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 utilizzati altri a seconda del tipo di cellula e esperimento). </li></ol> Esempio di gel ricetta <table border="1"><tbody><tr><td> Componenti </td><td> Concentrazione magazzino </td><td> Concentrazione finale </td><td> Aggiungere il volume </td></tr><tr><td> 10X PBS </td><td> 10X </td><td> 1X </td><td> 0,090 ml </td></tr><tr><td> Acqua sterile </td><td></td><td></td><td> 0,346 ml </td></tr><tr><td> NaOH 1N </td><td></td><td></td><td> 0,008 ml </td></tr><tr><td> Matrigel </td><td> 9,90 mg / ml </td><td> 1 mg / ml </td><td> 0,101 ml </td></tr><tr><td> Rat tail collagene di tipo I </td><td> 3,66 mg / ml </td><td> 1,3 mg / ml </td><td> 0,355 ml </td></tr></tbody></table><ol start="3"><li> Miscelare componenti del gel su ghiaccio nella stessa sequenza come sopra e incubare soluzione finale per 1 ora a 4 ° C. Nella nostra esperienza, a 1 ora di incubazione prima della semina cellulare risultati in una gelificazione più uniforme del collagene. Assicurarsi di mantenere Matrigel e collagene su ghiaccio in ogni momento e di lavorare il più velocemente possibile per evitare la gelificazione. </li><li> Inserire 12 mm di diametro 8 micron pori cultura inserisce in una cella di 12 pozzetti con pinze sterilizzate. </li><li> Contare le cellule e risospendere in mezzi privi di siero a 5 x 10 6 cellule / ml (volume totale dovrebbe essere 10% della soluzione di gel). </li><li> Aggiungere 100 ml di su cellularespensione a 900 microlitri di soluzione di gel (la concentrazione cellulare finale del 5 x 10 5 cellule / ml) e mescolare accuratamente pipettando delicatamente su e giù. </li><li> Aggiungere 150 pl della miscela finale di ciascun inserto e trasferimento a 37 ° C, 5% CO 2 incubatore per 30 minuti fino gel polimerizza. </li><li> Utilizzo di mezzi privi di siero, </li></ol><ul><li> Aggiungere 100 pl sulla superficie del gel e 1200 pl sotto l&#39;inserto per la condizione statica. I livelli di fluido all&#39;interno e all&#39;esterno l&#39;inserto nel pozzetto dovrebbe essere approssimativamente uguale, con conseguente differenza di pressione minima attraverso il gel e senza flusso interstiziale. </li><li> Aggiungere 100 ul sotto l&#39;inserto e 650 microlitri di sopra gel per la condizione FLOW. Fare attenzione ad evitare eventuali bolle d&#39;aria sotto l&#39;inserto, in quanto saranno impedire alle cellule di migrare attraverso la membrana in questi luoghi. La differenza di pressione generata da questi volumi è di circa 1,3 cm H 2 O (o 1 mm Hg). </li></ul><ol start="9"><li> Mettere in una piastra di 37° C, 5% CO 2 incubatore per 24 ore. </li></ol> 2. Colorazione della cellula e di conteggio <ol><li> Aggiungere 500 microlitri di PBS 1X per bene nella nuova piastra da 24 pozzetti, questo verrà utilizzato per lavare gli inserti. </li><li> Rimuovere il mezzo rimanente nella porzione superiore delle transwells flusso e determinare il volume. Calcolare il volume totale eluito sottraendo il volume rimanente del volume totale originariamente aggiunta, 650 pl (questo sarà utilizzato per i calcoli di portata, vedi sotto). Utilizzare tamponi di cotone per rimuovere il gel dalla gli inserti e per pulire la superficie superiore della membrana per rimuovere le cellule non-invaso. Posizionare gli inserti nella piastra da 24 pozzetti contenente 1X PBS per 15 s per lavare inserti. </li><li> Rimuovere PBS e aggiungere 500 microlitri del 4% paraformaldeide (PFA) sotto ciascun inserto e incubare per 30 minuti a temperatura ambiente per fissare le cellule trasmigrato. </li><li> Rimuovere il PFA e risciacquare una volta con 500 microlitri di PBS 1X per rimuovere residui di fissativo. </li><li> Aggiungere 500 ul of 0,5% Triton X-100 soluzione sotto gli inserti e incubare per 10 minuti a temperatura ambiente per permeabile alle cellule. </li><li> Tagliare membrane fuori dell&#39;inserto usando una lama di rasoio e posto in 100 microlitri di 2 ug / ml in soluzione DAPI 1X PBS, avendo cura di posizionare la parte inferiore della faccia membrana verso il basso (cellule trasmigrato faccia in giù). </li><li> Avvolgerla in foglio di alluminio e posto su un agitatore a 150 rpm per 30 minuti a temperatura