Questo lavoro è stato sostenuto da un finanziamento del Dipartimento della Difesa prostatico Programma di Ricerca sul Cancro (W81XWH-12-1-0124), U54CA143876 dal National Cancer Institute, e il Robert McCooey Genitourinary Oncology Research Fund. Vorremmo ringraziare il Dott. Annarita Lorenzo (Dipartimento di Patologia) per fornire gli anticorpi VE-caderina, e il dottor Marco Seandel (Dipartimento di Chirurgia) per la fornitura di HUVECs.

1. Coltura HUVECs su Microslides per interazioni Observing CTC-endoteliali <ol><li> Sotto il cofano di coltura tissutale, prima sciacquare la MicroSlide, larghezza del canale di 1 mm con PBS. Cappotto delicatamente il MicroSlide con 200 ml di 50 mg / ml fibronectina (sciolto in PBS) usando una siringa Luer-lock da 1 ml. </li><li> Coprire il MicroSlide con il coperchio e tenerlo all&#39;interno della cappa coltura tissutale per 30 min. Erogazione lenta del liquido nel MicroSlide impedisce la formazione di bolle nel canale. </li><li> Profumato 200 ml di acqua calda (37 ° C) HUVEC mezzo di crescita (M199 media, 1M HEPES, 20% FBS, 5 mg / ml di eparina, 100 pg / ml di fattore di crescita delle cellule endoteliali, e L-glutammina) sul MicroSlide e incubare per 20 min a RT. Durante la perfusione, preparare la sospensione di cellule HUVEC. </li><li> Risciacquare HUVECs con PBS e aggiungere 0,05% tripsina-EDTA per 1-2 minuti a temperatura ambiente. Centrifugare HUVEC in 2 ml di mezzo di crescita a 180 xg per 5 min. </li><li> Misurare la concentrazione cellulare utilizzando un neubaUER emocitometro e preparare 10 7 HUVEC cells/100 microlitri mezzo di crescita. Poi, rimuovere con attenzione il mezzo dall&#39;ingresso della MicroSlide utilizzando un puntale di 200 microlitri. </li><li> Portare il MicroSlide a livello degli occhi e l&#39;utilizzo di un 1 ml siringa Luer-lock, profumato delicatamente 200 ml di concentrazione preparata del HUVECs nel canale. Si richiede cautela in questa fase per evitare la formazione di bolle. Se compaiono le bolle, tenere perfusione per un tempo leggermente più lungo finché le bolle entrano nel canale di uscita. </li><li> Mettere un volume uguale (~ 80 microlitri) di supporto HUVEC sia l&#39;ingresso e l&#39;uscita del MicroSlide. Ciò impedisce il flusso di celle in entrambe le direzioni. </li><li> Coprire il vetrino e tenerlo in incubatore (37 ° C) per 1,5 ore. </li></ol> 2. Preparazione della camera di flusso dell&#39;Assemblea per Overnight cultura HUVEC su un MicroSlide <ol><li> Inserire una siringa sterile da 20 ml, connettori maschio e femmina Luer, tubi e una pompa a siringa in incubatrice per 15 kmn. </li><li> Per volume morto minimo, utilizzare il tubo con diametro interno di 0,04 pollici. Più piccolo diametro del tubo impedisce la formazione di bolle. </li><li> Riempite la siringa da 20 ml con acqua calda (37 ° C) HUVEC supporto (12 ml). Fissare il tubo con i connettori sulla siringa. Rimuovere le bolle. Collegare questo gruppo alla MicroSlide. </li><li> Riempire completamente l&#39;ingresso della MicroSlide con i media HUVEC. Portare la siringa preriempita da 20 ml collegata al connettore vicino al MicroSlide. Applicare delicatamente il connettore al MicroSlide. </li><li> Portare il set-up in incubatrice e collegarlo alla pompa siringa, fissato a 10 ml / min velocità di taglio. Lasciare le cellule