The authors would like to thank Joshua Kimani, clinical director of the research program at the University of Nairobi, for his assistance with mucosal immunology studies related to this protocol. The authors would like to acknowledge funding from CHVI grant MOP 86721.

Dichiarazione etica: la ricerca etica consigli di amministrazione di entrambe dell&#39;Università di Manitoba e Kenyatta Nazionale Ospedale / Università di Nairobi ha approvato questo studio e il consenso informato scritto è stato ottenuto da tutti i partecipanti allo studio. 1. Preparazione dei Media e CMC Collection Tubes <ol><li> Preparare la soluzione tampone fosfato (PBS) (137,93 mM NaCl, 2,67 mM KCl, 8.1 mM Na 2 HPO 4, 1.47 mM KH 2 PO 4). Autoclave per la sterilità. Questo può essere conservato a 4 ° C per diversi mesi. </li><li> Pre-aliquota 5 ml di soluzione in 50 ml di tubo falcon (uno per campione da raccogliere) e conservare a 4 ° C fino al momento di raccolta del campione. </li><li> Etichettare 2-4 cryovials o provette da 1,5 ml microcentrifuga per campione per raccogliere la lavanda per la conservazione. </li><li> Preparare cellule di coltura: RPMI 1640 + 1% di penicillina / streptomicina / amfotericina B (concentrazione finale 100 unità / ml, 100 pg / ml e 250 ng / ml, respectively, senza FCS / FBS aggiunto. </li></ol> 2. Insieme di Cytobrush Campioni Raccolta di campioni cytobrush è una procedura non invasiva che deve essere eseguita da un MD o ginecologo esperto. <ol><li> Raccogliere le cellule mononucleate del collo dell&#39;utero (CMC) utilizzando sia un cytobrush e un raschietto di legno o di plastica. Raccogliere CMC dal partecipante in esame speculum inserendo il raschietto e ruotando attorno al canale cervicale (Figura 1). Inserire il cytobrush nell&#39;OS endocervicale, ruotare di 360 °, e collocare immediatamente sia il cytobrush e raschietto nel tubo falcon da 50 ml contenente 5 ml di PBS. </li><li> Conservare i campioni in ghiaccio / a 4 ° C fino al trattamento. Per mantenere la vitalità cellulare, campioni di processo entro 2 ore dal prelievo. </li><li> Se possibile, raccogliere campioni di sangue compensate in vacutainer eparina top verde di cellule mononucleate del sangue periferico (PBMC) isolamento per servire come un comparatore / controllo per l&#39;analisi CMC. </li&gt; </ol> 3. Isolamento di CMC Eseguire l&#39;elaborazione del campione in un laboratorio di biosicurezza di livello 2, in un armadio biosicurezza sterile con doppi guanti. <ol><li> Durante la raccolta, i campioni CMC possono diventare contaminati con sangue. Escludere campioni con contaminazione sangue visibile dallo studio per evitare confusione inclusione di PBMC nel campione finale. A causa del basso numero di linfociti isolati da campioni CMC, la contaminazione del sangue minore può facilmente sopraffare il componente linfociti CMC. </li><li> Tubi Vortex falco contenenti sia il cytobrush e raschietto per 45 secondi di dissociare le cellule dalle spazzole. I campioni possono diventare spumosa durante questo processo; questo è normale. </li><li> Utilizzare il cytobrush per raschiare tutto il materiale residuo fuori il raschietto, e scartare il raschietto in una soluzione di candeggina. Per raccogliere le eventuali cellule rimanendo attaccati alla spazzola / raschietto, usare un guanto fresco per spremere il raschietto tra il pollice e l&#39;indice.Scivolare giù il pennello, permettendo alle cellule e PBS da rilevare nella stessa provetta 50 ml. Eliminare il cytobrush in una soluzione di candeggina, e gettare il guanto. IMPORTANTE: utilizzare un nuovo guanto per ogni campione. </li><li> Montare un 100 micron filtro cella nylon a un nuovo 50 ml tubo falcon. Usando una pipetta di trasferimento, raccogliere la sospensione PBS-cella dal tubo campione e filtrare attraverso il filtro nel tubo fresco. </li><li> Aggiungere 5 ml di RPMI 1640 alla provetta campione originale. Utilizzando la pipetta di trasferimento, lavare i lati del tubo campione con il RPMI e le trasferisce attraverso il filtro per il nuovo tubo di raccolta. Lavare la parte inferiore del filtro di nylon con il RPMI pure. </li><li> Centrifugare i campioni per 10 min a 514 x g. E &#39;fondamentale per liberare il freno centrifuga durante questa fase, al fine di evitare stacchi del pellet CMC. </li><li> Rimuovere delicatamente il surnatante dal tubo, facendo attenzione a non disturbare il pellet. Dimensione pellet sarà molto variabile; in modome campioni, il pellet è abbastanza grande per la presenza di un gran numero di cellule epiteliali, mentre in altri casi, il pellet è appena visibile ed altamente trasparente. </li><li> Risospendere il pellet da lieve agitazione del tubo falco. Aggiungere 5 ml di PBS per lavare il campione. </li><li> Centrifuga nuovamente per 10 min a 514 xg senza freno centrifuga. </li><li> Eliminare attentamente il supernatante e risospendere il pellet come sopra. Le cellule sono pronte sia per la crioconservazione (utilizzando un protocollo di crioconservazione PBMC), la stimolazione o citometria a flusso fenotipizzazione. </li></ol> 4. CMC colorazione superficiale