ambiente. </li><li> Lavare le membrane in 500 microlitri di PBS 1X su Shaker (ripetere 3 volte per 10 minuti ciascuno) per rimuovere DAPI libero. </li><li> Posizionare le membrane su vetrini con le cellule trasmigrato rivolti verso l&#39;alto, aggiungere la soluzione di montaggio e coprioggetto. </li></ol> 3. Analisi dei dati <ol><li> Contare DAPI macchiato di nuclei su 5 posizioni scelte a caso di ogni membrana (stare lontano dai bordi e utilizzare un 10X o 20X lente dell&#39;obiettivo). </li><li> Calcolare il numero medio di cellule e ottenere l&#39;invasione percentuale usando la seguente formula: Percentuale invasione x = 100% (conteggio medio superficie cellulare x membrana superficiale) / (area superficiale del numero x immagine al microscopio di cellule seminate) L&#39;invasione percentuale può essere normalizzato a una condizione di controllo (tipicamente la condizione statica) per consentire un confronto tra esperimenti indipendenti. </li><li> Calcolare la portata media: dividere il volume totale eluito nella condizione di flusso del tempo di incubazione (ad esempio, 24 ore). </li><li> Calcolare la velocità media di flusso: dividere la portata media della sezione trasversale del gel / membrana (in questo caso 60 mm 2). </li></ol> 4. Risultati rappresentativi Per misurare l&#39;invasione delle cellule tumorali in condizioni di flusso interstiziale, abbiamo effettuato il 3-D saggio di invasione flusso utilizzando MDA-MB-435s cellule di melanoma metastatico. Queste cellule sono state precedentemente dimostrato di invadere in risposta al flusso di fluido interstiziale 13-14. Le cellule sono state incorporate in una matrice composta di 1,3 mg / ml ratto tail tendine collagene di tipo I e 1 mg / ml di matrice basale Matrigel membrana ad una concentrazione cellulare finale di 5 x 10 5 cellule / ml. Due condizioni diverse sono stati confrontati: (1) flusso medio interstiziale = 0,1 micron s -1 e (2) = no condizione statica portata misurabile (Figura 1). Dopo 24 ore, le cellule che hanno invaso attraverso i pori della membrana sono stati colorati con DAPI per facilitare il conteggio delle cellule. La figura 2 mostra un&#39;immagine rappresentativa delle cellule invase. In brightfield, solo i pori della membrana sono visibili. Utilizzando fluorescenza, l&#39;DAPI macchiata nuclei sono stati utilizzati per la conta cellulare e le phalloidin macchiati F-actina strutture sono stati utilizzati per visualizzare il corpo cellulare (opzionale). Utilizzando un Student t-test assumendo varianze uguali, abbiamo dimostrato che il flusso interstiziale aumenta significativamente MDA-MB-435s invasione cella di 2,3 volte rispetto alle condizioni statiche (p = 0,003) (Figura 3). Questo corroborates risultati simili (ma con condizioni diverse matrice e quindi velocità di flusso) che utilizzano questa linea cellulare 13-14. <img alt="Figura 1" src="/files/ftp_upload/4159/4159fig1.jpg" /> Figura 1. Schematica del 3-D interstiziale saggio di invasione flusso di fluido. Prima preparare la soluzione gel con concentrazioni appropriate e volumi. Poi aggiungere celle a soluzione di gel e trasferire inserti di coltura cellulare. Infine aggiungete il volume appropriato di media per ogni condizione e incubare. Flusso di fluido interstiziale è guidato da una testa di pressione del fluido. <img alt="Figura 2" src="/files/ftp_upload/4159/4159fig2.jpg" /> Figura 2. Trasmigrato MDA-MB-435s celle di membrana. Le cellule hanno invaso sono state fissate dopo il nostro interstiziale saggio di invasione flusso del fluido e colorati con DAPI e Alexa Fluor 488-coniugato phalloidin per facilitare il conteggio delle cellule invase; A) Immagine della membrana in campo chiaro; B) DAPI STAnuclei ined (in blu); C) Alexa Fluor 488-phalloidin tinto F-actina (in verde). Barra della scala rappresenta 50 micron. <img alt="Figura 3" src="/files/ftp_upload/4159/4159fig3.jpg" /> Figura 3. Maggiore invasione di MDA-MB-435s cellule sotto flusso. Invasione delle cellule dopo 24 ore significativamente migliorata dal flusso interstiziale (p = 0,003). I risultati sono normalizzati alla condizione statica media e valori rappresentano la media ± SEM di inserti di coltura cellulare 6.