O / N in incubatrice a 37 ° C. </li><li> Il giorno dopo, smontare il set-up rimuovendo il connettore collegato all&#39;ingresso del MicroSlide. </li><li> Per aumentare l&#39;espressione E-selectina sulle cellule endoteliali, preparare il terreno di coltura fresco contenente IL-1β a 10 ng / ml in 4 ml di media. Aspirare i media in una siringa da 10 ml. Rimuovere tegli materiale dal ingresso del MicroSlide e collegare la siringa al vetrino. </li><li> Impostare la pompa a siringa 10 ml / min velocità di taglio per 4 ore in incubatrice. </li></ol> 3. Preparazione di Anti-PSMA (J591-488) con etichetta cellule tumorali della prostata <ol><li> Durante l&#39;IL-1β incubazione di HUVECs, preparare anti-PSMA J591-alexa488 etichettati cellule APC. Aggiungere 0,05% tripsina-EDTA a cellule MDA per 1 min. Non esporre le cellule di tripsina per tempi più lunghi in quanto può influenzare i glicopeptidi presenti sulla superficie cellulare. Enzima reagente dissociazione cellulare libero può anche essere usato al posto di tripsina. Centrifugare a 200 xg per 5 min. </li><li> Risospendere il pellet di cellule MDA in 1 ml di H / H tampone (Hanks salina bilanciata solution/0.1% HSA/10 mM HEPES / 1 mM CaCl 2). Aggiungi anticorpo J591-alexa488 anti-PSMA a 20 mg / ml per 30 min a RT in un luogo buio. Risospendere le cellule durante l&#39;incubazione. </li><li> Dopo 30 minuti, centrifugare la soluzione di cellule a 800 xg per 5 min. Aspivotare e risospendere il pellet in 1 ml di tampone H / H. Contare le cellule MDA etichettati e per portare la concentrazione finale di 1x10 6 cellule / ml. Riempire una siringa da 5 ml con J591-488 cellule MDA etichettati e rimuovere le bolle. </li></ol> 4. Preparazione di Anti-PSMA (J591-488) etichetta CTC arricchito da PCa pazienti <ol><li> Raccogliere 7,5 ml di sangue da pazienti affetti da cancro della prostata in un tubo tappo blu (contenente sodio citrato). Diluire delicatamente 1:01 sangue nel 0,1% BSA / 1 mM EDTA / PBS. </li><li> Aggiungere 5.3 ml di Ficoll-Paque più in una provetta da 50 ml. Strato diluito sangue sopra il Ficoll-Paque. Centrifugare a 400 xg per 30 min. </li><li> Pre-coat tutti i tubi o punte entrano in contatto con CTC con 2% FBS/RPMI-1640 / 1 mM CaCl 2/4 mM MgCl 2 (buffer R / S) per prevenire il legame non specifico delle CTC alle superfici. Mantenere 4 ° C di temperatura dei media e tamponi che vengono a contatto con CTC. </li><li> Raccogliere il cellule mononucleate del sangue periferico (PBMC) frazionecontenente CTC dall&#39;interfaccia in un&#39;altra provetta conica da 50 ml contenente 30 ml di tampone di R / S. Centrifugare a 400 xg per 8 min. </li><li> Risospendere il pellet e lavare di nuovo nel buffer di R / S a 400 xg per 8 min. Dopo centrifugazione, risospendere il pellet in tampone H / H. Aggiungere anti-PSMA J591-488 anticorpo a 20 ug / ml per 30 min a temperatura ambiente in un luogo buio. </li><li> Dopo 30 minuti, centrifugare le PBMC contenenti J591-488 CTC etichettati a 800 xg per 5 min. Risospendere in tampone H / H. Riempire una siringa da 1 ml (pre-rivestite con tampone R / S) con il campione. </li></ol> 5. Preparazione del microscopio, pompa a siringa e della Camera di flusso Assembly <ol><li> Accendere il microscopio invertito e impostare l&#39;illuminazione Kohler a 10X obiettivo. Portare la pompa a siringa