e Citometria a Flusso <ol><li> Risospendere il pellet dal punto 3.10 in 100 ml di soluzione bloccante e trasferire sia in una piastra a 96 pozzetti o il tubo FACS. La soluzione bloccante (per bloccare i recettori Fcy) ricetta è la seguente: IgG murine 1,8 ml (concentrazione finale 0,2 mg / mL), 5 microlitri FBS e 93,2 microlitri FACS lavaggio (PBS +2% FBS).La colorazione può essere eseguita in una piastra a 96 se formati pellet sono abbastanza piccole, ma può avere bisogno di essere eseguita in tubi FACS se pellets sono ordinariamente grande. E &#39;importante per titolare anticorpi conseguenza. </li><li> Bloccare i campioni per 10 min in ghiaccio / a 4 ° C. </li><li> Lavare le cellule con 100 ml di FACS lavaggio (PBS 2% FBS) in piastre a 96 pozzetti, o 500 ml in tubi FACS. Centrifugare a 600 xg per 10 min a 4 ° C con il freno a basso (o fuori, se una regolazione del freno basso non è disponibile). </li><li> Rimuovere il surnatante e risospendere il pellet cellulare mediante agitazione. Aggiungi vitalità Stain (Live Morto risolvibile vitalità colorante). Preparare la redditività colorante secondo le istruzioni del produttore. Potrebbe essere necessario titolato per l&#39;uso in ogni impostazione del laboratorio / citometro. Incubare per 30 minuti al buio a 4 ° C. </li><li> Lavare le cellule μl/500 100 ml di PBS (per i tubi targa / FACS 96 pozzetti). Centrifugare a 600 xg per 10 min a basso / senza freno. Rimuovere supernatant e risospendere le cellule. </li><li> Incubare le cellule con il cocktail di anticorpi marcatori di superficie, in un volume totale di 100 microlitri. IMPORTANTE: Ottimizzare e titolare ogni citometria a flusso pannello prima di assaggiare colorazione. Incubare le cellule per 30 minuti al buio a 4 ° C. </li><li> Ripetere passo 4.5. Se la colorazione in una piastra da 96 pozzetti, le cellule di trasferimento ad un tubo FACS e portare a un volume finale di 750 ml di FACS lavaggio contenente 1% paraformaldeide (PFA) (o CytoFix, diluito 1 a 4). Procedere all&#39;acquisizione dati su un citometro a flusso. </li></ol> 5. Raccolta e preparazione del collo dell&#39;utero vaginale del lavaggio (CVL) <ol><li> Prima cytobrush campionamento, utilizzare una siringa per lavare il endocervice con 2 ml di PBS sterile e aspirare il lavaggio del fornice posteriore. </li><li> Raccogliere il campione di lavaggio aspirato in un tubo da 15 ml falco e tenere in ghiaccio / a 4 ° C fino al trattamento. </li><li> Campione centrifugare a 400 xg per 7 minuti per rimuovere detriti cellulari. </li><li> Collect il surnatante, un&#39;aliquota del campione in 1 ml o 500 microlitri aliquote e conservare a -70 ° C. Aliquote possono essere scongelati per l&#39;ELISA o l&#39;analisi di matrice cordone di proteine ​​CVL, ma evitano cicli multipli di congelamento / scongelamento. </li><li> Se lo si desidera, risospendere il pellet di RNA detriti cellulari in seguito a misurare l&#39;espressione di RNA seguendo il protocollo del produttore. </li></ol> 6. Acquisizione Dati <ol><li> Preparare una serie di tubi di compensazione che corrisponde al pannello di anticorpi. Eseguire i tubi di compensazione per regolare sovrapposizione spettrale. Eseguire i campioni su una qualsiasi flusso multicolore citometro che è configurato per gli anticorpi coniugati con fluorocromi utilizzati nel pannello. </li><li> Acquisire un campione PBMC prima di regolare Forward Scatter (FSC) e Side Scatter (SSC) della tensione, al fine di rilevare la popolazione linfocitaria. Cercate di tenerli a 100 su ciascun asse su una scala lineare. Esecuzione di un controllo PBMC renderà molto più facile trovare la popolazione linfocitaria CMC. </li><li> La soglia FSC per campioni CMC può essere necessario aumentare rispetto ai campioni di PBMC causa spalmare fino all&#39;asse SSC a bassi valori FSC. </li><li> Agitare ogni provetta prima di acquistare. Acquisire l&#39;intero tubo per massimizzare il numero di eventi cancello linfociti raccolti. Monitorare attentamente la macchina per evitare intasamenti. </li><li> Esportare i dati in un programma di analisi di citometria a flusso, come FlowJo. </li></ol> 7. Strategia di gating <ol><li> Selezionare Forward scatter lato alto (FSC-H) / Forward Area side scatter (FSC-A) per determinare la popolazione singoletto. </li><li> Selezionare Ora contro marcatore fluorescente (come ad esempio FITC) per controllare la qualità di acquisizione. Variazione della portata può introdurre artefatti nei dati. Escludere qualsiasi area che mostra discrepanza nei portata. </li><li> Identificare la popolazione linfocitaria su SSC-A/FSC-A trama. Utilizzare un controllo PBMC per facilitare l&#39;identificazione. Si noti che la popolazione linfocitaria CMC potrebbe non essere chiaro come il controllo PBMC. &lt;/ Li&gt; <li> Gate SSC-A/viability tintura di escludere le cellule morte da cellule vive. Le cellule morte possono non specificamente incorporare gli anticorpi, che introdurrà artefatti. </li><li> Porta sul SSC-A/CD3 per identificare la popolazione di cellule T. Da questa porta, individuare le popolazioni CD4 + e CD8 +. </li></ol>