<ol>
	<li>Cichon, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microenvironmental+influences+that+drive+progression+from+benign+breast+disease+to+invasive+breast+cancer.">Microenvironmental influences that drive progression from benign breast disease to invasive breast cancer.</a> J. Mammary Gland. Biol. Neoplasia. 15, 389-3897 (2010).</li><li>Proia, D. A., Kuperwasser, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stroma:+tumor+agonist+or+antagonist.">Stroma: tumor agonist or antagonist.</a> Cell Cycle. 4, 1022-1025 (2005).</li><li>Dvorak, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+microenvironment+and+progression.">Tumor microenvironment and progression.</a> J .Surg. Oncol. 103, 468-474 (2011).</li><li>Provenzano, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+density+promotes+mammary+tumor+initiation+and+progression.">Collagen density promotes mammary tumor initiation and progression.</a> BMC Med. 6, 11 (2008).</li><li>Engler, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+elasticity+directs+stem+cell+lineage+specification.">Matrix elasticity directs stem cell lineage specification.</a> Cell. 126, 677-689 (2006).</li><li>Paszek, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tensional+homeostasis+and+the+malignant+phenotype.">Tensional homeostasis and the malignant phenotype.</a> Cancer Cell. 8, 241-254 (2005).</li><li>Levental, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+crosslinking+forces+tumor+progression+by+enhancing+integrin+signaling.">Matrix crosslinking forces tumor progression by enhancing integrin signaling.</a> Cell. 139, 891-906 (2009).</li><li>Butler, T. P., Grantham, F. H., Gullino, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bulk+transfer+of+fluid+in+the+interstitial+compartment+of+mammary+tumors.">Bulk transfer of fluid in the interstitial compartment of mammary tumors.</a> Cancer Res. 35, 3084-3088 (1975).</li><li>Dafni, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+vascular+endothelial+growth+factor+165+drives+peritumor+interstitial+convection+and+induces+lymphatic+drain:+magnetic+resonance+imaging,+confocal+microscopy,+and+histological+tracking+of+triple-labeled+albumin.">Overexpression of vascular endothelial growth factor 165 drives peritumor interstitial convection and induces lymphatic drain: magnetic resonance imaging, confocal microscopy, and histological tracking of triple-labeled albumin.</a> Cancer Res. 62, 6731-6739 (2002).</li><li>Chary, S. R., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+measurement+of+interstitial+convection+and+diffusion+of+albumin+in+normal+and+neoplastic+tissues+by+fluorescence+photobleaching.">Direct measurement of interstitial convection and diffusion of albumin in normal and neoplastic tissues by fluorescence photobleaching.</a> Proc. Natl. Acad. Sci. U.S.A. 86, 5385-5389 (1989).</li><li>Heldin, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+interstitial+fluid+pressure+-+an+obstacle+in+cancer+therapy.">High interstitial fluid pressure - an obstacle in cancer therapy.</a> Nat. Rev. Cancer. 4, 806-813 (2004).</li><li>Fukumura, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+microvasculature+and+microenvironment:+novel+insights+through+intravital+imaging+in+pre-clinical+models.">Tumor microvasculature and microenvironment: novel insights through intravital imaging in pre-clinical models.</a> Microcirculation. 17, 206-225 (2010).</li><li>Shields, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+chemotaxis+as+a+mechanism+of+tumor+cell+homing+to+lymphatics+via+interstitial+flow+and+autocrine+CCR7+signaling.">Autologous chemotaxis as a mechanism of tumor cell homing to lymphatics via interstitial flow and autocrine CCR7 signaling.