1. Ensayo de Set-up <ol><li> Descongelar una alícuota pequeña (&lt;500 l) de Matrigel en hielo a 4 ° C (aproximadamente 2 horas). </li><li> Prepare la receta de gel (véase el volumen de ejemplo en la tabla a continuación): 10x PBS (1x en el volumen total), hidróxido de sodio 1N (equivalente a 0.023 volúmenes de colágeno añadido, o por las recomendaciones del fabricante del colágeno, según corresponda), Matrigel y el colágeno de tipo I a la las concentraciones finales de 1 mg / ml y 1,3 mg / ml, respectivamente (otras formulaciones de la matriz pueden ser utilizados en función del tipo de célula y el experimento). </li></ol> Ejemplo de receta de gel de <table border="1"><tbody><tr><td> Componentes </td><td> Concentración de archivo </td><td> La concentración final </td><td> Añadir un volumen </td></tr><tr><td> PBS 10 veces </td><td> 10 veces </td><td> 1X </td><td> 0.090 ml </td></tr><tr><td> Agua estéril </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> Cola de rata colágeno 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> Mezclar los componentes del gel sobre hielo en la misma secuencia que el anterior y solución final se incuba durante 1 hora a 4 ° C. En nuestra experiencia, una incubación de 1 hora antes de la siembra de células resultados en una gelificación más uniforme del colágeno. Asegúrese de mantener Matrigel y el colágeno en el hielo en todo momento y para trabajar lo más rápido posible para evitar la gelificación. </li><li> Colocar 12 mm de diámetro 8 micras de poro insertos de cultivo de células en una placa de 12 pocillos usando pinzas esterilizadas. </li><li> Contar las células y resuspender en medios libres de suero a 5 x 10 6 células / ml (volumen total debe ser de 10% de la solución de gel). </li><li> Añadir 100 l de Su celularspension a 900 l de solución de gel (concentración final de células de 5 x 10 5 células / ml) y mezclar bien con la pipeta suavemente hacia arriba y hacia abajo. </li><li> Añadir 150 ml de la mezcla final de cada inserto y la transferencia a un 37 ° C, 5% de CO 2 incubadora durante 30 minutos hasta que el gel se polimeriza. </li><li> Utilizando medios libres de suero, </li></ol><ul><li> Añadir 100 l en la parte superior del gel y l 1200 bajo el inserto para la condición estática. Los niveles de fluido en el interior del inserto y en el exterior, así debe ser aproximadamente igual, lo que resulta en la diferencia de presión mínima a través del gel y no hay flujo intersticial. </li><li> Añadir 100 l bajo el inserto y por encima de 650 l de gel para la condición de flujo. Tenga cuidado para evitar las burbujas de aire debajo de la inserción, ya que impiden que las células migren a través de la membrana en estos lugares. La diferencia de presión generada por estos volúmenes es de aproximadamente 1,3 cm de H 2 O (o 1 mm de Hg). </li></ul><ol start="9"><li> Coloque la placa en un 37° C, 5% de CO 2 incubadora durante 24 horas. </li></ol> 2. Tinción celular y recuento <ol><li> Añadir 500 l de 1X PBS por pocillo en la placa de nuevo 24-bien, lo que se utiliza para lavar los insertos. </li><li> Eliminar el medio restante en la porción superior de los transwells flujo y determinar el volumen. Calcular el volumen total de eluido restando el volumen restante del volumen total originalmente añadido, 650 l (esto se usa para los cálculos de velocidad de flujo, ver más abajo). Utilizar bastoncillos de algodón para quitar el gel de los insertos y para limpiar la superficie superior de la membrana para eliminar las células no invadidas. Coloque los insertos en la placa de 24 pocillos que contenía 1X PBS durante 15 s para lavar insertos. </li><li> Retire PBS y añadir 500 l de paraformaldehído al 4% (PFA) debajo de cada inserto y se incuba durante 30 minutos a temperatura ambiente para fijar las células transmigrados. </li><li> Retire la PFA y enjuagar una vez con 500 l 1X PBS para eliminar el fijador residual. </li><li> Añadir 500 l of 0,5% Triton X-100 solución por debajo de las inserciones y se incuban durante 10 minutos a temperatura ambiente para permeabilizar las células. </li><li> Cortar las membranas de la inserción utilizando una cuchilla de afeitar y el lugar en 100 l de 2 g / ml DAPI en 1X solución de PBS, teniendo cuidado de colocar la parte inferior de la cara membrana hacia abajo (células transmigrados boca