<ol>
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The authors have nothing to disclose.

Date le grandi lacune nella conoscenza per quanto riguarda l&#39;immunità al tratto genitale femminile (FGT), analisi fenotipica di CMC in grado di fornire una vasta gamma di intuizioni più popolazioni linfocitarie alla cervice. Accoppiato con l&#39;analisi proteomica e le misurazioni della carica virale di lavanda cervicale, l&#39;immunità alle infezioni sessualmente trasmissibili (IST) s ed altri agenti patogeni può essere sezionato in varie popolazioni. Considerazioni tecniche...

La maggior parte delle nuove infezioni da HIV in tutto il mondo nascono attraverso la trasmissione eterosessuale, con le donne rappresentano il 47% delle nuove infezioni nel 2011 (UNAIDS 1). Comprendere il tratto genitale femminile (FGT), uno dei principali portali di ingresso per l&#39;HIV e di altri agenti patogeni a trasmissione sessuale, è di grande importanza nel cammino per trovare strategie efficaci per prevenire l&#39;infezione. Risposte immunitarie alla mucosa genitale sono chiaramente unico e differiscono da quelli misurati nel sangue periferico 2. Tuttavia, l&#39;attuale conoscenza delle dinamiche immunodeficienza al FGT è limitata al meglio. Ad oggi, gli studi di ambiente immunitario della mucosa hanno in gran parte concentrata sui tessuti linfoidi dell&#39;intestino associato (GALT), dove è diventato chiaro che i primi eventi nei tessuti della mucosa seguenti infezioni hanno un forte impatto sulla successiva progressione della malattia 3,4. Raccolta dei campioni dalla mucosa genitale rappresenta una grande sfida ed è almeno parzialmente responsabile del lack di comprensione della immunologia del FGT. Risolvere il puzzle della dinamica immunitario tra ospite e patogeno nel contesto dell&#39;ambiente distinto che è il FGT richiede metodi efficienti per raccogliere e analizzare campioni di questo locale. Il FGT è divisa in due sezioni: il tratto riproduttivo superiore che comprende le tube di Falloppio, endometrio e endocervice, e il tratto inferiore che contiene la portio e la vagina (recensito da Kaushic et al 5). Non è ancora chiaro quanto il contributo relativo di questi diversi siti è all&#39;HIV, ma si ritiene che entrambi i siti possono contribuire a voce HIV 6. Cellule T rappresentano il 40-50% dei leucociti nei tratti riproduttivi superiori e inferiori, mentre macrofagi costituiscono circa il 10% (recensito in Rodriguez-Garcia et al 2). Cellule T possono essere rilevati nella vagina, cervice e dell&#39;endometrio. I macrofagi sono più fortemente Localized nell&#39;endometrio e tessuto connettivo miometrio rispetto alla cervice, anche se possono essere rilevati in entrambi i tessuti. Infine, le cellule dendritiche plasmacitoidi (PDC) e cellule di Langerhans possono essere rilevati anche nei tessuti FGT. Il fenotipo e proporzioni delle popolazioni immunitarie e la loro suscettibilità alle infezioni da HIV possono variare importante secondo cicli ormonali, l&#39;uso di contraccettivi ormonali, vaginosi batterica o attività sessuali 5,7-9. Diversi metodi sono stati sviluppati per studiare le popolazioni immunitarie e l&#39;ambiente del FGT. Biopsia cervicale, cytobrushes cervicale e lavaggi cervico-vaginale (CVL), 10-12 sono i più comunemente utilizzato in tutto il letteratura. Collezione CVL da PBS lavanda è il metodo più semplice e permette lo studio delle proteine ​​modulatori immunitari, ma i risultati del rendimento delle cellule estremamente basso, e quindi non è adatto