</a> Cancer Cell. 11, 526-538 (2007).</li><li>Shieh, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cell+invasion+is+promoted+by+interstitial+flow-induced+matrix+priming+by+stromal+fibroblasts.">Tumor cell invasion is promoted by interstitial flow-induced matrix priming by stromal fibroblasts.</a> Cancer Res. 71, 790-800 (2011).</li><li>Shi, Z. D., Wang, H., Tarbell, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparan+sulfate+proteoglycans+mediate+interstitial+flow+mechanotransduction+regulating+MMP-13+expression+and+cell+motility+via+FAK-ERK+in+3D+collagen.">Heparan sulfate proteoglycans mediate interstitial flow mechanotransduction regulating MMP-13 expression and cell motility via FAK-ERK in 3D collagen.</a> PLoS One. 6, e15956 (2011).</li><li>Fleury, M. E., Boardman, K. C., Swartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+morphogen+gradients+by+subtle+interstitial+flow+and+matrix+interactions.">Autologous morphogen gradients by subtle interstitial flow and matrix interactions.</a> Biophys J. 91, 113-121 (2006).</li><li>Miteva, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmural+flow+modulates+cell+and+fluid+transport+functions+of+lymphatic+endothelium.">Transmural flow modulates cell and fluid transport functions of lymphatic endothelium.</a> Circ. Res. 106, 920-931 (2010).</li><li>Ng, C. P., Hinz, B., Swartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interstitial+fluid+flow+induces+myofibroblast+differentiation+and+collagen+alignment+in+vitro.">Interstitial fluid flow induces myofibroblast differentiation and collagen alignment in vitro.</a> J. Cell. Sci. 118, 4731-4739 (2005).</li><li>Kunder, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+cell-derived+particles+deliver+peripheral+signals+to+remote+lymph+nodes.">Mast cell-derived particles deliver peripheral signals to remote lymph nodes.</a> J. Exp. Med. 206, 2455-2467 (2009).</li><li>Helm, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergy+between+interstitial+flow+and+VEGF+directs+capillary+morphogenesis+in+vitro+through+a+gradient+amplification+mechanism.">Synergy between interstitial flow and VEGF directs capillary morphogenesis in vitro through a gradient amplification mechanism.</a> Proc. Natl. Acad. Sci. U.S.A. 102, 15779-15784 (2005).</li><li>McGuire, P. G., Seeds, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interaction+of+plasminogen+activator+with+a+reconstituted+basement+membrane+matrix+and+extracellular+macromolecules+produced+by+cultured+epithelial+cells.">The interaction of plasminogen activator with a reconstituted basement membrane matrix and extracellular macromolecules produced by cultured epithelial cells.</a> J Cell Biochem. 40, 215-227 (1989).</li><li>Kleinman, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+type+IV+procollagen,+laminin,+and+heparan+sulfate+proteoglycan+from+the+EHS+sarcoma.">Isolation and characterization of type IV procollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma.</a> Biochemistry. 21, 6188-6193 (1982).</li><li>Haessler, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+dynamics+of+breast+cancer+cells+in+a+tunable+3D+interstitial+flow+chamber.">Migration dynamics of breast cancer cells in a tunable 3D interstitial flow chamber.</a> Integr. Biol. (Camb). , (2011).</li><li>Polacheck, W. J., Charest, J. L., Kamm, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interstitial+flow+influences+direction+of+tumor+cell+migration+through+competing+mechanisms.">Interstitial flow influences direction of tumor cell migration through competing mechanisms.</a> Proc. Natl. Acad. Sci. U.S.A. 108, 11115-11120 (2011).</li></ol>