allo stesso livello della fase del campione del microscopio e impostarlo a 1 dyn / cm 2 shear stress (~ 10 microlitri / min). </li><li> Aprire il software Zeiss AxioVision. Selezionare l&#39;obiettivo sullo schermo del computer. Creare un nuovocartella sotto l&#39;opzione strumenti nel software. </li><li> Aprire l&#39;opzione esperimenti intelligenti in AxioVision e regolare le impostazioni di registrare brevi video di 30 secondi per 30 minuti. </li><li> Per il video fluorescente dal vivo, impostare le opzioni-12 msec immagine di esposizione, 2 x 2 bin, 5 Gain. Questi parametri aiutano a raggiungere i video vicino al frame rate video (~ 23 fotogrammi al secondo) con la fotocamera Zeiss MRM. </li><li> Per visualizzare tutta la larghezza del canale di flusso a 10 X obiettivo sullo schermo del computer, utilizzare un adattatore C-mount (0.63 xf/60 mm Interface). </li><li> Accendere la lampada a mercurio. </li><li> Porre la siringa e il connettore contenente le cellule sulla pompa siringa. </li><li> Portare IL-1β stimolato HUVECs dal termostato. Collegare il connettore al canale di ingresso pieno della MicroSlide contenente HUVECs. </li><li> Collegare il canale di uscita con il connettore collegato al tubo. Mettere il tubo in un piatto o tubo 15 ml conica per raccogliere il flusso attraverso. </li><li> Avviare il Infusiattraverso il MicroSlide a 10 microlitri / min. Osservare le interazioni tra cellule endoteliali e cellule MDA etichettati o etichettati CTC derivati ​​da pazienti al di sotto del filtro 488 nm sul microscopio a epifluorescenza. </li><li> Avviare la registrazione l&#39;esperimento come 30 sec brevi video. Durante l&#39;analisi riproduzione, misurare la velocità di rotazione. Velocità di rotazione si misura dividendo la distanza percorsa dalle cellule nel tempo. </li></ol> 6. Immunocolorazione del MicroSlide <ol><li> Rimuovere il supporto dal ingresso e l&#39;uscita del MicroSlide dopo perfusione cellule MDA su IL-1β-stimolata HUVEC per 10 min. </li><li> Utilizzando una punta di 200 microlitri, mettere calda (37 ° C) PBS contenente calcio e magnesio nella presa del MicroSlide per 5 min. Inclinare il MicroSlide usando il coperchio come un puntello in panchina. Mantenere la MicroSlide in una posizione inclinata per tutte le incubazioni durante immunocolorazione. </li><li> Fissare HUVECs mediante perfusione caldo 2% di formaldeide in ingresso </li><li&gt; incubare per 20 minuti a temperatura ambiente. Risciacquare due volte con PBS per 5 minuti ciascuno. </li><li> Aggiungere triton X-100 (0,1%) in 5% BSA in PBS per 10 minuti a RT. </li><li> Risciacquare due volte con PBS per 5 minuti ciascuno. Blocco con 5% BSA in PBS per 30 min. </li><li> Incubare con anticorpo primario di capra anti-VE-caderina (1:100) nel 2,5% BSA O / N a 4 ° C. </li><li> Il giorno dopo, lavare due volte con PBS per 5 minuti ciascuno. Aggiungi asino secondario anti-goat 647 anticorpo per 45 min. Lavare due volte con PBS per 5 min ciascuno. </li><li> Incubare a RT con umanizzato anticorpo coniugato J591-alexa488 per 1 ora. </li><li> Lavare due volte con PBS per 5 min ciascuno. Aggiungere DAPI per 5 min. Lavare con acqua distillata per 5 minuti. </li><li> Aggiungi PBS (o Mowiol per la conservazione a lungo termine). Visualizza la MicroSlide sotto il microscopio confocale. </li></ol>