per studiare le popolazioni di cellule immunitarie del FGT 13. Campioni CVL sono, invece, very utili per valutare l&#39;ambiente immunitario del FGT misurando l&#39;espressione di diverse citochine, chemochine o fattori antimicrobici usando metodi quali ELISA, citochine branello matrice 14 o spettrometria di massa 15,16. Caratterizzazione delle frequenze di cellule immunitarie, fenotipi e funzioni può essere ottenuto mediante prelievo di cellule mononucleate cervicali (CMC) da cytobrush cervicale o mediante campionamento biopsia cervicale. Campionamento biopsia cervicale è un metodo invasivo che aumenta il disagio e rischio di sanguinamento e richiede 2 a 11 giorni per guarire seguendo la procedura a seconda dello stato immunitario della donna 12. D&#39;altra parte, cytobrushes cervicali, nonostante la minore resa di cellule raccolte, è un metodo meno invasivo e più conveniente per raccogliere le cellule immunitarie dal FGT. Entrambi i metodi possono raggiungere la stessa resa di leucociti CD45 +, ma due cytobrushes cervicali sequenziali sono necessari per avere la stessa quantità di cellule contenute in una biopsia 13. Tuttavia, il campionamento cytobrush fornisce ancora un numero accettabile di cellule (cellule + circa 5.000 CD45 / cytobrush) per ulteriore ex vivo fenotipizzazione mediante citometria di flusso 14. Inoltre, la caratterizzazione funzionale può essere effettuata su questi campioni, la stimolazione e flusso intracellulare citometria o qPCR sono state effettuate utilizzando CMC cytobrush derivate per identificare HIV-specifiche risposte immunitarie 17 o polarizzazione cellulare Th 18. L&#39;espansione della popolazione di cellule T può anche facilitare gli studi funzionali con CMC 19. È importante notare che le biopsie e cytobrushes campionare porzioni distinte della FGT. Biopsie sono derivati ​​dalla porzione superiore del epitelio e stroma della portio 12,13, mentre cytobrushes cervicali campionare della cervice, raccogliendo cellule derivate dall&#39;epitelio di endocervice e presumibilmente la zona di trasformazione. Campioni Cytobrush quindi degustare un regione composto da un unico strato di epitelio colonnare, mentre biopsie, comprendono una regione rivestita da un epitelio squamoso stratificato 5. Di conseguenza, la natura delle popolazioni di leucociti raccolti da biopsia cervicale e cytobrush differisce. Biopsie raccolgono una percentuale maggiore di cellule T CD3 +, mentre cytobrushes comportano la raccolta di una maggiore percentuale di monociti CD14 + / macrofagi 13. Studiare la immunologia della FGT è stato un interesse per molti anni 20-22 e abbiamo accumulato una grande esperienza con lo studio della CMC cytobrush-derivati. I nostri studi si concentrano principalmente sullo studio del virus HIV-infetti, non infetto e HIV-esposti sieronegativi (HESN) i lavoratori di sesso femminile da Nairobi, Kenya. HIV si replica preferenzialmente nelle cellule T attivate 23 e inferiore numero di cellule attivate che possono essere avviati da HIV nel FGT potrebbero contribuire alla protezione contro l&#39;acquisizione HIV. In linea con questa ipotesi, diversi studies hanno descritto attivazione immunitaria più bassa tra i lavoratori di sesso HESN che sono altamente esposti al virus HIV ancora rimanere infetto 24,25, e questo fenotipo quiescente si osserva anche nel FGT 14. Qui, descriviamo la metodologia per l&#39;elaborazione e la valutazione cellule T di attivazione nei campioni CMC derivati ​​da cytobrushes cervicali da ex vivo citometria a flusso.

