1. El establecimiento de tres dimensiones Co-culturas <ol><li> El día antes de establecer la co-culturas, retire el Matrigel del congelador y descongele el hielo en un refrigerador de 4 ° C durante la noche. </li><li> En el día del experimento, preparar el medio para el co-cultivo, que corresponde al medio para las células epiteliales que se utilizarán (medio HMEC en este ejemplo: DME/F12 medio con 10% de suero bovino ternero, 5 ug / ml de insulina , 1 mg / ml de hidrocortisona, y 1 ng / ml de EGF). Mantener el medio en hielo al menos 1 hora antes de mezclar con Matrigel. </li><li> Para diluir el Matrigel descongelado, determinar la cantidad total deseada de Matrigel: medio (300 ml por pocillo de placas de 24 pocillos) y agregue la mitad que el volumen de helado a medio HMEC a un tubo estéril sobre hielo. Luego se agrega el mismo medio-volumen total de Matrigel descongelado al tubo para lograr una dilución 1:1. Mantenga el tubo en hielo. </li><li> Colocar 50 l de Matrigel: mezcla medio mediados de bien en pozos designados de una placa de 24 pocillos yincubar durante 10 min en una incubadora a 37 ° C con aire humidificado y 5% de CO 2. </li><li> Añadir un adicional de 450 l de Matrigel: mezcla de medio a los pocillos y se incuba durante otros 60 minutos. </li><li> Durante la incubación, preparar una suspensión 1:1 de los fibroblastos (aquí h-TER-inmortalizadas fibroblastos reducción mamaria expresan GFP) y células epiteliales (aquí h-TER-inmortalizada humano línea de células epiteliales mamarias expresar SDP) a una concentración de 0,5 x 10 5 células cada una en 0,5 ml de medio HMEC. </li><li> Después de la etapa de incubación de 60 minutos, suavemente caer la suspensión celular en la parte superior del gel solidificado, y devolver la cultura a la incubadora. </li><li> No moleste a los cultivos durante 2 días. Después de esto, medio puede ser cambiado cada 2-3 días por medio de aspiración muy suavemente desde el lado del pozo y suavemente reemplazar con medio fresco HMEC. </li><li> Supervisar la formación esferoide día bajo el microscopio. Las culturas son generalmente madura después de 10-14 días. La imagen comoapropiado usando la luz o microscopía de fluorescencia. El tamaño y la longitud del esferoide de proyección se puede medir con microscopía de luz. </li></ol> 2. Aislamiento de células de Esferoide Co-culturas <ol><li> Muy suavemente, pipetear la cultura y 10x hacia abajo con una solución estéril de 2 ml pipeta de vidrio. Por otra parte, cortar la punta de una punta de pipeta de plástico de 1000 microlitros con unas tijeras esterilizadas en autoclave o por inmersión en etanol al 70% con el fin de la pipeta de la cultura. Transferir el cultivo, ya sea con la pipeta de vidrio o la punta de corte a una pipeta de plástico estéril 15 ml tubo de centrífuga y se centrifuga durante 5 min a 2000 rpm a temperatura ambiente. Los esferoides celulares se sedimento en el fondo. </li><li> Utilizar una punta de pipeta Pasteur estéril para eliminar suavemente el Matrigel interrumpido desde la parte superior del tubo se centrifugó. </li><li> Añadir 1 ml de medio HMEC a los esferoides granuladas, pipetear hacia arriba y abajo 2-3 veces con una pipeta estéril de 2 ml de vidrio, y transferir la cultura de un pozo en un 24 yplaca de cultivo de tejidos. </li><li> Permita que los esferoides para insertarse en el plato por lo menos 48 horas antes de cambiar el medio. Con el tiempo las células se dejan las estructuras esferoidales y crecer como monocapas. </li><li> Pasaje de la célula culturas cuando las células alcanzan el 50% de confluencia. En primer lugar ampliar el cultivo a una placa de 35 mm (aproximadamente 2-4 días), y luego a una cápsula de 100 mm (aproximadamente 3-5 días). Inmediatamente antes de cada paso paso, utilizar una pipeta Pasteur estéril para eliminar manualmente como muchos &quot;esferoides fantasma&quot; residual como sea posible. </li><li> Analice las células por citometría de flujo para la pureza de la población con adecuados marcadores celulares de superficie y otras poblaciones distintas de células diferentes de células de clasificación de fluorescencia, si es necesario. Las células aisladas son ahora listo para su posterior análisis como se desee. </li></ol> 3. Los resultados representativos En tres dimensiones esferoide co-cultivos, los fibroblastos con ingeniería Tiam1 silenciamiento inducir la invasión aumentó en matrix por las células epiteliales mamarias (Figura 1, comparar D, E, F y A, B, C). Los fibroblastos con ingeniería sobre-expresión de Tiam1 inducir invasión disminuyó matriz por la línea celular de cáncer de mama SUM159 (Figura 1, comparar J, K, L a G, H, I). Una vez que la co-cultivadas esferoides se han establecido, las poblaciones de células pueden ser aisladas de esferoides por re-chapado en un contexto de dos dimensiones, como se muestra en la Figura 2. Dependiendo de las tasas relativas de crecimiento de las células de la prueba, las poblaciones puras pueden surgir a través del paso de serie (como se muestra en la Figura 3) o se pueden clasificar utilizando marcadores apropiados de células. Después del aislamiento de 3D co-cultivo, las células epiteliales retener las propiedades conferidas por co-cultivo con fibroblastos durante períodos prolongados de tiempo, permitiendo una amplia oportunidad para el análisis. En este ejemplo, la exposición a Tiam1 deficientes en fibroblastos migración inducida por el aumento en la co-cultivadas HMECs que persistió durante semanas unaD espués de aislamiento de 3D ​​co-cultivo (Figura 4). <img alt="Figura 1" src="/files/ftp_upload/3760/3760fig1.jpg" /> Figura 1. Imagen del esferoide estableció co-cultivos con microscopía de luz y fluorescencia. Culturas esferoides se establecen con líneas de células epiteliales mamarias y de los fibroblastos y se dejan crecer en cultivo durante 10-14 días. AF: epiteliales = HMEC-mCherry células. GL: las células epiteliales = SUM159-mCherry células. AC, GI: = fibroblastos de control RMF-GFP células. DF: fibroblastos = RMF-GFP con Tiam1 silenciamiento. JL: fibroblastos = RMF-GFP con Tiam1 sobre-expresión. Las flechas indican las proyecciones epiteliales invasoras en la matriz. <img alt="Figura 2" src="/files/ftp_upload/3760/3760fig2.jpg" /> Figura 2. Serie de imágenes de las culturas con microscopía de luz y fluorescencia después del aislamiento de la cultura Matrigel. Día 0 representa imágenes inmediatamente después del aislamiento; días 1, 2 y5 indican días después del aislamiento. La corona de células que salen de los aumentos esferoidal en el tiempo. Finalmente, un &quot;fantasma&quot; esferoide con muy pocas células sigue siendo (flecha). Esferoides quedan muy pocos después de la eliminación de fantasmas y el primer paso. <img alt="Figura 3" src="/files/ftp_upload/3760/3760fig3.jpg" /> Figura 3. Análisis de citometría de flujo de las células después del aislamiento de Matrigel co-cultivo. Después de esferoide co-cultivos han madurado, las células se aíslan de la 3D co-cultivo con pipetas y de serie de pases. Alícuotas de las muestras se analizaron por citometría de flujo (en este ejemplo, el uso de fluorescencia de fluorescencia verde y rojo para identificar las buenas prácticas agrarias que expresan los fibroblastos y mCherry que expresan las células epiteliales, respectivamente) para determinar la población relativa de las células epiteliales y fibroblastos. <img alt="Figura 4" src="/files/ftp_upload/3760/3760fig4.jpg" /> Figura 4. La migración Transwell de HMECs persiste después de isolatien Matrigel de co-cultivo. Una vez que las poblaciones puras se han obtenido mediante el cultivo de células de clasificación o de fluorescencia, las células pueden ser analizados como se desee. Aquí la migración de HMECs co-cultivadas con cualquiera de los controles (C) o Tiam1 deficientes (SHT) a través de los fibroblastos transwells perforadas se evaluó en las semanas 2, 4 y 8 después del aislamiento de co-cultivo.

