Gli autori ringraziano le famiglie dei donatori e le Organizzazioni Organ Procurement coinvolti in questa ricerca e di Maria Martino e Irina Kusmartseva per la loro assistenza di un esperto. Questo lavoro è stato finanziato dalla Juvenile Diabetes Research Foundation (MC-T., MA) a sostegno della rete per i donatori di organi del pancreas con diabete.

1. Set-up <ol><li> Cassette etichette per i blocchi di paraffina manualmente con una matita, o automaticamente con una stampante cassette. Includere l&#39;identificatore donatore (CaseID), tipo di campione, il numero di aliquote, e qualsiasi ulteriore identificazione univoco (foglio di lavoro Case, <a href="http://jove.com/files/ftp_upload/4039/4039appendix1.doc" target="_blank">appendice 1</a> ). </li><li> Label stampi PTOM come per cassette con un pennarello indelebile. Ritagliare rettangoli di cm 5-10 di fogli di alluminio. </li><li> Stampa o manualmente un&#39;etichetta con pennarello indelebile tutti i flaconi e tubi. Includere fiale sufficienti per i campioni congelati a scatto tritati e tubi con i media RPMI per spedizioni freschi. Per le fiale a scatto congelati con RNAlater, aggiungere 1 mL RNAlater ad ogni esageratamente. </li><li> Set-up schede dissezione in materia di biosicurezza cappa e piani di lavoro con strumenti di dissezione sterilizzati (pinze, bisturi, lame, compresse di garza (4x4)) così come cassette e muffe PTOM disposti in ordine di utilizzo. </li><li> Preparare un bagno di congelamento mettendoghiaccio secco in un mezzo scatola dimensioni polistirolo (10x10 cm) ad una profondità di almeno 3 cm e aggiungere isopentano finché il ghiaccio secco è completamente coperto. </li><li> Opzionale: Riempire un piccolo contenitore Dewar con azoto liquido. </li><li> Se il pancreas è necessario per la microscopia elettronica (EM), preparazione fissativo TAAB con l&#39;aggiunta di 1,25 mL di paraformaldeide 16%, 1,25 mL glutaraldeide 8%, e 7,5 mL 0,1 M PBS. Preparare fissativo Lowicryl con l&#39;aggiunta di 2,5 mL di paraformaldeide 16% a 7,5 mL 0,1 M PBS. </li><li> Preparare soluzioni impiegate per isolare cellule. Per flacone da 500 ml del DMEF (con glutammina), seguite tecniche asettiche, mentre aggiungendo quanto segue: <ol><li> Siero fetale bovino (FBS): aggiungere 50 ml, 10% finale. </li><li> Penicillina / Streptomicina (Pen / Strep, 10.000 U / mL / 10.000 ug / mL stock) - aggiungere 5 ml Pen / Strep stock RPMI per la concentrazione finale di 100 U / mL / 100ug/mL. Anche aggiungere 5 ml in un flacone da 500 ml di DPBS quando viene utilizzato per lo svolgimento di linfonodi o campioni milza. </li></ol></li></ol> 2. Personale <ol&gt; <li> Procedura è ottimamente realizzato con tre membri dello staff Tuttavia, la procedura può essere condotte con un individuo ben addestrato. Due individui possono completare la procedura generalmente in 90 minuti. Durata effettiva dipende dal numero di tessuti ricevuti e la facilità di trovare linfonodi pancreatici. I campioni per isolamenti cellule sono tenuti a 4 ° C fino al completamento della dissezione tessuto mentre campioni di siero sono elaborati in parallelo. </li><li> Patologo, assistente di patologia, o equivalente, un membro del personale esperto conduce la manipolazione dei tessuti e svolge dissezioni importanti, tra cui la divisione (s) della testa del pancreas e di raccolta dei linfonodi pancreatici. Gli sforzi iniziali sono diretti verso il pancreas seguita da esame di peri-pancreatica grasso per linfonodi. </li><li> Tecnico di laboratorio o equivalente-membro del personale addestrato nel tessuto taglio la cui prima responsabilità sono a pesare pancreas e le regioni e tessuti assetto per blocchi fissi e congelati e surgelati a scatto vIALS. Riceve duodeno e milza da assistente patologia ed elabora tali organi mentre l&#39;assistente patologia è l&#39;elaborazione del pancreas. </li><li> Assistente di laboratorio-membro del personale che assiste gli altri tra cui blocco e l&#39;etichettatura vial, il campione per tritare cryovials, e la trasformazione del siero. </li></ol> 3. Pancreas Dissection <ol><li> Rimuovere il duodeno e la milza e pulire il pancreas di grassi estranei, vasi e nervi con dissezione smussa e taglienti </li><li> Posizionare il pancreas pulito su una tavola di dissezione coperta con carta da cucina. Immergere un bastoncino di cotone con punta applicatore in inchiostro blu e la diffusione sulla superficie anteriore del pancreas. Asciugare l&#39;eccesso. </li><li> Optional: Immergere un nuovo applicatore in inchiostro nero e la diffusione sulla superficie posteriore del pancreas. Torna pancreas all&#39;orientamento normale con superficie anteriore rivolta verso prosector. </li><li> Tagliare il pancreas in 3 regioni: testa, corpo e coda (Figura 1). Tagliare la giunzione tra HEAd corpo e poi dividere la parte restante in ~ parti uguali per il corpo e coda. </li><li> Pesare ogni regione di pesi pancreas e registrare (Causa foglio workup ( <a href="http://jove.com/files/ftp_upload/4039/4039appendix1.doc" target="_blank">appendice 1</a> )). </li><li> Sezione (circa 0,5 cm) ogni regione del pancreas in una trasversale &quot;pagnotta&quot; modo (Figura 1). </li><li> Utilizzare le sezioni di entrambe le giunzioni tra le regioni per i campioni tritate. A seconda delle dimensioni pancreas, un campione dal corpo-coda regione di giunzione può essere difficile da ottenere in tal caso il numero di blocchi di paraffina può essere diminuita per ottenere campioni macinati o campioni solo la regione pancreas testa corpo può essere ottenuta. Cryovials un&#39;etichetta in base a testa o il corpo per indicare regione prossimale a livello della giunzione. <ol><li> Tritate pezzi e si dividono equamente tra cryovials (≥ 1 grammo per flacone). Rapidamente congelare fiale senza RNAlater in azoto liquido o ghiaccio secco - slurry isopentano poi il trasferimento toa -80 ° C freezer. </li><li> Tenere flaconcini con RNAlater a temperatura ambiente per 30 minuti per consentire equilibrio, remix e rapidamente congelare in azoto liquido o in ghiaccio secco-isopentano impasto poi trasferire in un congelatore a -80 ° C. </li></ol></li><li> Rimuovere le sezioni alterne e blocchi di paraffina OCT che iniziano con paraffina (Figura 1). </li><li> Appoggiare tutte le sezioni trasversali a destra. </li><li> Luogo ~ 1,5 x 1,5 x 0,5 cm sezioni nelle cassette. Se le sezioni trasversali sono troppo grandi, tagliata a metà (Figura 2b). Cassette di etichette per il blu metà anteriore come &quot;A&quot; e la metà posteriore, &quot;B&quot;. </li><li> Se i pezzi sono ancora troppo grandi, tagliare ciascuna sezione perpendicolare al precedente taglio (figura 2c). Cassette Label AD in senso orario. Tagliare pezzi ulteriormente necessarie per adattarsi all&#39;interno di cassette. </li><li> Serie di numeri in sequenza all&#39;interno della regione, a partire dalla parte più mediale. </li><li> Per i blocchi di paraffina, la posizione cassettes così l&#39;etichetta è a sinistra e posto tessuto nel cassetto, mantenendo il suo orientamento. Chiudere il coperchio cassetta e trasferimento in un contenitore con 500 ml ~ 10% formalina tamponata neutra (NBF). Avviare la fissazione tempi quando la cassetta ultimo posto in fissativo. </li><li> Per i blocchi PTOM, versare uno strato sottile di ottobre i media nello stampo prelabeled poi stendere il tessuto nello stampo facendo attenzione a mantenere l&#39;orientamento e coprire con il tessuto ottobre Rapidamente immergere gli stampi in un PTOM ghiaccio secco - slurry isopentano per ~ 1 minuto. Togliere dal liquame e il luogo in ghiaccio secco per lo stoccaggio temporaneo o in freezer -80 ° C per una conservazione a lungo termine. </li><li> Ottenere ~ 0.5 x 0.5 cm fette dalla testa-corpo di giunzione per la microscopia elettronica (EM). Mettere una fetta nel fissativo TAAB e l&#39;altra fetta del fissativo Lowicryl. Conservare entrambi i tipi di fissativo con i campioni a 4 ° C per almeno 48 ore e / o a tempo indeterminato fino al procedimento successivo per EM. </li></ol> 4. Milza, dissezione linfonodalezioni e dissezioni Mucosa duodenale <ol><li> Isolare linfonodi pancreatici (PLN) da grassi e tagliare tutto il tessuto connettivo alla capsula nodo. Mettere in un piatto sterile coltura cellulare contenente DMEF. A seconda del numero totale isolati, dividere PLN in aliquote per le spedizioni fresche, isolamenti cellulari crioconservati, paraffina e / o blocchi PTOM, e fiale. Tritare i nodi di grandi dimensioni in pezzi non più grandi di 0,5 cm. </li><li> Tritare non-pancreatiche linfonodi e nella milza in piccoli (circa 0,5 cm) cubi. Luogo in fissativo, ottobre media, e per le spedizioni o DMEM campioni freschi o crioconservati isolamenti cellulari in base alle quantità di materie prime. </li><li> Aprire il duodeno e pulire delicatamente la superficie della mucosa pulita di muco con un tampone di garza inumidito. Tagliare sezioni della mucosa di paraffina e / o blocchi congelati PTOM e campioni macinate per flaconi, con e senza RNAlater. Congelare come per altri campioni. </li></ol> 5. Wrap blocchi PTOM in pre-etichettati foglio di alluminio e di collocare i blocchi PTOM e<html> fiale in scatole per la conservazione a lungo termine a -80 ° C. 6. Inviare i campioni fissati per l&#39;elaborazione di paraffina <ol><li> Fissare i tessuti per 16 ore (range 20 ± 4 ore) con un elaboratore automatico di paraffina o con cronometraggio manuale. Processo per i blocchi di paraffina usando un elaboratore automatico ( <a href="http://jove.com/files/ftp_upload/4039/4039appendix2.doc" target="_blank">Appendice 2</a> ). </li><li> Incorporare sezioni nell&#39;orientamento esatto immesso dal prosector con l&#39;etichetta cassetta a sinistra. Incorporare la mucosa duodenale con la superficie di taglio verso il basso per consentire l&#39;esame della mucosa perpendicolare alla sottomucosa. </li></ol> 7. Pulire e disinfettare le zone di dissezione. Lavare gli strumenti di dissezione e ri-sterilizzare. Posizionare i tessuti rimanenti umani in fissativo formalina per lo stoccaggio di rifiuti biomedico. Smaltire i rifiuti biomedica conformità alla normativa vigente entro un mese. 8. Aggiornare il sistema di inventario del campione ve_title &quot;&gt; 9. rappresentativi Risultati Questa procedura consente di elaborare un pancreas umano con intatta campione rappresentativo per tutta l&#39;organo tra cui delimitazione delle tre regioni principali, vale a dire, testa, corpo e coda entro 2 ore. La regione uncinato, che si trova nella regione posteriore della testa, è inclusa nella dissezione testa. Il peso medio del pancreas in donatori di organi senza diabete era 82.4 grammi (52,7 - 139,0 grammi). Pesi pancreas regionali sono approssimativamente uguali (figura 3). Questa procedura può anche permettere per la ricostruzione del pancreas intero utilizzando vetrini colorati da paraffina sequenziale e blocchi PTOM. Più formati sono possibili consentendo il massimo utilizzo per le tecnologie attuali e future. Il foglio di lavoro corrente nPOD caso è previsto ( <a href="http://jove.com/files/ftp_upload/4039/4039appendix1.doc" target="_blank">appendice 1</a> ) che viene utilizzato per documentare i campioni recuperati e proc successivaessing aliquote per tipo e quantità. <img alt="Figura 1" src="/files/ftp_upload/4039/4039fig1.jpg" /> Figura 1. Schema generale della procedura. L&#39;intero pancreas viene elaborato, pur mantenendo orientamenti anatomiche. La regione testa è più lunga nella direzione superiore-inferiore a causa della presenza del lobo ventrale pancreatico nella regione posteriore che comprende la regione uncinato. Zona tratteggiata in corrispondenza dei nodi indicano regioni utilizzate per i campioni in cryovials tritate. P, paraffina, O, ottobre <img alt="Figura 2" src="/files/ftp_upload/4039/4039fig2.jpg" /> Figura 2. Schema di Lettering per le sezioni del pancreas. Fette trasversali pancreas può essere ulteriormente divisa in due metà o quarti e lettere in senso orario. <img alt="Figura 3" src="/files/ftp_upload/4039/4039fig3.jpg" /> Figura 3. Pesi pancreas di donatori di organi senza diabete. Ipancreas intatta da donatori adulti (&gt; 17 anni) è stato pesato poi diviso in regioni e pesi regionali ottenuti. I dati sono medie ± SEM (n = 15).