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Dr. Bander è l&#39;inventore di brevetti che vengono assegnati a Cornell Research Foundation (&quot;CRF&quot;) per l&#39;anticorpo J591 utilizzato in questo articolo. Dr. Bander è un consulente per e possiede azioni in BZL Biologics, la società a cui i brevetti sono stati concessi in licenza da CRF per ulteriore ricerca e sviluppo.

A causa del basso numero di CTC tra cellule del sangue, è difficile isolare CTC come una popolazione pura di cellule. Al fine di studiare le interazioni CTC / CE, la popolazione rara e impura delle CTC pone due sfide importanti: a) identificazione di CTC tra le cellule del sangue; b) Osservazione delle interazioni CTC / CE. Per superare la prima limitazione di identificare CTC prostata tra le cellule del sangue, abbiamo approfittato del fatto che praticamente tut...

Metastasi è un complesso processo multi-step che rimane poco conosciuta. L&#39;asse ligando E-selectin/selectin ha dimostrato di giocare un ruolo importante nella metastasi del tumore promuovendo interazioni adesive principali tra l&#39;endotelio vascolare e cellule tumorali 1,2. Endoteliale (E)-selectina è una proteina transmembrana espressa dalle cellule endoteliali attivate, mentre differente ligando E-selectina (s) sono espressi dalle cellule tumorali 3. Numerosi approcci in vitro sono stati impiegati con successo per modellare interazioni ligando E-selectin/selectin tra cellule tumorali e cellule endoteliali (EC) 1. Per studiare queste interazioni, diversi sistemi di camera di flusso vengono impiegati per simulare sistema vascolare sanguigno. Tra assembly camera di flusso, a piatti paralleli camera di flusso (PPFC) in combinazione con EC è abitualmente utilizzato come modello in vitro simulando in condizioni di stress taglio vivo. In questometodo, EC sono coltivate su un piatto da 35 mm e dopo aver ottenuto un monostrato, EC sono attaccati alla PPFC e gli esperimenti shear stress sulla base vengono eseguite. Tuttavia, PPFC e altri sistemi attuali presentano molte limitazioni per studiare le interazioni adesive tra cellule tumorali circolanti (CTC) derivate da pazienti e EC, in primo luogo, perché CTC sono una rara popolazione di cellule, versato dal tumore primario, che circola tra milioni di cellule del sangue (1 CTC per 10 9 cellule ematiche) 4. Quindi, a differenza di fornitura illimitata di linee cellulari in coltura, bassa conta CTC portare ad interazioni molto poche e rare CTC / CE, che richiede un&#39;adeguata larghezza di canale di flusso per registrare le interazioni per l&#39;analisi di riproduzione. Inoltre, dal momento che pazienti CTC derivati ​​sono una popolazione impura, quindi un marcatore di identificazione è necessaria per tenere traccia CTC in specifico. Per risolvere questo problema, abbiamo sviluppato un nuovo metodo per identificare il cancro alla prostata (PCA) CTCs approfittando del fatto che la quasi totalità di questi CTC esprimono prostatico specifico antigene di membrana (PSMA) sulla loro superficie cellulare 5,6. In questa relazione, abbiamo utilizzato la linea di cellule di cancro alla prostata, MDA PCa2b (MDA), per dimostrare la potenziale utilità del nostro nuovo sistema per lo studio della prostata interazioni CTC con EC, alla fine di capire il meccanismo di metastasi. La nostra metodologia può essere applicata per vari esperimenti di taglio basato simulano nel sistema di vivo vascolare 7-9. Oltre ad esaminare le interazioni PCa CTC / CE, l&#39;attuale sistema di camera di flusso potrebbe essere facilmente adattato per l&#39;analisi di cellule mononucleari del sangue periferico o interazioni cellule tumorali &quot;con EC. La facilità di smontaggio e rimontaggio della camera di flusso, un MicroSlide III (0,1) (in appresso MicroSlide), permette la coltura EC sotto perfusione e stimolante EC con differenti citochine di indurre protein espressione. Inoltre, colta EC, proteine ​​ricombinanti come la P-selectina-E e possono essere rivestiti sul MicroSlide e le interazioni con le cellule tumorali possano essere rispettati in condizioni di flusso laminare 10.

<table border="0" cellpadding="0" cellspacing="0" width="744">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Microslide</td>
			<td style="width:192px;">Ibidi</td>
			<td style="width:119px;">80331</td>
			<td style="width:189px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Fibronectin</td>
			<td style="width:192px;">Millipore</td>
			<td style="width:119px;">FC010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Plastic tubing</td>
			<td style="width:192px;">Tygon</td>
			<td style="width:119px;">AAQ04103</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Male luer adapter</td>
			<td style="width:192px;">GlycoTech</td>
			<td style="width:119px;">31-001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Female luer adapter</td>
			<td style="width:192px;">GlycoTech</td>
			<td style="width:119px;">31-001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Syringe pump</td>
			<td style="width:192px;">Chemyx Inc</td>
			<td style="width:119px;">Fusion 100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Luer-lock syringe</td>
			<td style="width:192px;">BD Biosciences</td>
			<td style="width:119px;">309628</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">M199 medium</td>
			<td style="width:192px;">Sigma</td>
			<td style="width:119px;">M7653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Endothelial Mitogen</td>
			<td style="width:192px;">Biomedical Technologies</td>
			<td style="width:119px;">BT-203</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">HBSS</td>
			<td style="width:192px;">Sigma</td>
			<td style="width:119px;">H9269</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:244px;">Anti-PSMA J591-488</td>
			<td style="width:192px;">Weill Cornell Medical College-Lab of Urologic Oncology</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Interleukin-1 beta</td>
			<td style="width:192px;">Peprotech</td>
			<td style="width:119px;">200-01B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Trypsin</td>
			<td style="width:192px;">Millipore</td>
			<td style="width:119px;">SM-2002-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Heparin</td>
			<td style="width:192px;">Sigma</td>
			<td style="width:119px;">H-3149</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:244px;">HUVECs</td>
			<td style="width:192px;">Weill Cornell Medical College-Department of Surgery</td>
			<td style="width:119px;"></td>
			<td>provided by Marco Seandel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">VE-Cadherin</td>
			<td style="width:192px;">Santa Cruz</td>
			<td style="width:119px;">sc-5648</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">10x objective</td>
			<td style="width:192px;">Zeiss Plan Neofluar</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:244px;">Enzyme free cell dissociation reagent</td>
			<td style="width:192px;">Millipore</td>
			<td style="width:119px;">S-004-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">RPMI-1640</td>
			<td style="width:192px;">Lonza</td>
			<td style="width:119px;">12-702-F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Ficoll-paque plus</td>
			<td style="width:192px;">GE healthcare</td>
			<td style="width:119px;">17-1440-02</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro method to observe e-selectin-mediated interactions between prostate circulating tumor cells derived from patients and human endothelial cells

La metastasi è un processo in cui le cellule tumorali capannone dal tumore primario intravasate vascolare sangue e del sistema linfatico, in tal modo, l&#39;accesso a estravasare e formare una nicchia secondaria. Il stravaso delle cellule tumorali dal sistema vascolare sanguigno può essere studiata utilizzando cellule endoteliali (EC) e cellule tumorali ottenute da linee cellulari differenti. Gli studi iniziali sono stati condotti utilizzando condizioni statiche, ma si è ben documentato che i CE si comportano in modo diverso in condizioni di flusso fisiologiche. Pertanto, diversi gruppi da camera di flusso sono attualmente utilizzate per studiare le interazioni delle cellule cancro con EC. Assemblee camera di flusso attuali offrono risultati riproducibili utilizzando sia linee cellulari diversi o fluido a differenti condizioni di stress di taglio. Tuttavia, per osservare e studiare le interazioni con cellule rare come cellule tumorali circolanti (CTC), sono necessarie alcune modifiche da apportare al gruppo di camera di flusso convenzionale. CTCsono una popolazione di cellule rare tra milioni di cellule del sangue. Di conseguenza, è difficile ottenere una popolazione pura di CTC. Contaminazione delle CTC con diversi tipi di cellule che normalmente si trovano in circolazione è inevitabile utilizzare presente arricchimento o tecniche di esaurimento. Nel presente rapporto, descriviamo un metodo unico per etichette circolanti cellule tumorali della prostata modo fluorescente e studiare le loro interazioni con EC in un sistema di camera di flusso auto-assemblati. Questa tecnica può essere ulteriormente applicato ad osservare le interazioni tra CTC prostata e qualsiasi proteina di interesse.