abajo). </li><li> Envolver la placa en papel de aluminio y el lugar en un agitador a 150 rpm durante 30 minutos a temperatura ambiente. </li><li> Lave las membranas en 500 l de PBS 1X en el agitador (repetir 3 veces durante 10 minutos cada uno) para eliminar DAPI libre. </li><li> Coloque las membranas en portaobjetos de vidrio con células transmigrados hacia arriba, agregar la solución de montaje y cubreobjetos. </li></ol> 3. Análisis de Datos <ol><li> Cuente con DAPI núcleos teñidos en 5 lugares seleccionados al azar de cada membrana (mantenerse alejado de los bordes y utilizar un 10X o 20X lente del objetivo). </li><li> Calcular el recuento promedio de células y obtener la invasión por ciento mediante la siguiente fórmula: Invasión Porcentaje = x 100% (recuento medio de células x membrana de superficie) / (área de superficie del número microscopio x imagen de células sembradas) La invasión por ciento puede ser normalizado en cierta condición de control (típicamente la condición estática) para permitir la comparación entre los experimentos independientes. </li><li> Calcular el caudal medio: dividir el volumen total eluida en la condición de flujo por el tiempo de incubación (por ejemplo, 24 h). </li><li> Calcular la velocidad de flujo media: dividir el caudal medio por el área transversal del gel / membrana (en este caso de 60 mm 2). </li></ol> 4. Los resultados representativos Para medir la invasión de células tumorales bajo flujo intersticial, hemos realizado nuestra 3-D ensayo de invasión de flujo utilizando MDA-MB-435S células de melanoma metastásico. Estas células han sido demostrado para invadir en respuesta al flujo de fluido intersticial 13-14. Las células fueron incorporados en una matriz compuesta de 1,3 mg / ml de rata taitendón l colágeno de tipo I y 1 mg / ml de matriz sótano Matrigel membrana celular a una concentración final de 5 x 10 5 células / ml. Dos condiciones se compararon diferentes: (1) el flujo intersticial media = 0,1 m s-1 y (2) condición estática = sin flujo mensurable (Figura 1). Después de 24 horas, las células que invaden a través de los poros de la membrana se tiñeron con DAPI para facilitar el recuento de células. La figura 2 muestra una imagen representativa de las células invadidas. Bajo claro, sólo los poros de la membrana son visibles. Uso de fluorescencia, los núcleos con DAPI-manchado fueron utilizados para el recuento de células y las estructuras manchadas de phalloidin F-actina se utiliza para visualizar el cuerpo celular (opcional). El uso de un de t de Student asumiendo varianzas iguales, se demostró que el flujo intersticial aumenta significativamente la MDA-MB-435S invasión de las células en 2,3 veces en condiciones estáticas (p = 0,003) (Figura 3). Esta corroboraciónTes resultados similares (pero con diferentes condiciones de la matriz y por lo tanto las velocidades de flujo) utilizando esta línea celular 13-14. <img alt="Figura 1" src="/files/ftp_upload/4159/4159fig1.jpg" /> Figura 1. Esquemática del ensayo de 3-D invasión intersticial de flujo de fluido. En primer lugar preparar la solución en gel utilizando concentraciones apropiadas y volúmenes. Luego añadir las células a la solución de gel y transferir a los insertos de cultivo celular. Por último agregar el volumen adecuado de los medios de comunicación a cada condición y se incuba. El flujo de fluido intersticial es impulsado por una cabeza de presión de fluido. <img alt="Figura 2" src="/files/ftp_upload/4159/4159fig2.jpg" /> Figura 2. Transmigrado MDA-MB-435S celdas de membrana. Células invadidas fueron fijadas después de nuestra invasión de ensayo de flujo del líquido intersticial y se tiñeron con DAPI y Alexa Fluor 488-conjugado phalloidin para facilitar el recuento de las células invadidas, una imagen) de la membrana en campo claro; B) DAPI estaciónnúcleos INED (en azul), C) Alexa Fluor 488-phalloidin manchas F-actina (en verde). Barra de escala representa 50 micras. <img alt="Figura 3" src="/files/ftp_upload/4159/4159fig3.jpg" /> Figura 3. El aumento de la invasión de MDA-MB-435S células bajo flujo. Invasión de la célula después de 24 h mejorado significativamente por el flujo intersticial (p = 0,003). Los resultados se normalizan a la condición estática y el promedio de los valores representan la media ± SEM de 6 inserciones de cultivo celular.

