Name: three-dimensional cell culture model for measuring the effects of interstitial fluid flow on tumor cell invasion
Uploaded: 2012-07-25T12:00:00
Description: Watch this Scientific Journal Video about Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion at JoVE.com

1. Assay Set-up <ol><li> Scongelare una piccola aliquota (&lt;500 pl) di Matrigel in ghiaccio a 4 ° C (circa 2 ore). </li><li> Preparare la ricetta gel (veda che i volumi di esempio nella tabella sottostante): 10x PBS (1x in volume totale), idrossido di sodio 1N (equivalente a 0.023 volumi di collagene aggiunta, o le raccomandazioni del costruttore del collagene, se del caso), Matrigel e collagene di tipo I a concentrazione finale di 1 mg / ml e 1,3 mg / ml, rispettivamente (formulazioni di matrice possono essere uti...

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Qui abbiamo descritto un metodo per quantificare l&#39;effetto del flusso interstiziale invasione delle cellule tumorali, utilizzando cellule annegate in una matrice 3-D in un inserto di coltura cellulare. Questi metodi e simili sono stati usati per studiare l&#39;effetto del flusso interstiziale su una varietà di tipi cellulari 13-15, 17. Il nostro approccio imita parzialmente il microambiente matrice del tumore mediante collagene di tipo I e Matrigel che contengono proteine ​​che si trovano nella membr...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> Collagene (coda di topo) </td><td> BD </td><td> 354236 </td><td> Tenere sterile </td></tr><tr><td> Millicell cellule inserto cultura </td><td> Millipore </td><td> PI8P01250 </td><td> 8 diametro dei pori um, policarbonato membrana </td></tr><tr><td> Matrigel </td><td> BD </td><td> 354234 </td><td> Tenere sterile </td></tr><tr><td> PBS </td><td> Sigma Aldrich </td><td> 100M-8202 </td><td> 10x per la preparazione di soluzione di gel, 1x per i lavaggi </td></tr><tr><td> Idrossido di sodio, soluzione 1.0N </td><td> Sigma Aldrich </td><td> S2770 </td><td> Tenere sterile </td></tr><tr><td> DMEM 1X </td><td> Cellgro </td><td> 10-013-CV </td><td> Tenere sterile </td></tr><tr><td> Siero fetale bovino </td><td> Atlanta Biologicals </td><td> 511150 </td><td> Tenere sterile </td></tr><tr><td> Penicillina Streptomicina </td><td> Cellgro </td><td> 30002CI </td><td> Tenere sterile </td></tr><tr><td> Triton X-100 </td><td> Sigma Aldrich </td><td> X100-500 ml </td><td> 0,5% in PBS </td></tr><tr><td> Paraformaldeide </td><td> Fisher Scientific </td><td> 04042-500 </td><td> 4% in PBS </td></tr><tr><td> Acqua deionizzata </td><td></td><td></td><td> Tenere sterile </td></tr><tr><td> 4 &#39;,6-diaminido-2-fenilindolo (DAPI) </td><td> MP Biomedicals </td><td> 0215757401 </td><td> 1 mg / ml di soluzione concentrata </td></tr><tr><td> Soluzione di montaggio </td><td> Thermo Scientific </td><td> TA-030-FM </td><td></td></tr><tr><td> Tripsina-EDTA </td><td> Cellgro </td><td> 25-052-CI </td><td> Tenere sterile </td></tr></tbody> &lt;/ Table&gt;

three-dimensional cell culture model for measuring the effects of interstitial fluid flow on tumor cell invasion

La crescita e la progressione della maggior parte dei tumori solidi dipendono dalla trasformazione iniziale delle cellule tumorali e la loro risposta a stroma segnalazione associata nel microambiente tumorale 1. In precedenza, la ricerca sul microambiente tumorale si è concentrata principalmente sui tumori stromali interazioni 1-2. Tuttavia, il microambiente tumorale include anche una varietà di forze biofisiche, i cui effetti sono scarsamente compreso. Queste forze sono le conseguenze biomeccaniche della crescita tumorale che portano a cambiamenti nell&#39;espressione genica, la divisione cellulare, la differenziazione e l&#39;invasione 3. Densità Matrix 4, rigidità 5-6, 6-7 e la struttura, la pressione del fluido interstiziale 8, e il flusso di fluido interstiziale 8 sono modificati durante la progressione del cancro. Flusso di fluido interstiziale in particolare è maggiore nei tumori rispetto ai normali tessuti 8-10. La stima fl liquido interstizialevelocità ow stati misurato e trovato essere nell&#39;intervallo di 0,1-3 um s -1, a seconda delle dimensioni del tumore e differenziazione 9, 11. Ciò è dovuto alla elevata pressione del fluido interstiziale causata da angiogenesi indotta da tumore e aumento della permeabilità vascolare 12. Flusso di fluido interstiziale ha dimostrato di aumentare l&#39;invasione delle cellule tumorali 13-14 vascolari, fibroblasti e cellule muscolari lisce 15. Questa invasione può essere dovuto a gradienti chemiotattici autologhe create attorno cellule in 3-D 16 o maggiore di metalloproteinasi di matrice (MMP) espressione 15, secrezione di chemochine e l&#39;adesione delle cellule espressione molecola di 17. Tuttavia, il meccanismo con cui le cellule rilevare il flusso di fluido non è ben compreso. Oltre a modificare il comportamento delle cellule tumorali, flusso di fluido interstiziale modula l&#39;attività di altre cellule nel microambiente tumorale. E &#39;associato con (a) differenziazione dei fibroblasti di guida in tumore di promozione dei myofibroblasts 18, (b) trasporto di antigeni e di altri fattori solubili ai linfonodi 19, e (c) modulante morfogenesi delle cellule endoteliali linfatico 20. La tecnica qui presentata impone flusso di liquido interstiziale su cellule in vitro e quantifica suoi effetti sulla invasione (Figura 1). Questo metodo è stato pubblicato in diversi studi per misurare gli effetti del flusso di fluido sulla stromali e invasione cellulare del cancro 13-15, 17. Variando la composizione della matrice, tipo di cellula, e la concentrazione cellulare, questo metodo può essere applicato ad altre malattie e sistemi fisiologici per studiare gli effetti del flusso interstiziale processi cellulari come l&#39;invasione, la differenziazione, proliferazione, e l&#39;espressione genica.

Watch this Scientific Journal Video about Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion at JoVE.com

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

Flusso di fluido interstiziale è elevata in tumori solidi e in grado di modulare l&#39;invasione delle cellule tumorali. Qui si descrive una tecnica per applicare il flusso di fluido interstiziale alle cellule incorporati in una matrice e quindi misurare i suoi effetti sulla invasione cellulare. Questa tecnica può essere facilmente adattato per studiare altri sistemi.

Tridimensionale Cultura modello cellulare per misurare gli effetti del flusso del fluido interstiziale sulla Invasion cellule tumorali

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