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			<td height="21" style="height:21px;width:295px;">Name of Material/ Equipment</td>
			<td style="width:136px;">Company</td>
			<td style="width:133px;">Catalog Number</td>
			<td style="width:205px;">Comments/Description</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:295px;">100uM Cell Strainer for 50 ml Falcon tube</td>
			<td style="width:136px;">BD</td>
			<td style="width:133px;">352360</td>
			<td>CMC processing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">RPMI 1640</td>
			<td style="width:136px;">Hyclone</td>
			<td style="width:133px;">SH30027.01</td>
			<td>CMC processing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Fetal Bovine serum&#160;</td>
			<td style="width:136px;">Life technology</td>
			<td style="width:133px;">16000044</td>
			<td>CMC processing</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Fungizone</td>
			<td style="width:136px;">Life technology</td>
			<td style="width:133px;">15290-018</td>
			<td>CMC processing</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Penicillin/streptomycin</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:133px;">P4333-20ml</td>
			<td>CMC processing</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:295px;">50ml Falcon tube</td>
			<td style="width:136px;">Fisher</td>
			<td style="width:133px;">14-959-49A</td>
			<td>CMC processing</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Blood Bank disposable transfer pipette</td>
			<td style="width:136px;">Fisher&#160;</td>
			<td style="width:133px;">13-711-6M</td>
			<td>CMC processing</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Cytobrush plus</td>
			<td style="width:136px;">Cooper surgical</td>
			<td style="width:133px;">C0121</td>
			<td>CMC sampling</td>
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			<td height="21" style="height:21px;width:295px;">Disposable cervical scraper</td>
			<td style="width:136px;">Quick medical</td>
			<td style="width:133px;">2183</td>
			<td>CMC sampling</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:295px;">15 ml Falcon tube&#160;</td>
			<td style="width:136px;">Fisher&#160;</td>
			<td style="width:133px;">14-959-70c</td>
			<td>CVL processsing</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:295px;">1.5ml tube ependroff</td>
			<td style="width:136px;">Fisher</td>
			<td style="width:133px;">05-402-18</td>
			<td>CVL storage</td>
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		<tr height="21">
			<td height="21" style="height:21px;">LIVE/DEAD Fixable Cell Stain Kit</td>
			<td style="width:136px;">Invitrogen</td>
			<td>Various</td>
			<td>Flow cytometry reagent</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Fixation Buffer (4% PFA)</td>
			<td style="width:136px;">BD</td>
			<td>554655</td>
			<td>Flow cytometry regeant&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:295px;">IgG mouse&#160;</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:133px;">I8765</td>
			<td>Flow cytometry regeant&#160;</td>
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</table>

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.

