proteggere i tessuti da umidità e sbiancamento, gli altri due sono stati immediatamente microdissezione. Questa procedura è stata eseguita utilizzando un PALM MicroBeam strumento (Zeiss) che impiega il laser a pressione Auto catapultando (AutoLPC) modalità. Il completamento di cellule beta / dissezione isolotto da quattro criosezioni necessari non più di 40-60 min. Le cellule sono state raccolte in un unico AdhesiveCap e lisate con 10 microlitri di tampone di lisi. Ogni singolo campione di RNA per analisi trascrittomica stata ottenuta combinando 10 campioni cellulari microdissezione, seguita da estrazione RNA utilizzando il Pico Pure RNA Isolation Kit (Arcturus). Questo protocollo migliora l&#39;autofluorescenza intrinseca delle cellule beta umane, facilitando così la loro rilevazione rapida e precisa e Collezione. Ulteriore miglioramento di questa procedura potrebbe consentire la dissezione delle cellule beta fenotipicamente diversi, con possibili implicazioni per una migliore comprensione dei cambiamenti associati al diabete di tipo 2.

Come mostrato in figura 1, il protocollo modificato disidratante portato ad un miglioramento di autofluorescenza cellule beta rispetto al precedente protocollo pubblicato 6. L&#39;applicazione del protocollo descritto, ciascuno dei 39 campioni chirurgici pancreatiche è stato usato per generare 40 criosezioni seriali per una media di 31&#39;544&#39;704 micron 3 tessuto / pancreatico campione (range: 8&#39;742&#39;390 - 81&#39;522&#39;153 micron 3) come mostrato nella <st...

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

Microdissezione laser è una tecnica che consente il recupero di cellule scelte da piccole quantità di parenchima. Qui si descrive un protocollo per l&#39;acquisizione di isole pancreatiche umane da campioni chirurgici da utilizzare per gli studi di trascrittomica. Il nostro protocollo migliora l&#39;autofluorescenza intrinseca delle cellule beta umane, facilitando così la loro collezione.

Protocollo migliorato per microdissezione laser di isole pancreatiche umane da campioni chirurgici

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 Views.  Paul Langerhans Institute Dresden. Microdissezione laser è una tecnica che consente il recupero di cellule scelte da piccole quantità di parenchima. Qui si descrive un protocollo per l'acquisizione di isole pancreatiche umane da campioni chirurgici da utilizzare per gli studi di trascrittomica. Il nostro protocollo migliora l'autofluorescenza intrinseca delle cellule beta umane, facilitando così la loro collezione.

Video: 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.

Metodo per Novel Anti-Cancer Drug Development utilizzando Espianti Tumore di campioni chirurgici


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

Ricerca

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 di isole umane da pazienti parzialmente Pancreatectomized

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.

Protocollo di raccolta per pancreas umano

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.

Protocolli di colorazione di isole pancreatiche umane

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.

Procedure chirurgiche per un modello di Rat parziale trapianto di fegato con la ricostruzione arteriosa epatica

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.

Analisi trascrittomica di campioni chirurgici retiniche umane Utilizzo ufficiale

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.

Ottimizzazione della coltura cellulare glioma di alto grado da campioni chirurgici per l'uso in modelli animali clinicamente rilevanti e immunochimica 3D

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;

Due metodi per stabilire primarie cellule umane endometriale stromali da isterectomia campioni

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-capture Microdissezione di Human prostatica Epitelio per l&#39;analisi di RNA

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: Uno strumento per la misurazione dell'isolotto pancreatico Vasomotion microvascolare In Vivo

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