Non ci sono conflitti di interesse dichiarati.

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

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

three-dimensional-cell-culture-model-for-measuring-effects

Research

JoVE Journal

Bioengineering

16.4K Views.  Drexel University. Flusso di fluido interstiziale è elevata in tumori solidi e in grado di modulare l'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.

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

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

Idrogeli di alginato per tridimensionale Cultura Organo di ovaie e tube

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

Ricerca

Bioingegneria

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

Investigare la separazione tridimensionale del flusso indotta da un modello di polipo piega vocale

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

Ingegneria dei tessuti del tumore stromale Microenvironment con l&#39;applicazione di Invasion Cancer Cell

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

Analisi della migrazione delle cellule all&#39;interno di una tridimensionale di collagene Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

Gene Transfection verso Celle sferoide su lastre micropatterned coltura per trapianto di cellule geneticamente modificati

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

Fabbricazione di invertito colloidale cristallo poli (glicole etilenico) Scaffold: una piattaforma Cell Culture tridimensionale per il fegato Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

Che caratterizzano la migrazione delle cellule all'interno del tridimensionale In Vitro ferita ambienti

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

Valutazione della proliferazione dei cheratinociti su bi - e tri - dimensionale substrati di collagene di tipo I

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...

Using Multilayered Hydrogel Bioink in Three-Dimensional Bioprinting for Homogeneous Cell Distribution

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by shear stress and improve the survival of the encapsulated cells. However, rapid gravity-driven cell sedimentation in the reservoir could lead to an inhomogeneous cell distribution in bioprinted structures and therefore hinder the application of liquid-like bioinks. Here, we developed a novel multilayered modified strategy for liquid-like bioinks (e.g., gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells. Multiple liquid interfaces were manipulated in the multilayered bioink to provide interfacial retention. Consequently, the cell sedimentation action going across adjacent layers in the multilayered system was retarded in the bioink reservoir. It was found that the interfacial retention was much higher than the sedimental pull of cells, demonstrating a critical role of the interfacial retention in preventing cell sedimentation and promoting a more homogeneous dispersion of cells in the multilayered bioink.

Here, we developed a novel multilayered modified strategy for liquid-like bioinks (gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells.

Utilizzo di Bioinchiostro idrogel multistrato nella biostampa tridimensionale per la distribuzione omogenea delle cellule

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by ...

Primary Clarification of CHO Harvested Cell Culture Fluid using an Acoustic Separator

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested cell culture fluid. While traditional methods like centrifugation or filtration are widely implemented for cell removal, the equipment for these processes have large footprints and operation can involve contamination risks and filter fouling. Additionally, traditional methods may not be ideal for continuous bioprocessing schemes for primary clarification. Thus, an alternate application using acoustic (sound) waves was investigated to continuously separate cells from the cell culture fluid. Presented in this study is a detailed protocol for using a bench-scale acoustic wave separator (AWS) for the primary separation of culture fluid containing a monoclonal IgG1 antibody from a CHO cell bioreactor harvest. Representative data are presented from the AWS and demonstrate how to achieve effective cell clarification and product recovery. Finally, potential applications for AWS in continuous bioprocessing are discussed. Overall, this study provides a practical and general protocol for the implementation of AWS in primary clarification for CHO cell cultures and further describes its application potential in continuous bioprocessing.

Presented here is a protocol for the primary clarification of CHO cell culture using an acoustic separator. This protocol can be used for the primary clarification of shake flask cultures or bioreactor harvests and has the potential application for continuous clarification of the cell bleed material during perfusion bioreactor operations.

Chiarificazione primaria del fluido di coltura cellulare raccolto da CHO utilizzando un separatore acustico

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested ...