1. Essai Set-up <ol><li> Décongeler une aliquote de petite taille (&lt;500 pi) de Matrigel sur la glace à 4 ° C (environ 2 h). </li><li> Préparez la recette du gel (voir les volumes, par exemple dans le tableau ci-dessous): 10x PBS (1x dans le volume total), l&#39;hydroxyde de sodium 1N (équivalent à 0,023 volumes de collagène ajoutée, ou par les recommandations du fabricant du collagène, le cas échéant), le Matrigel et du collagène de type I à des concentrations finales de 1 mg / ml et 1,3 mg / ml, respectivement (formulations de la matrice d&#39;autres peuvent être utilisés en fonction du type de cellule et de l&#39;expérience). </li></ol> Recette gel Exemple <table border="1"><tbody><tr><td> Composants </td><td> Concentration d&#39;images </td><td> Concentration finale </td><td> Ajouter le volume </td></tr><tr><td> PBS 10X </td><td> 10X </td><td> 1X </td><td> 0,090 ml </td></tr><tr><td> L&#39;eau stérile </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> La queue de type I de rat collagène </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> Mélanger les composants du gel sur la glace dans la même séquence que ci-dessus et incuber la solution finale pendant 1 h à 4 ° C. Dans notre expérience, une incubation de 1 h avant l&#39;ensemencement des résultats de cellules dans une gélification plus uniforme du collagène. Assurez-vous de garder Matrigel et du collagène sur la glace en tout temps et à travailler aussi vite que possible pour empêcher la gélification. </li><li> Placer les 12 mm de diamètre 8 um pores inserts de culture cellulaire dans une plaque de 12 puits en utilisant des pinces stérilisées. </li><li> Compter les cellules et remettre en suspension dans un milieu sans sérum à 5 x 10 6 cellules / ml (devraient être volume total de 10% de la solution de gel). </li><li> Ajouter 100 ul de cellules suspension à 900 pi de solution de gel (concentration cellulaire finale de 5 x 10 5 cellules / ml) et bien mélanger par pipetage doucement de haut en bas. </li><li> Ajouter 150 ul du mélange final à chaque insertion et le transfert à un 37 ° C, 5% de CO 2 incubateur pendant 30 minutes jusqu&#39;à ce que gel polymérise. </li><li> Utilisation de milieux sans sérum, </li></ol><ul><li> Ajouter 100 pi au-dessus du gel et 1200 pi sous l&#39;insert pour l&#39;état statique. Les niveaux de liquide à l&#39;intérieur de l&#39;insert et à l&#39;extérieur dans le puits doit être approximativement égale, résultant en la différence de pression minimale dans le gel et aucun flux interstitiel. </li><li> Ajouter 100 pi sous l&#39;insert et 650 pi au-dessus de gel pour la condition de flux. Soyez prudent pour éviter les bulles d&#39;air sous l&#39;insert, car ils empêchent les cellules de migrer à travers la membrane à ces endroits. La différence de pression générée par ces volumes est d&#39;environ 1,3 cm H 2 O (ou 1 mm Hg). </li></ul><ol start="9"><li> Placer la plaque dans un 37° C, 5% de CO 2 incubateur pendant 24 heures. </li></ol> 2. Cellule de coloration et de comptage <ol><li> Ajouter 500 ul de PBS 1X par puits dans de nouvelles de 24 puits de plaque, ce qui sera utilisée pour laver les inserts. </li><li> Retirer le milieu restant dans la partie supérieure des transwells écoulement et déterminer le volume. Calculer le volume total éluée en soustrayant le volume restant du volume total initialement ajouté, 650 pi (il sera utilisé pour les calculs de débit, voir ci-dessous). Utilisez des cotons-tiges pour enlever le gel des inserts et d&#39;essuyer la surface supérieure de la membrane pour éliminer les cellules non-envahis. Placez les inserts dans la plaque de 24 puits contenant du PBS 1X pendant 15 s pour laver inserts. </li><li> Retirez du PBS et ajouter 500 ul de paraformaldéhyde à 4% (PFA) en dessous de chaque insert et incuber pendant 30 minutes à température ambiante pour fixer les cellules transmigré. </li><li> Retirez la PFA et rincer une fois avec 500 pl 1X PBS pour éliminer fixateur résiduel. </li><li> Ajouter 500 ul of 0,5% Triton X-100 solution sous les inserts et les incuber pendant 10 minutes à température ambiante pour perméabiliser les cellules. </li><li> Couper les membranes de l&#39;insert à l&#39;aide d&#39;une lame de rasoir et le placer dans 100 pi de 2 ug / ml dans du PBS 1X DAPI solution, en prenant soin de placer la partie inférieure du visage membrane vers le bas (cellules transmigré face vers le bas). </li><li> Entourer la plaque