Flusso multiparametrica citometria è un potente strumento per analizzare i fenotipi e le funzioni dei sottoinsiemi di cellule in tessuti precedentemente non caratterizzati. Analisi dei campioni CMC può dare informazioni sia su linfociti e monociti popolazioni con le strategie di gating adeguate. Un rappresentante CMC strategia gating, rispetto ad un profilo PBMC abbinato, è mostrato in Figura 2. L&#39;FSC-A piuttosto FSC-H trama consente l&#39;esclusione di doppietti cell...

Watch this Scientific Journal Video about Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples at JoVE.com

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

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 ...

collection-isolation-flow-cytometric-analysis-human-endocervical

Research

JoVE Journal

Medicine

19.0K Views.  University of Manitoba. 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. 

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

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 ...

Ricerca

Medicina

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 ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

Isolamento, Cultura e Imaging di Human Fetal pancreatici gruppi di cellule

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

Isolamento e caratterizzazione funzionale di Human ventricolare Cardiomyocytes da campioni freschi chirurgici

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

Isolamento di cancro cellule staminali da campioni umani di cancro alla prostata

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both with and without preexisting conditions. Rapid processing of these samples is essential to avoid degradation of key cellular components. Included here are methods for simultaneous peripheral blood mononuclear cell (PBMC), DNA, RNA and serum isolation from a single blood draw performed in the homes of consenting participants across a metropolitan area, with processing initiated within 2 hr of collection. We have used these techniques to process over 1,600 blood specimens yielding consistent, high quality material, which has subsequently been used in successful DNA methylation, genotyping, gene expression and flow cytometry analyses. Some of the methods employed are standard; however, when combined in the described manner, they enable efficient processing of samples from participants of population- and/or community-based studies who would not normally be evaluated in a clinical setting. Therefore, this protocol has the potential to obtain samples (and subsequently data) that are more representative of the general population.

We outline a methodology for the processing of whole blood to obtain a variety of components for further analysis. We have optimized a streamlined protocol that enables rapid, high-throughput simultaneous processing of whole blood samples in a non-clinical setting.

Frazionamento rapida e isolamento dei componenti sangue intero in campioni ottenuti da una impostazione basata su Comunità

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both ...

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.
Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 &#176;C.

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

Isolamento e Profiling di MicroRNA contenenti Esosomi da bile umana

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central nervous system (CNS). However, further optimization of these technologies would benefit from methods allowing rapid and streamline determination of the extent of CNS and cell-specific targeting upon administration of viral vectors or nanoparticles in the body. Here, we present a protocol that takes advantage of the high throughput and multiplexing capabilities of flow cytometry to allow a straightforward quantification of different cell subtypes isolated from mouse brain or spinal cord, namely microglia/macrophages, lymphocytes, astrocytes, oligodendrocytes, neurons and endothelial cells. We apply this approach to highlight critical differences between two tissue homogenization methods in terms of cell yield, viability and composition. This could instruct the user to choose the best method depending on the cell type(s) of interest and the specific application. This method is not suited for analysis of anatomical distribution, since the tissue is homogenized to generate a single-cell suspension. However, it allows to work with viable cells and it can be combined with cell-sorting, opening the way for several applications that could expand the repertoire of tools in the hands of the neuroscientist, ranging from establishment of primary cultures derived from pure cell populations, to gene-expression analyses and biochemical or functional assays on well-defined cell subtypes in the context of neurodegenerative diseases, upon pharmacological treatment or gene therapy.

We present a flow cytometry method to identify simultaneously different cell types retrieved from mouse brain or spinal cord. This method could be exploited to isolate or characterize pure cell populations in neurodegenerative diseases or to quantify the extent of cell targeting upon in vivo administration of viral vectors or nanoparticles.

Caratterizzazione citometrica a flusso simultaneo di più tipi di cellule recuperate dal cervello del mouse/cavo spinale attraverso diversi metodi di omogeneizzazione

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central ...

Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the body&#39;s circulatory system demands a reliable and multiparametric approach that evaluates the possible hematologic complications caused by these devices (i.e., activation and destruction of blood components). Comprehensive in vitro hemocompatibility testing of blood-contacting implants is the first step towards successful in vivo implementation. Therefore, extensive analysis according to the International Organization for Standardization 10993-4 (ISO 10993-4) is mandatory prior to clinical application. The presented flow loop describes a sensitive model to analyze the hemostatic performance of stents (in this case, neurovascular) and reveal adverse effects. The use of fresh human whole blood and gentle blood sampling are essential to avoid the preactivation of blood. The blood is perfused through a heparinized tubing containing the test specimen by using a peristaltic pump at a rate of 150 mL/min at 37 &#176;C for 60 min. Before and after perfusion, hematologic markers (i.e., blood cell count, hemoglobin, hematocrit, and plasmatic markers) indicating the activation of leukocytes (polymorphonuclear [PMN]-elastase), platelets (&#946;-thromboglobulin [&#946;-TG]), the coagulation system (thombin-antithrombin III [TAT]), and the complement cascade (SC5b-9) are analyzed. In conclusion, we present an essential and reliable model for extensive hemocompatibility testing of stents and other blood-contacting devices prior to clinical application.

This protocol describes a comprehensive hemocompatibility evaluation of blood-contacting devices using laser-cut neurovascular implants. A flow loop model with fresh, heparinized human blood is applied to mimic blood flow. After perfusion, various hematologic markers are analyzed and compared to the values gained directly after blood collection for hemocompatibility evaluation of the tested devices.

Test di emocompatibilità degli impianti che si contattono con il sangue in un modello di flusso a ciclo continuo che imita il flusso sanguigno umano

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the ...

Validated LC-MS/MS Panel for Quantifying 11 Drug-Resistant TB Medications in Small Hair Samples

Drug resistant-tuberculosis (DR-TB) is a growing public health threat, and assessment of therapeutic drug levels may have important clinical benefits. Plasma drug levels are the current gold standard assessment, but require phlebotomy and a cold chain, and capture only very recent adherence. Our method uses hair, a matrix that is easily collected and reflective of long-term adherence, to test for 11 anti-TB medications. Previous work by our group shows that antiretroviral drug levels in hair are associated with HIV outcomes. Our method for DR-TB drugs uses 2 mg of hair (3 cm proximal to the root), which is pulverized and extracted in methanol. Samples are analyzed with a single LC-MS/MS method, quantifying 11 drugs in a 16 min run. Lower limits of quantification (LLOQs) for the 11 drugs range from 0.01 ng/mg to 1 ng/mg. Drug presence is confirmed by comparing ratios of two mass spectrometry transitions. Samples are quantified using the area ratio of the drug to the deuterated, 15N-, or 13C-labeled drug isotopologue. We used a calibration curve ranging from 0.001-100 ng/mg. Application of the method to a convenience sample of hair samples collected from DR-TB patients on directly observed therapy (DOT) indicated drug levels in hair within the linear dynamic range of nine of the eleven drugs (isoniazid, pyrazinamide, ethambutol, linezolid, levofloxacin, moxifloxacin, clofazimine, bedaquiline, pretomanid). No patient was on prothionamide, and the measured levels for ethionamide were close to its LLOQ (with further work instead examining the suitability of ethionamide&#8217;s metabolite for monitoring exposure). In summary, we describe the development of a multi-analyte panel for DR-TB drugs in hair as a technique for therapeutic drug monitoring during drug-resistant TB treatment.

Current methods of analyzing patients&#8217; adherence to complex drug resistant-tuberculosis (DR-TB) regimens can be inaccurate and resource-intensive. Our method analyzes hair, an easily collected and stored matrix, for concentrations of 11 DR-TB medications. Using LC-MS/MS, we can determine sub-nanogram drug levels that can be utilized to better understand drug adherence.

Pannello LC-MS/MS convalidato per quantificare 11 farmaci TB resistenti ai farmaci in piccoli campioni di capelli

Drug resistant-tuberculosis (DR-TB) is a growing public health threat, and assessment of therapeutic drug levels may have important clinical benefits. ...