<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>

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Aquí hemos descrito una metodología para cuantificar el efecto del flujo intersticial en la invasión de células tumorales, utilizando células embebidas en una matriz de 3-D dentro de un inserto de cultivo celular. Este y otros métodos se han utilizado para estudiar el efecto del flujo intersticial en una variedad de tipos celulares 13-15, 17. Nuestro enfoque parte imita la matriz de microambiente del tumor mediante el uso de colágeno tipo I y Matrigel que contienen proteínas que se encuentran en la me...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Colágeno (cola de rata) </td><td> BD </td><td> 354236 </td><td> Mantener estériles </td></tr><tr><td> Celular Millicell la cultura de inserción </td><td> Millipore </td><td> PI8P01250 </td><td> 8 micras de diámetro de poro, policarbonato membrana </td></tr><tr><td> Matrigel </td><td> BD </td><td> 354234 </td><td> Mantener estériles </td></tr><tr><td> PBS </td><td> Sigma Aldrich </td><td> 100M-8202 </td><td> 10 veces para preparar solución de gel, 1x para el lavado de los pasos </td></tr><tr><td> Hidróxido de sodio, solución 1,0 N </td><td> Sigma Aldrich </td><td> S2770 </td><td> Mantener estériles </td></tr><tr><td> DMEM 1X </td><td> CellGro </td><td> 10 a 013 CV </td><td> Mantener estériles </td></tr><tr><td> Suero Bovino Fetal </td><td> Atlanta Productos Biológicos </td><td> 511150 </td><td> Mantener estériles </td></tr><tr><td> La penicilina estreptomicina </td><td> CellGro </td><td> 30002CI </td><td> Mantener estériles </td></tr><tr><td> Triton X-100 </td><td> Sigma Aldrich </td><td> X100-500 ml </td><td> 0,5% en PBS </td></tr><tr><td> Paraformaldehído </td><td> Fisher Scientific </td><td> 04042-500 </td><td> 4% en PBS </td></tr><tr><td> Agua desionizada </td><td></td><td></td><td> Mantener estériles </td></tr><tr><td> 4 &#39;,6-diaminido-2-phenylindole (DAPI) </td><td> MP Biomedicals </td><td> 0215757401 </td><td> 1 mg / ml de solución de stock </td></tr><tr><td> Solución de montaje </td><td> Thermo Scientific </td><td> TA-030-FM </td><td></td></tr><tr><td> Tripsina-EDTA </td><td> CellGro </td><td> 25 a 052-CI </td><td> Mantener estériles </td></tr></tbody> &lt;/ Table&gt;