<ol>
	<li>Brissova, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+human+pancreatic+islet+architecture+and+composition+by+laser+scanning+confocal+microscopy.">Assessment of human pancreatic islet architecture and composition by laser scanning confocal microscopy.</a> J. Histochem. Cytochem. 53, 1087-1097 (2005).</li><li>Rahier, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pancreatic+polypeptide+cells+in+the+human+pancreas:+the+effects+of+age+and+diabetes.">The pancreatic polypeptide cells in the human pancreas: the effects of age and diabetes.</a> J. Clin. Endocrinol. Metab. 56, 441-444 (1983).</li><li>Uchida, T., Takada, T., Ammori, B. J., Suda, K., Takahashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+reconstruction+of+the+ventral+and+dorsal+pancreas:+a+new+insight+into+anatomy+and+embryonic+development.">Three-dimensional reconstruction of the ventral and dorsal pancreas: a new insight into anatomy and embryonic development.</a> J. Hepatobiliary Pancreat. Surg. 6, 176-180 (1999).</li><li>Wittingen, J., Frey, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+concentration+in+the+head,+body,+tail+and+uncinate+process+of+the+pancreas.">Islet concentration in the head, body, tail and uncinate process of the pancreas.</a> Ann. Surg. 179, 412-414 (1974).</li><li>Rahier, J., Goebbels, R., Henquin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+composition+of+the+human+diabetic+pancreas.">Cellular composition of the human diabetic pancreas.</a> Diabetologia. 24, 366-371 (1983).</li><li>Vaught, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+NCI+perspective+on+creating+sustainable+biospecimen+resources.">An NCI perspective on creating sustainable biospecimen resources.</a> J. Natl. Cancer Inst. Monogr. 42, 1-7 (2011).</li><li>Gianani, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+results+of+screening+of+nondiabetic+organ+donors+for+expression+of+islet+autoantibodies.">Initial results of screening of nondiabetic organ donors for expression of islet autoantibodies.</a> J. Clin. Endocrinol. Metab. 91, 1855-1861 (2006).</li><li>Tauriainen, S., Salmela, K., Rantala, I., Knip, M., Hy&#246;ty, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collecting+high-quality+pancreatic+tissue+for+experimental+study+from+organ+donors+with+signs+of+&#946;-cell+autoimmunity.">Collecting high-quality pancreatic tissue for experimental study from organ donors with signs of &#946;-cell autoimmunity.</a> Diabetes Metab. Res. Rev. 26, 585-592 (2010).</li><li>In't Veld, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+insulitis+in+adult+autoantibody-positive+organ+donors.">Screening for insulitis in adult autoantibody-positive organ donors.</a> Diabetes. 56, 2400-2404 (2007).</li><li>T&#246;ns, H. A., Terpstra, O. T., Bouwman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+human+pancreata+in+perspective+of+the+isolation+of+the+islets+of+langerhans.">Heterogeneity of human pancreata in perspective of the isolation of the islets of langerhans.</a> Transplant Proc. 40, 367-369 (2008).</li></ol>

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L&#39;obiettivo di questa procedura è quello di definire uno standard di trattamento del pancreas che può essere armonizzato in tutta siti di raccolta multiple e consentire in tal modo il confronto dei risultati ottenuti con diversi gruppi. Il metodo di dissezione è basata su standard, pratiche di Patologia Chirurgica, con ulteriori passaggi per l&#39;annotazione delle regioni più importanti del pancreas seguita da intra-regionali suddivisioni. Standardizzazione dei metodi di trasformazione biospecimen è necessario...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> Strumenti di dissezione, inclusi bisturi con 22 lame </td><td> Vario </td><td> Vario </td><td></td></tr><tr><td> Provette (15 e 50 ml) </td><td> Fisher Scientific </td><td> 05-539-5, 430.828 </td><td> Sterile </td></tr><tr><td> Criotubi (1,8 ml) </td><td> VWR Nord America </td><td> 89004-300 </td><td> Sterili, codici a barre opzionale </td></tr><tr><td> RNAlater </td><td> Ambion </td><td> AM7021 </td><td></td></tr><tr><td> Patologia Chirurgica Ink </td><td></td><td></td><td></td></tr><tr><td> Cassette </td><td> Mercedes Medical </td><td> CAS ECO109 </td><td></td></tr><tr><td> 10% formalina tamponata neutra </td><td> Fisher Scientific </td><td> 23-245-685 </td><td></td></tr><tr><td> PTOM congelamento media </td><td> TissueTek </td><td> 4583 </td><td></td></tr><tr><td> Stampi </td><td> ThermoScientific </td><td> 58952 </td><td> Varie dimensioni </td></tr><tr><td> Isopentano </td><td> Fisher Scientific </td><td> O3551-4 </td><td> Ottobre bagno agghiacciante </td></tr><tr><td> Il ghiaccio secco </td><td></td><td></td><td> Ottobre bagno agghiacciante </td></tr><tr><td> L&#39;azoto liquido in vaso Dewar </td><td></td><td></td><td> Snap cryovials congelamento </td></tr><tr><td> Piatto di coltura cellulare (100mm) </td><td> Corning </td><td> 430167 </td><td> Sterile </td></tr><tr><td> Hyclone DMEF </td><td> ThermoScientific </td><td> SH30023.01 </td><td> Sterile, conservare in frigorifero, glutammina inclusa </td></tr><tr> &lt;td&gt; siero fetale bovino </td><td> Cellgro </td><td> 35-010-CV </td><td> Sterili, aliquote congelare </td></tr><tr><td> Penicillina / streptomicina / antifungina </td><td> GIBCO </td><td> 15240-062 </td><td> Sterili, aliquote congelare </td></tr><tr><td> Tipo 9 paraffina </td><td> Richard-Allan Scientific </td><td> 8337 </td><td></td></tr><tr><td> 1X D-PBS </td><td> GIBCO </td><td> 14190-144 </td><td> Sterile </td></tr><tr><td> 16% paraformaldeide </td><td> EM Scienze </td><td> 15710 </td><td> Fai fresco, refrigerato negozio </td></tr><tr><td> 8% glutaraldeide </td><td> EM Scienze </td><td> 16019 </td><td> Fai fresco, refrigerato negozio </td></tr><tr><td> Livello di biosicurezza 2 cappuccio </td><td> Vario </td><td></td><td></td></tr><tr><td> Ultra-low freezer </td><td> Vario </td><td></td><td></td></tr><tr><td> Cassette stampante Sakura </td><td> Sakura </td><td></td><td></td></tr><tr><td> Sakura VIP300 </td><td> Sakura </td><td></td><td> Paraffina elaboratore automatico </td></tr><tr><td> Paraffina Embedding Stazione </td><td> ThermoFisher </td><td></td><td></td></tr></tbody></table> Tabella 1. Reagenti e attrezzature specifiche.

collection protocol for human pancreas

La dissezione e procedura di campionamento è stato sviluppato per la Rete per i donatori di organi del pancreas con diabete (nPOD) programma di standardizzare la preparazione del pancreas recuperati da donatori di organi da cadavere. Il pancreas è diviso in 3 regioni principali (testa, corpo, coda) seguiti da sezioni trasversali seriali in tutto il mediale all&#39;asse laterale. Sezioni alternate sono utilizzati per paraffina fisso e blocchi congelati freschi e campioni rimanenti vengono macinate per moschettoni preparati campioni congelati, con o senza RNasi inibitori, per DNA, RNA, o isolamento delle proteine. L&#39;obiettivo generale della procedura di dissezione pancreas è quello di campionare l&#39;intero pancreas, pur mantenendo l&#39;orientamento anatomico. Eterogeneità delle cellule endocrine in termini di composizione delle isole, le dimensioni e il numero è riportato per isole umane rispetto alle isole roditori 1. La maggior parte delle isole pancreatiche dalla testa pancreas, regioni di body e di coda sono costituiti da insulino-containing cellule beta seguita da minori proporzioni di glucagone contenenti a-cellule e somatostatina-contenenti delta-cellule. Polipeptide pancreatico contenenti cellule PP e grelina contenenti cellule epsilon sono presenti, ma in piccole quantità. Al contrario, la regione uncinato contiene isolotti che sono principalmente composti di polipeptide pancreatico cellule contenenti PP 2. Queste variazioni isole regionali derivano dalle differenze di sviluppo. Il pancreas sviluppa dai germogli pancreatiche ventrale e dorsale del foregut e dopo la rotazione dello stomaco e del duodeno, si muove lobo ventrale e si fondono con la dorsale 3. Il lobo ventrale costituisce la parte posteriore della testa compreso il processo uncinato mentre il lobo dorsale dà luogo al resto dell&#39;organo. Variazione del pancreas regionale è riportato anche con la regione della coda avente una densità isolotto superiore rispetto ad altre regioni e le dorsali del lobo componenti derivati ​​dalla fase di atrofia selettiva nel diabete tipo 1 <sup&gt; 4,5. Altri organi e tessuti sono spesso recuperato dai donatori di organi e includono i linfonodi, della milza e pancreas non-pancreatiche linfonodi. Questi campioni sono recuperati con formati analoghi a quelli per il pancreas con l&#39;aggiunta di isolamento di cellule crioconservate. Quando il duodeno prossimale è incluso con il pancreas, mucosa duodenale possono essere raccolti per i blocchi di paraffina e congelati e preparati a scatto tritati surgelati.

Watch this Scientific Journal Video about Collection Protocol for Human Pancreas at JoVE.com

Collection Protocol for Human Pancreas

Questo video mostra una procedura di dissezione per l&#39;elaborazione di pancreas umano in diversi formati di memorizzazione. Orientamento anatomico viene mantenuta in tutte le regioni del pancreas per consentire la definizione della composizione delle isole regionale e la densità.

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 ...

Research

JoVE Journal

Medicine

14.7K Views.  University of Florida. Questo video mostra una procedura di dissezione per l'elaborazione di pancreas umano in diversi formati di memorizzazione. Orientamento anatomico viene mantenuta in tutte le regioni del pancreas per consentire la definizione della composizione delle isole regionale e la densità.

Video: Collection Protocol for Human Pancreas

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

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

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 ...

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Medicina

Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.

The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays.

Raccolta, isolamento e analisi citofluorimetrica di campioni endocervicali umano

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female ...

A Detailed Protocol for Perspiration Monitoring Using a Novel, Small, Wireless Device

Perspiration monitoring can be utilized for the detection of certain diseases, such as thermoregulation and mental disorders, particularly when the patients are unaware of such disorders or are having difficulty expressing their symptoms. Until now, several devices for perspiration monitoring have been developed; however, such devices tend to have a relatively large exterior, considerable power consumption, and/or less sensitivity.
Recently, we developed a small, wireless device for perspiration monitoring. The device consists of a temperature/relative humidity (T/RH) sensor, battery-driven small data logger, and silica gel as a desiccant in a small cylindrical exterior. The T/RH sensor is placed between the detection windows (through which the water vapor from the skin enters) and the silica gel. The underlying principle of the perspiration monitoring device is based on Fick&#39;s law of diffusion, which means that water vapor flux from the skin to the silica gel (i.e.&#160;transepidermal water loss and perspiration) can be captured by change in humidity at the T/RH sensor. In addition, a baseline subtraction method was adopted to distinguish perspiration and transepidermal water loss.
As shown in the previous report, the developed device can monitor the perspiration at any sites of the body in an easy, wireless manner. However, detailed methods of how to use the device have not been disclosed yet. In this article, therefore, we would like to show the point-by-point tutorials of how to use the device for perspiration monitoring, by showing the sympathetic activity test with the sympathetic skin response monitoring as an example.

Recently, we developed a small wireless device for perspiration monitoring. In this article, we present detailed protocols on how to use the device for perspiration monitoring with an example of the sympathetic activity test.

Un protocollo dettagliato per la sudorazione monitoraggio Utilizzando un romanzo, Piccolo, dispositivo wireless

Perspiration monitoring can be utilized for the detection of certain diseases, such as thermoregulation and mental disorders, particularly when the ...

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the availability of the widest range of molecular probes and reagents to perform in vivo as well as in vitro studies. Based on our experience with various murine transplantation models, we developed a heterotopic pancreas transplantation model in mice with the intent to analyze mechanisms underlying severe ischemia reperfusion injury-associated early graft damage. In contrast to previously described techniques using suture techniques, herein we describe a new procedure using a non-suture cuff technique. 
In recent years, we have performed more than 300 pancreas transplantations in mice with an overall success rate of &gt;90%, a success rate never described before in mouse pancreas transplantation. The backbone of this non-suture cuff technique for graft revascularization consists of two major steps: (I) pulling the recipient vessel over a polyethylene/polyamide cuff and fixing it with a circumferential ligature, and (II) placing the donor vessel over the everted recipient vessel and fixing it with a second circumferential ligature. The resultant continuity of the endothelial layer results in less thrombogenic lesions with high patency rates and, finally, high success rates.
In this model, arterial anastomosis is achieved by pulling the abdominal aorta of the donor graft over the everted common carotid artery of the recipient animal. Venous drainage of the graft is achieved by pulling the portal vein of the graft over the everted external jugular vein of the recipient. This manuscript provides details and crucial steps of the organ recovery and organ implantation procedures, which will allow researchers with microsurgical skills to perform the transplantation successfully in their laboratories.

Among abdominal solid organ transplantation, pancreatic grafts are prone to develop severe ischemia reperfusion injury-associated graft damage, leading eventually to early graft loss. This protocol describes a model of murine pancreas transplantation using a non-suture cuff technique, ideally suited for analyzing these early, deleterious damages.

Modello di topo per il trapianto di Pancreas usando una tecnica modificata polsino

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the ...

Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at the 12 codon (Ptf1acre/+;LSL-KrasG12D/+) mouse strain as a model of human pancreatic cancer. The goal of our current studies is to identify novel metabolic biomarkers of pancreatic cancer progression. We have performed metabolic profiling of urine, feces, blood, and pancreas tissue extracts, as well as histological analyses of the pancreas to stage the cancer progression. The mouse pancreas is not a well-defined solid organ like in humans, but rather is a diffusely distributed soft tissue that is not easily identified by individuals unfamiliar with mouse internal anatomy or by individuals that have little or no experience performing mouse organ dissections. The purpose of this article is to provide a detailed step-wise visual demonstration to guide novices in the removal of the mouse pancreas by dissection. This article should be especially valuable to students and investigators new to research that requires harvesting of the mouse pancreas by dissection for metabolic profiling or histological analyses.

This video article provides a detailed demonstration of the procedures required to successfully remove the pancreas from a mouse by dissection for histological analysis and metabolic profiling.

Dissezione del Pancreas del Mouse per l'analisi istologica e Profiling metabolico

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at ...

Robotic Lateral Pancreaticojejunostomy for Chronic Pancreatitis

Lateral pancreaticojejunostomy (LPJ) has shown good postoperative outcomes in patients with painful, morphine dependent, chronic pancreatitis (CP). The recent rise of robotic and laparoscopic pancreatic surgery has found benefits such as reduced time to functional recovery. Few studies have reported on the feasibility, technique and outcome of robotic LPJ, especially including transection of the gastroduodenal artery. The present study describes the main steps for robotic LPJ in a patient with painful chronic pancreatitis with a dilated main pancreatic duct. The patient underwent robotic LPJ. The LPJ anastomosis is performed using a running suture technique in a longitudinal side-to-side manner. Routinely, the gastroduodenal artery is transected to drain the entire length of the main pancreatic duct. The patient is in French position; 7 trocars are placed (4 robotic, 2 laparoscopic assistants, 1 liver-retractor). After docking of the robot system, the omental bursa is opened, and the right gastroepiploic artery and vein are ligated at their base at the lower border of the pancreas. Intraoperative ultrasonography is performed in order to determine trajectory of the dilated main pancreatic duct which is opened for its entire length after the gastroduodenal artery has been suture ligated both cranially and caudally from the main pancreatic duct. A Roux limb is created, and a latero-lateral PJ is fashioned using several 3-0 barbed sutures. A stapled jejuno-jejunostomy is created at sufficient distance from the pancreatic anastomosis, aided by a 50 cm suture. The described technique for robotic LPJ is a complex but feasible operation for patients with treatment refractory CP and a dilated main pancreatic duct. Due to its complexity, implementation in high volume centers with extensive experience with CP surgery may improve outcomes.

Robotic lateral pancreaticojejunostomy (RLPJ) may be used in patients with painful, morphine dependent, chronic pancreatitis and a dilated main pancreatic duct. We describe a standardized and reproducible technique for RLPJ, which includes transection of the gastroduodenal artery.

Pancreaticojejutostomia laterale robotica per la pancreatite cronica

Lateral pancreaticojejunostomy (LPJ) has shown good postoperative outcomes in patients with painful, morphine dependent, chronic pancreatitis (CP). The ...

Robot-Assisted Kidney Transplantation

This paper describes robot-assisted kidney transplantation (RAKT) from a living donor. The robot is docked between the parted legs of the patient, placed in the supine Trendelenburg position. Kidney allografts are provided by a living donor. Before vascular anastomosis, the kidney allograft is prepared by inserting a double-J stent in the ureter, and the temperature for the anastomosis is lowered by wrapping it in an ice-packed gauze. A 12 mm or 8 mm port for the robotic camera and three 8 mm ports for robotic arms are placed. A peritoneal pouch is created for the kidney allograft by raising the peritoneal flaps on both sides over the psoas muscle before dissecting the iliac vessels and bladder. A 6 cm Pfannenstiel incision is made to insert the kidney into the peritoneal pouch, lateral to the right iliac vessels. 
After clamping the external iliac vein with Bulldogs clamps, a venotomy is performed, and the graft renal vein is anastomosed to the external iliac vein in an end-to-side continuous manner with a 6/0 polytetrafluoroethylene suture. After clamping the graft renal vein, the iliac vein is declamped. This is followed by clamping of the external iliac artery, arteriotomy, arterial anastomosis with a 6/0 polytetrafluoroethylene suture, clamping of the graft renal artery, and declamping of the external iliac artery. Reperfusion is then carried out, and ureteroneocystostomy is performed using the Lich-Gregoir technique. The peritoneum is closed at a few locations with polymer locking clips, and a closed-suction drain is placed through one of the working ports. After deflating the pneumoperitoneum, all incisions are closed.

This paper provides technical details for robot-assisted kidney transplantation from a living donor.

Trapianto di rene assistito da robot

This paper describes robot-assisted kidney transplantation (RAKT) from a living donor. The robot is docked between the parted legs of the patient, placed ...

Collection, Expansion, and Differentiation of Primary Human Nasal Epithelial Cell Models for Quantification of Cilia Beat Frequency

Measurements of cilia function (beat frequency, pattern) have been established as diagnostic tools for respiratory diseases such as primary ciliary dyskinesia. However, the wider application of these techniques is limited by the extreme susceptibility of ciliary function to changes in environmental factors e.g., temperature, humidity, and pH. In the airway of patients with Cystic Fibrosis (CF), mucus accumulation impedes cilia beating. Cilia function has been investigated in primary airway cell models as an indicator of CF Transmembrane conductance Regulator (CFTR) channel activity. However, considerable patient-to-patient variability in cilia beating frequency has been found in response to CFTR-modulating drugs, even for patients with the same CFTR mutations. Furthermore, the impact of dysfunctional CFTR-regulated chloride secretion on ciliary function is poorly understood. There is currently no comprehensive protocol demonstrating sample preparation of in vitro airway models, image acquisition, and analysis of Cilia Beat Frequency (CBF). Standardized culture conditions and image acquisition performed in an environmentally controlled condition would enable consistent, reproducible quantification of CBF between individuals and in response to CFTR-modulating drugs. This protocol describes the quantification of CBF in three different airway epithelial cell model systems: 1) native epithelial sheets, 2) air-liquid interface models imaged on permeable support inserts, and 3) extracellular matrix-embedded three-dimensional organoids. The latter two replicate in vivo lung physiology, with beating cilia and production of mucus. The ciliary function is captured using a high-speed video camera in an environment-controlled chamber. Custom-built scripts are used for the analysis of CBF. Translation of CBF measurements to the clinic is envisioned to be an important clinical tool for predicting response to CFTR-modulating drugs on a per-patient basis.

This protocol describes nasal epithelial cell collection, expansion, and differentiation to organotypic airway epithelial cell models and quantification of cilia beat frequency via live-cell imaging and custom-built scripts.

Raccolta, espansione e differenziazione di modelli primari di cellule epiteliali nasali umane per la quantificazione della frequenza del battito delle ciglia

Measurements of cilia function (beat frequency, pattern) have been established as diagnostic tools for respiratory diseases such as primary ciliary ...

Normothermic Ex Vivo Pancreas Perfusion for the Preservation of Pancreas Allografts before Transplantation

Pancreas transplantation (PTx) is a curative treatment for people who live with the burden of a diagnosis of diabetes mellitus (DM). However, due to organ shortages and increasing numbers of patients being listed for PTx, new strategies are needed to increase the number of available grafts for transplantation. 
Static cold storage (SCS) is considered the gold standard for standard criteria organs. However, standard criteria donors (SCD) are becoming scarce and new strategies that can increase the rate of organ acceptance from extended criteria donors (ECD) are urgently needed. 
Normothermic ex vivo perfusion (NEVP) is one of the strategies that has become increasingly popular over the past couple of decades. This preservation method has already been used successfully in other organs (liver, kidneys, and lungs) but has been minimally explored in pancreas transplantation. The few papers that describe the method for pancreas show little success, edema being one of the major issues. The following manuscript describes the successful NEVP method and setup developed by our group to perfuse swine pancreas.

Normothermic Ex Vivo Pancreas Perfusion for the Preservation of Pancreas Allografts before Transplantation

Normothermic ex vivo machine perfusion (NEVP) has scarcely been explored for the preservation of pancreas allografts. We present an innovative preservation technique for pancreas allografts before transplantation.

Perfusione Pancreatica Ex Vivo Normothermic per la Conservazione degli Allotrapianti Pancreatici prima del Trapianto

Pancreas transplantation (PTx) is a curative treatment for people who live with the burden of a diagnosis of diabetes mellitus (DM). However, due to organ ...

Surgical Tips and Tricks for Performing Porcine Pancreas Transplantation

Despite the promising results of pancreas transplantation in type 1 diabetes mellitus and metabolic syndrome, the biggest concern around this state-of-the-art technique remains the paucity of organs deemed fit for transplantation. High intravascular resistance, delicate intraparenchymal capillary framework, and complex lobular anatomy around the mesenteric vasculature are what make this organ more susceptible to injury and less tolerant to trivial trauma compared to organs such as the liver and kidney. Meticulous surgical dissection and judicious tissue handling form the cornerstone of the entire exercise of pancreas transplantation. Owing to morphological similarity between the anatomy of the porcine pancreas to the surrounding mesenteric vessels and the organs when compared to the human anatomy, demonstration of the technique in the porcine model could help to most accurately extrapolate this to a human setting. The present article aims to outline the essential surgical tips and tricks that need to be followed, in order to ensure a higher success rate of transplantation of this highly susceptible organ in a porcine 3-day survival model.

The video article summarizes the technique of pancreatectomy and pancreas allotransplantation in a porcine 3-day survival model with a step-by-step description of the method and emphasis on the surgical tips and tricks to deal with the precarious and delicate porcine visceral anatomy.