dans de l&#39;aluminium et le placer sur un agitateur à 150 tours par minute pendant 30 minutes à température ambiante. </li><li> Laver les membranes dans 500 ul de PBS 1X sur agitateur (répéter 3 fois pendant 10 minutes chaque) pour enlever DAPI libre. </li><li> Membranes placer sur des lames de verre avec des cellules transmigré vers le haut, ajouter la solution de montage et de lamelle. </li></ol> 3. Analyse des données <ol><li> Comptez DAPI-noyaux colorés sur 5 sites choisis au hasard de chaque membrane (rester à l&#39;écart des bords et utilisez un 10X ou 20X objectif). </li><li> Calculer la moyenne et le nombre de cellules obtenir l&#39;invasion pour cent en utilisant la formule suivante: Invasions = x pour cent à 100% (moyenne numération cellulaire x surface membranaire) / (surface de l&#39;image de microscope nombre x de cellules ensemencées) L&#39;invasion pour cent peut être normalisée à une condition de contrôle (généralement à l&#39;état statique) pour permettre la comparaison entre les expériences indépendantes. </li><li> Calculer le débit moyen: diviser le volume total éluée dans la condition d&#39;écoulement par le temps d&#39;incubation (par exemple, 24 h). </li><li> Calculer la vitesse d&#39;écoulement moyenne: diviser le débit moyen par l&#39;aire de section transversale du gel / membrane (dans ce cas à 60 mm 2). </li></ol> 4. Les résultats représentatifs Pour mesurer l&#39;invasion des cellules tumorales sous flux interstitiel, nous avons effectué notre test 3-D invasion de flux en utilisant MDA-MB-435S cellules de mélanome métastatique. Ces cellules ont déjà été montré à envahir en réponse à l&#39;écoulement du fluide interstitiel 13-14. Les cellules ont été noyées dans une matrice composée de 1,3 mg / ml de rat taiType de l collagène de tendon I et 1 mg / ml de Matrigel matrice de la membrane sous-sol à une concentration cellulaire finale de 5 x 10 5 cellules / ml. Deux conditions différentes ont été comparées: (1) débit moyen interstitielle = 0,1 um s -1 et (2) condition statique = pas de débit mesurable (figure 1). Après 24 h, les cellules qui ont envahi à travers les pores de la membrane ont été colorées avec du DAPI pour faciliter le comptage des cellules. La figure 2 montre une image représentative des cellules envahies. Sous fond clair, que les pores de la membrane sont visibles. Utilisation de la fluorescence, les noyaux au DAPI teinté ont été utilisés pour le comptage des cellules et les phalloïdine colorés F-actine structures ont été utilisés pour visualiser le corps de la cellule (en option). L&#39;utilisation d&#39;un de test t de Student en supposant des variances égales, nous avons montré que l&#39;écoulement interstitiel augmente significativement l&#39;invasion des cellules MDA-MB-435S de 2,3 fois par rapport à des conditions statiques (p = 0,003) (figure 3). Cette corroboraTes résultats similaires (mais avec des conditions différentes de la matrice et par conséquent la vitesse d&#39;écoulement) en utilisant cette ligne 13-14 cellule. <img alt="Figure 1" src="/files/ftp_upload/4159/4159fig1.jpg" /> Figure 1. Schéma de la 3-D dosage de liquide interstitiel invasion de flux. D&#39;abord préparer la solution de gel en utilisant des concentrations appropriées et des volumes. Puis ajouter des cellules à la solution de gel et transfert à inserts de culture cellulaire. Enfin, ajoutez un volume approprié des médias pour chaque condition et incuber. L&#39;écoulement de fluide interstitiel est entraîné par une tête de pression de fluide. <img alt="Figure 2" src="/files/ftp_upload/4159/4159fig2.jpg" /> Figure 2. Transmigré MDA-MB-435S cellules sur la membrane. Cellules envahies ont été fixés après notre test de fluide interstitiel invasion débit et colorées avec du DAPI et Alexa Fluor 488-conjugué phalloïdine pour faciliter le comptage des cellules envahies; une image) de la membrane sous champ lumineux; B) DAPI stanoyaux Ined (en bleu); C) Alexa Fluor 488-phalloïdine teinté F-actine (en vert). La barre d&#39;échelle représente 50 um. <img alt="Figure 3" src="/files/ftp_upload/4159/4159fig3.jpg" /> Figure 3. Augmentation invasion de cellules MDA-MB-435S cellules sous écoulement. L&#39;invasion cellulaire après 24 h significativement renforcée par écoulement interstitiel (p = 0,003). Les résultats sont normalisés à l&#39;état statique moyenne et les valeurs représentent la moyenne ± SEM de 6 inserts de culture cellulaire.

<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>

Pas de conflits d&#39;intérêt déclarés.

Ici, nous avons décrit une méthodologie pour quantifier l&#39;effet de l&#39;écoulement interstitiel sur l&#39;invasion des cellules tumorales, en utilisant des cellules noyées dans une matrice 3-D au sein d&#39;un insert de culture cellulaire. Méthodes et d&#39;autres semblables ont été utilisés pour étudier l&#39;effet de l&#39;écoulement interstitiel sur une variété de types de cellules 13-15, 17. Notre approche reproduit partiellement le microenvironnement de la tumeur matrice en utilisant col...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> Collagène (queue de rat) </td><td> BD </td><td> 354236 </td><td> Gardez stérile </td></tr><tr><td> Insert Millicell culture cellulaire </td><td> Millipore </td><td> PI8P01250 </td><td> 8 diamètre de pore um, membrane de polycarbonate </td></tr><tr><td> Matrigel </td><td> BD </td><td> 354234 </td><td> Gardez stérile </td></tr><tr><td> PBS </td><td> Sigma-Aldrich </td><td> 100M-8202 </td><td> 10x pour préparer la solution de gel, 1x pour les étapes de lavage </td></tr><tr><td> Hydroxyde de sodium, solution 1.0N </td><td> Sigma-Aldrich </td><td> S2770 </td><td> Gardez stérile </td></tr><tr><td> DMEM 1X </td><td> Cellgro </td><td> 10-013-CV </td><td> Gardez stérile </td></tr><tr><td> Sérum fœtal bovin </td><td> Atlanta Biologicals </td><td> 511150 </td><td> Gardez stérile </td></tr><tr><td> Streptomycine Pénicilline </td><td> Cellgro </td><td> 30002CI </td><td> Gardez stérile </td></tr><tr><td> Triton X-100 </td><td> Sigma-Aldrich </td><td> X100-500 ml </td><td> 0,5% dans du PBS </td></tr><tr><td> Paraformaldéhyde </td><td> Fisher Scientific </td><td> 04042-500 </td><td> 4% dans du PBS </td></tr><tr><td> Eau déminéralisée </td><td></td><td></td><td> Gardez stérile </td></tr><tr><td> 4 &#39;,6-diaminido-2-phénylindole (DAPI) </td><td> MP Biomedicals </td><td> 0215757401 </td><td> 1 mg / ml de solution mère </td></tr><tr><td> Solution de montage </td><td> Thermo Scientific </td><td> TA-030-FM </td><td></td></tr><tr><td> Trypsine-EDTA </td><td> Cellgro </td><td> 25-052-CI </td><td> Gardez stérile </td></tr></tbody> &lt;/ Table&gt;