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

El crecimiento y la progresión de la mayoría de los tumores sólidos depende de la transformación inicial de las células cancerosas y su respuesta a estroma asociada a la señalización en el microambiente del tumor 1. Anteriormente, la investigación sobre el microambiente del tumor se ha centrado principalmente en tumores del estroma interacciones 1-2. Sin embargo, el microambiente del tumor también incluye una variedad de fuerzas biofísicas, cuyos efectos siguen siendo poco conocidos. Estas fuerzas son las consecuencias biomecánicas de crecimiento de los tumores que conducen a cambios en la expresión génica, la división celular, la diferenciación y la invasión 3. Densidad de matriz 4, rigidez 5-6, 6-7 y la estructura, la presión del fluido intersticial 8, y el flujo de fluido intersticial 8 están alterados durante la progresión del cáncer. El flujo de fluido intersticial en particular es mayor en los tumores en comparación con 8-10 tejidos normales. La baja estima que el líquido intersticialvelocidades ow se midieron y se encontró que en el intervalo de 0,1-3 micras s -1, dependiendo del tamaño del tumor y la diferenciación 9, 11. Esto es debido a la presión elevada del líquido intersticial causada por el tumor angiogénesis inducida por aumento de la permeabilidad vascular y 12. El flujo de fluido intersticial se ha demostrado que aumenta la invasión de las células cancerosas 13-14, fibroblastos vasculares y células musculares lisas 15. Esta invasión puede ser debido a los gradientes quimiotácticos autólogas creadas en torno a las células en 3-D de 16 o mayor metaloproteinasas de matriz (MMP) la expresión 15, la secreción de quimioquinas y la adhesión celular expresión de la molécula 17. Sin embargo, el mecanismo por el cual las células detectar el flujo de fluido no se entiende bien. Además de alterar el comportamiento de las células tumorales, el flujo de líquido intersticial modula la actividad de otras células en el microambiente tumoral. Está asociada con la diferenciación de conducción (a) de fibroblastos en promotor de tumores myofibroblasts 18, (b) al transporte de los antígenos y otros factores solubles a los ganglios linfáticos 19, y (c) modulación de morfogénesis linfático de células endoteliales 20. La técnica que aquí se presenta impone el flujo de fluido intersticial en células in vitro y cuantifica sus efectos sobre la invasión (Figura 1). Este método ha sido publicada en múltiples estudios para medir los efectos de flujo de fluido en estroma y la invasión de las células cancerosas 13-15, 17. Al cambiar la composición de la matriz, el tipo de célula, y la concentración celular, este método puede ser aplicado a otras enfermedades y sistemas fisiológicos para estudiar los efectos del flujo intersticial en los procesos celulares tales como la invasión, la diferenciación, proliferación y la expresión génica.

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

El flujo de fluido intersticial es elevada en los tumores sólidos y puede modular la invasión de células tumorales. Aquí se describe una técnica para aplicar el flujo de fluido intersticial a las células embebidas en una matriz y luego medir sus efectos sobre la invasión de células. Esta técnica se puede adaptar fácilmente para estudiar otros sistemas.

Tridimensional de la célula modelo de cultivo para medir los efectos del flujo del fluido intersticial en la invasión de células tumorales

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 vistas.  Drexel University. El flujo de fluido intersticial es elevada en los tumores sólidos y puede modular la invasión de células tumorales. Aquí se describe una técnica para aplicar el flujo de fluido intersticial a las células embebidas en una matriz y luego medir sus efectos sobre la invasión de células. Esta técnica se puede adaptar fácilmente para estudiar otros sistemas.

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.

Los hidrogeles de alginato para la cultura del órgano en tres dimensiones de los ovarios y oviductos

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 ...

Investigación

Bioingeniería

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.

Investigación De La Separación De Flujo Tridimensional Inducida Por Un Pólipo Modelo De Pliegue Vocal

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.

Ingeniería de tejidos de tumor del estroma Microambiente con Aplicación a la invasión de células cancerosas

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.

Análisis de la migración de células dentro de una matriz de colágeno tridimensional

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.

La transfección de genes hacia células Esferoide sobre placas micropatterned Cultura para el trasplante de células genéticamente modificadas

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.

La fabricación de cristal invertida coloidal poli (etilenglicol) Andamios: Una célula Plataforma de cultivo tridimensional para ingeniería de tejidos de hígado

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.

Caracterización de la migración de la célula en tres dimensiones In Vitro herida ambientes

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.

Evaluación de la proliferación del Keratinocyte en dos y tres dimensiones substratos de colágeno de 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.

Uso de Bioink de hidrogel multicapa en bioimpresión tridimensional para distribución celular homogénea

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.

Clarificación primaria del fluido de cultivo celular cosechado con CHO utilizando un separador acústico

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