La Figura 1 mostra una cultura O / N di un monostrato di cellule endoteliali sulla MicroSlide. I valori di scala sulla figura 1A mostra che il 100% del MicroSlide è visibile usando obiettivo 5X mentre il 70% è visibile usando un obiettivo 10X (Figura 1B). Per interazioni E-selectina mediata, cellule rotolamento ai bordi non sono considerati che rende più del 70% del MicroSlide disponibile per il video-registrazione e analisi riproduzione. Nella nostra esperienza, ini...

Watch this Scientific Journal Video about In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells at JoVE

In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

Il nostro rapporto descrive un metodo unico per visualizzare e analizzare le interazioni CTC / CE del cancro della prostata in condizioni di flusso fisiologiche.

Nel metodo in vitro per osservare le interazioni E-selectina-mediata Tra prostata cellule tumorali circolanti derivate da pazienti e Cellule endoteliali umane

Metastasis is a process in which tumor cells shed from the primary tumor intravasate blood vascular and lymphatic system, thereby, gaining access to ...

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Research

JoVE Journal

Medicine

In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

9.0K Views.  Weill Cornell Medical College.  Il nostro rapporto descrive un metodo unico per visualizzare e analizzare le interazioni CTC / CE del cancro della prostata in condizioni di flusso fisiologiche. 

Video: In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Differenziazione Isolamento, caratterizzazione e comparata delle cellule umane Dental Pulp staminali derivate da denti permanenti mediante due diversi metodi

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Ricerca

Medicina

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Paziente coltura cellulare derivato e isolamento di CD133<sup&gt; +</sup&gt; Putativi staminali alle cellule tumorali di melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

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 ...

Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with muscular dystrophy is an invaluable cellular source for in&#160;vitro disease model systems and can be used for drug screening studies. Patient-derived urine cells have been used in successful reprogramming into induced pluripotent stem cells in order to model dystrophic cardiomyopathy1. Addressing the safety concerns of integrating vector systems, we present a protocol using a non-integrating Sendai virus vector for transduction of Yamanaka factors into urine cells collected from patients with muscular dystrophy. This protocol generates fully reprogrammed clones within 2&#8211;3 weeks. The pluripotent cells are vector-free by passage-13. These dystrophic iPSCs can be differentiated into cardiomyocytes and used either to study disease mechanisms or for drug screening.

This protocol entails detailed procedures for isolation of urine derived cells from muscular dystrophy patients; their efficient and rapid reprogramming through Sendai virus transduction.

Generazione di pluripotenti indotte cellule staminali da distrofia muscolare di pazienti: Efficient Riprogrammazione senza integrazione di origine urinaria Cells

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with ...

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent &#8216;floater&#8217; cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

In Arricchimento vitro di Ovarian Cancer Cells-tumorali avvio

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Studiare cancro del pancreas staminali caratteristiche cellulari per lo sviluppo di nuove strategie di trattamento

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

Isolamento e propagazione di cellule tumorali circolanti da un mouse cancro modello

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

Isolamento e cultura espansione delle cellule endoteliali tumore-specifici

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

Generazione di cancro alla prostata del paziente Derivato modelli di xenotrapianto di cellule tumorali circolanti

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

L&#39;esaurimento delle cellule di topo da xenotrapianti tumorali umani migliora in modo significativo Analisi A valle delle cellule bersaglio

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...