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

La croissance et la progression de la plupart des tumeurs solides dépendra de la transformation initiale des cellules cancéreuses et leur réponse aux stroma associé à la signalisation dans le microenvironnement de la tumeur 1. Auparavant, la recherche sur le microenvironnement de la tumeur s&#39;est principalement concentrée sur la tumeur du stroma interactions 1-2. Toutefois, le microenvironnement tumoral comprend également une variété de forces, dont les effets biophysiques encore mal compris. Ces forces sont les conséquences biomécaniques de croissance de la tumeur qui entraînent des changements dans l&#39;expression des gènes, la division cellulaire, la différenciation et l&#39;invasion 3. La densité de la matrice 4, la raideur 5-6, et la structure 6-7, la pression du fluide interstitiel 8, et l&#39;écoulement du fluide interstitiel 8 sont tous modifiés lors de la progression du cancer. L&#39;écoulement du fluide interstitiel en particulier est plus élevé dans les tumeurs par rapport à la normale 8-10 tissus. Le fl fluide interstitiel estiméevitesses oe ont été mesurés et jugés dans la gamme de 0,1-3 um s -1, en ​​fonction de la taille tumorale et la différenciation 9, 11. Cela est dû à la pression du fluide interstitiel élevée causée par une tumeur induite par l&#39;angiogenèse et la perméabilité vasculaire accrue 12. L&#39;écoulement du fluide interstitiel a été montré pour augmenter l&#39;invasion des cellules cancéreuses 13-14, les fibroblastes et les cellules musculaires vasculaires lisses 15. Cette invasion peut être due à des gradients chimiotactiques autologues créés autour des cellules en 3-D 16 ou augmenté métalloprotéinases matricielles (MMP) l&#39;expression 15, la sécrétion de chimiokines et de l&#39;expression des molécules d&#39;adhésion cellulaire 17. Cependant, le mécanisme par lequel les cellules détectent l&#39;écoulement du fluide n&#39;est pas bien comprise. En plus de modifier le comportement des cellules tumorales, l&#39;écoulement du fluide interstitiel module l&#39;activité d&#39;autres cellules dans le microenvironnement tumoral. Il est associé à la différenciation de conduite (a) des fibroblastes en promotion de tumeurs myofibroblasts 18, (b) le transport des antigènes solubles et d&#39;autres facteurs de ganglions lymphatiques 19, et (c) moduler la morphogenèse des cellules endothéliales lymphatique 20. La technique présentée ici impose l&#39;écoulement du fluide interstitiel sur des cellules in vitro et quantifie les effets sur l&#39;invasion (figure 1). Cette méthode a été publié dans de nombreuses études pour mesurer les effets de l&#39;écoulement du fluide sur stroma et l&#39;invasion des cellules cancéreuses 13-15, 17. En changeant la composition de la matrice, le type de cellule, et la concentration cellulaire, cette méthode peut être appliquée à d&#39;autres maladies et des systèmes physiologiques d&#39;étudier les effets de l&#39;écoulement interstitiel sur les processus cellulaires tels que l&#39;invasion, la différenciation, la prolifération et l&#39;expression des gènes.

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

L&#39;écoulement du fluide interstitiel est élevée dans les tumeurs solides et peut moduler l&#39;invasion des cellules tumorales. Nous décrivons ici une technique d&#39;appliquer l&#39;écoulement du fluide interstitiel aux cellules noyées dans une matrice, puis de mesurer ses effets sur l&#39;invasion des cellules. Cette technique peut être facilement adapté pour étudier d&#39;autres systèmes.

Tridimensionnelle modèle de culture cellulaire pour mesurer les effets de l&#39;écoulement des fluides interstitiels sur l&#39;invasion tumorale

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 vues.  Drexel University. L'écoulement du fluide interstitiel est élevée dans les tumeurs solides et peut moduler l'invasion des cellules tumorales. Nous décrivons ici une technique d'appliquer l'écoulement du fluide interstitiel aux cellules noyées dans une matrice, puis de mesurer ses effets sur l'invasion des cellules. Cette technique peut être facilement adapté pour étudier d'autres systèmes.

Vidéo: 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.

Les hydrogels d&#39;alginate de culture d&#39;organe en trois dimensions des ovaires et des trompes

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 ...

Recherche

Bio-ingénierie

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.

Étude de la séparation d’écoulement tridimensionnelle induite par un polype de pli vocal modèle

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.

Ingénierie tissulaire de la tumeur stromale Microenvironnement avec application de 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 ...

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.

Analyse de la migration cellulaire dans un tridimensionnelle Matrice Collagène

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.

Transfection génique vers les cellules Spheroid sur microélectrodes plaques de culture destinés à la transplantation de cellules génétiquement modifiées

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.

Fabrication de Inverted colloïdal cristal Poly (éthylène glycol) Échafaudages: Une cellule plate-forme en trois dimensions de la culture pour l&#39;ingénierie tissulaire du foie

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.

Caractérisation de la Migration cellulaire dans les trois dimensions In Vitro plaie environnements

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.

Évaluation de la prolifération des kératinocytes sur deux et trois dimensions des substrats de collagène de Type 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.

Utilisation de Bioink Hydrogel multicouches dans la bioimpression tridimensionnelle pour la distribution homogène des cellules

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.

Clarification primaire du fluide de culture cellulaire récolté par CHO à l’aide d’un séparateur acoustique

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