<ol>
	<li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hallmarks+of+cancer.">The hallmarks of cancer.</a> Cell. 100, 57-70 (2000).</li><li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+cancer:+the+next+generation.">Hallmarks of cancer: the next generation.</a> Cell. 144, 646-674 (2011).</li><li>Kim, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+tissue+culture+models+in+cancer+biology.">Three-dimensional tissue culture models in cancer biology.</a> Semin. Cancer Biol. 15, 365-377 (2005).</li><li>Li, H., Fan, X., Houghton, J. <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:+the+role+of+the+tumor+stroma+in+cancer.">Tumor microenvironment: the role of the tumor stroma in cancer.</a> J. Cell Biochem. 101, 805-815 (2007).</li><li>Tse, J. C., Kalluri, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+metastasis:+epithelial-to-mesenchymal+transition+and+contribution+of+tumor+microenvironment.">Mechanisms of metastasis: epithelial-to-mesenchymal transition and contribution of tumor microenvironment.</a> J. Cell Biochem. 101, 816-829 (2007).</li><li>Bhowmick, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta+signaling+in+fibroblasts+modulates+the+oncogenic+potential+of+adjacent+epithelia.">TGF-beta signaling in fibroblasts modulates the oncogenic potential of adjacent epithelia.</a> Science. 303, 848-851 (2004).</li><li>Bhowmick, N. A., Moses, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-stroma+interactions.">Tumor-stroma interactions.</a> Curr. Opin. Genet. Dev. 15, 97-101 (2005).</li><li>Havlickova, B., Biro, T., Mescalchin, A., Arenberger, P., Paus, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+optimization+of+an+organotypic+assay+system+that+imitates+human+hair+follicle-like+epithelial-mesenchymal+interactions.">Towards optimization of an organotypic assay system that imitates human hair follicle-like epithelial-mesenchymal interactions.</a> Br. J. Dermatol. 151, 753-765 (2004).</li><li>Kilani, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+cytotoxicity+of+gemcitabine+in+bladder+cancer+cell+lines.">Selective cytotoxicity of gemcitabine in bladder cancer cell lines.</a> Anticancer Drugs. 13, 557-566 (2002).</li><li>Seidl, P., Huettinger, R., Knuechel, R., Kunz-Schughart, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+fibroblast-tumor+cell+interaction+causes+downregulation+of+RACK1+mRNA+expression+in+breast+cancer+cells+in+vitro.">Three-dimensional fibroblast-tumor cell interaction causes downregulation of RACK1 mRNA expression in breast cancer cells in vitro.</a> Int. J. Cancer. 102, 129-136 (2002).</li><li>Silzle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-associated+fibroblasts+recruit+blood+monocytes+into+tumor+tissue.">Tumor-associated fibroblasts recruit blood monocytes into tumor tissue.</a> Eur. J. Immunol. 33, 1311-1320 (2003).</li><li>Bartling, B., Demling, N., Silber, R. E., Simm, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferative+stimulus+of+lung+fibroblasts+on+lung+cancer+cells+is+impaired+by+the+receptor+for+advanced+glycation+end-products.">Proliferative stimulus of lung fibroblasts on lung cancer cells is impaired by the receptor for advanced glycation end-products.</a> Am. J. Respir. Cell Mol. Biol. 34, 83-91 (2006).</li><li>Kunz-Schughart, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+of+fibroblasts+to+regulate+the+formation+of+three-dimensional+vessel-like+structures+from+endothelial+cells+in+vitro.">Potential of fibroblasts to regulate the formation of three-dimensional vessel-like structures from endothelial cells in vitro.</a> Am. J. Physiol. Cell Physiol. 290, C1385-C1398 (2006).</li><li>Liu, T., Lin, B., Qin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carcinoma-associated+fibroblasts+promoted+tumor+spheroid+invasion+on+a+microfluidic+3D+co-culture+device.">Carcinoma-associated fibroblasts promoted tumor spheroid invasion on a microfluidic 3D co-culture device.</a> Lab Chip. 10, 1671-1677 (2010).</li><li>Zengel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multimodal+therapy+for+synergic+inhibition+of+tumour+cell+invasion+and+tumour-induced+angiogenesis.">Multimodal therapy for synergic inhibition of tumour cell invasion and tumour-induced angiogenesis.</a> BMC Cancer. 10, 92-92 (2010).</li><li>Kunz-Schughart, L. A., Heyder, P., Schroeder, J., Knuechel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+heterologous+3-D+coculture+model+of+breast+tumor+cells+and+fibroblasts+to+study+tumor-associated+fibroblast+differentiation.">A heterologous 3-D coculture model of breast tumor cells and fibroblasts to study tumor-associated fibroblast differentiation.</a> Exp. Cell Res. 266, 74-86 (2001).</li><li>Djordjevic, B., Lange, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-cell+interactions+in+spheroids+maintained+in+suspension.">Cell-cell interactions in spheroids maintained in suspension.</a> Acta Oncol. 45, 412-420 (2006).</li><li>Hirschhaeuser, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicellular+tumor+spheroids:+an+underestimated+tool+is+catching+up+again.">Multicellular tumor spheroids: an underestimated tool is catching up again.</a> J. Biotechnol. 148, 3-15 (2010).</li><li>Hoevel, T., Macek, R., Swisshelm, K., Kubbies, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reexpression+of+the+TJ+protein+CLDN1+induces+apoptosis+in+breast+tumor+spheroids.">Reexpression of the TJ protein CLDN1 induces apoptosis in breast tumor spheroids.</a> Int. J. Cancer. 108, 374-383 (2004).</li><li>Paduch, R., Kandefer-Szerszen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitamin+D,+tamoxifen+and+beta-estradiol+modulate+breast+cancer+cell+growth+and+interleukin-6+and+metalloproteinase-2+production+in+three-dimensional+co-cultures+of+tumor+cell+spheroids+with+endothelium.">Vitamin D, tamoxifen and beta-estradiol modulate breast cancer cell growth and interleukin-6 and metalloproteinase-2 production in three-dimensional co-cultures of tumor cell spheroids with endothelium.</a> Cell. Biol. Toxicol. 21, 247-256 (2005).</li><li>Timmins, N. E., Nielsen, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+multicellular+tumor+spheroids+by+the+hanging-drop+method.">Generation of multicellular tumor spheroids by the hanging-drop method.</a> Methods Mol. Med. 140, 141-151 (2007).</li><li>Foty, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Hanging+Drop+Cell+Culture+Protocol+for+Generation+of+3D+Spheroids.">A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids.</a> J. Vis. Exp. (51), e2720-e2720 (2011).</li><li>Xu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+fibroblast+Tiam1+in+tumor+cell+invasion+and+metastasis.">The role of fibroblast Tiam1 in tumor cell invasion and metastasis.</a> Oncogene. 29, 6533-6542 (2010).</li></ol>

No hay conflictos de interés declarado.

El método aquí presentado representa un método sencillo para analizar los efectos de los fibroblastos del estroma de las células epiteliales, incluyendo las células tumorales. Permite directo célula-célula interacción dentro de un contexto tridimensional apoyada por la matriz extracelular de la membrana basal, al tiempo que permite una fácil extracción de las células de la matriz para su posterior análisis. Esto se puede aplicar al estudio de la estroma asociado al tumor, como fibroblastos de líneas puede s...

<table border="1"><tbody><tr><td> Nombre de reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> BD Matrigel </td><td> BD Biosciences </td><td> 356237 </td><td> El fenol rojo libre de </td></tr><tr><td> Hyclone clásico medio líquido: DMEM F12 </td><td> ThermoScientific </td><td> SH30023.01 </td><td></td></tr><tr><td> Hyclone bovina de suero de ternera </td><td> ThermoScientific </td><td> SH3007303 </td><td></td></tr><tr><td> Insulina </td><td> Merck Chemicals </td><td> 407709 </td><td> Disolver en HCl 0.005N; 0,22 mM filtro </td></tr><tr><td> La hidrocortisona </td><td> Sigma-Aldrich </td><td> H4001 </td><td> Disolver en 50% de EtOH; 0,22 mM filtro </td></tr><tr><td> EGF </td><td> Sigma-Aldrich </td><td> E9644 </td><td> Disolver en ácido acético 10 mM; filtro de 0,22 um </td></tr></tbody></table>

isolation of mammary epithelial cells from three-dimensional mixed-cell spheroid co-culture

Mientras que los enormes esfuerzos han ido a la identificación de vías de señalización y las moléculas que intervienen en los comportamientos de células normales y malignas 1-2, gran parte de este trabajo se ha hecho con clásicos bidimensionales modelos de cultivo celular, que permiten la manipulación de células fácil. Ha quedado claro que las vías de señalización intracelulares se ven afectados por las fuerzas extracelulares, incluyendo la dimensionalidad y la tensión de la superficie celular 3-4. Múltiples enfoques se han tomado para desarrollar modelos en tres dimensiones que representan con mayor precisión la arquitectura biológica del tejido 3. Si bien estos modelos incorporan la multidimensionalidad y las tensiones arquitectónicas, el estudio de los consiguientes efectos sobre las células es menos fácil que en el cultivo de tejidos en dos dimensiones debido a las limitaciones de los modelos y la dificultad en la extracción de las células para su posterior análisis. El importante papel del microambiente alrededor de los tumores en la tumorigénesis y el comportamiento del tumors cada vez más reconocido 4. Estroma del tumor se compone de múltiples tipos de células y moléculas extracelulares. Durante el desarrollo del tumor hay señales bidireccionales entre las células tumorales y las células del estroma 5. Aunque algunos de los factores que participan en el tumor de estroma-co-evolución han sido identificados, todavía hay una necesidad de desarrollar técnicas sencillas para identificar de forma sistemática y estudiar toda la gama de estos 6 señales. Los fibroblastos son el tipo de célula más abundante en los tejidos normales o estroma del tumor asociado, y contribuir a la deposición y el mantenimiento de la membrana basal y factores de crecimiento paracrinos 7. Muchos grupos han utilizado tres sistemas de cultivo de dimensiones para estudiar el papel de los fibroblastos en varias funciones celulares, incluyendo la respuesta del tumor a los tratamientos, el reclutamiento de células inmunes, las moléculas de señalización, proliferación, la apoptosis, la angiogénesis y la invasión de 8-15. Hemos optimizado un método sencillo para el culoessing los efectos de los fibroblastos mamarias en células epiteliales mamarias utilizando un modelo de matriz extracelular comercialmente disponible para crear tridimensionales cultivos de las poblaciones de células mixtas (co-cultivos) 16-22. Con continua co-cultivo de las células formar esferoides con la agrupación fibroblastos en el interior y las células epiteliales en gran medida en el exterior de los esferoides y formando multicelulares proyecciones en la matriz. La manipulación de los fibroblastos que conduce a la invasividad de las células epiteliales alterados pueden ser fácilmente cuantificados por cambios en el número y la longitud de las proyecciones epiteliales 23. Además, hemos ideado un método para aislar células epiteliales de tres dimensiones de co-cultivo que facilita el análisis de los efectos de la exposición de fibroblastos en el comportamiento epitelial. Hemos encontrado que los efectos de co-cultivo persistir durante semanas después del aislamiento de células epiteliales, permitiendo tiempo suficiente para realizar ensayos múltiples. Este método es adaptable a las células devariable potencial maligno y no requiere equipo especializado. Esta técnica permite la evaluación rápida de los modelos de células in vitro bajo condiciones múltiples, y los resultados correspondientes se pueden comparar con los modelos in vivo de tejidos animales, así como muestras de tejidos humanos.

Watch this Scientific Journal Video about Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture at JoVE.com

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

Un método simple se describe para analizar los efectos de los fibroblastos del tejido en las células epiteliales asociados. La combinación de este método y tridimensional cultivo de tejidos puede facilitar el análisis de las células después del aislamiento de 3D. La técnica es aplicable a las células de diferentes potencial maligno, permitiendo el estudio sistemático de los efectos de estroma asociado al tumor en las células tumorales.

Aislamiento de células epiteliales mamarias de tridimensional mixto de células Esferoide Co-cultura

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

isolation-mammary-epithelial-cells-from-three-dimensional-mixed-cell

Research

JoVE Journal

Biology

16.0K vistas.  Tufts Medical Center. Un método simple se describe para analizar los efectos de los fibroblastos del tejido en las células epiteliales asociados. La combinación de este método y tridimensional cultivo de tejidos puede facilitar el análisis de las células después del aislamiento de 3D. La técnica es aplicable a las células de diferentes potencial maligno, permitiendo el estudio sistemático de los efectos de estroma asociado al tumor en las células tumorales.

Video: Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo 2. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

Células epiteliales mamarias de ratón forma Mammospheres durante la diferenciación lactogénica

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

Investigación

Biología

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

Microfluidos Co-cultivo de células epiteliales y bacterias para la Investigación de la señal mediada Soluble Interacciones

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

Mammary Epithelial Transplant Procedure

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, followed by the mammary epithelial transplant procedure. The mammary transplant procedure is widely used by mammary biologists because it takes advantage of the fact that significant development of the mammary epithelium doesn't occur until after puberty. At 3 weeks of age, growth of the mammary epithelial tree is confined to the vicinity of the nipple and the fat pad is largely devoid of mammary epithelium, but by 7 weeks of age the epithelial ductal tree extends throughout the entire fat pad. Therefore, if this small portion of the fat pad containing epithelium, the region between the nipple and the lymph node, is removed at 3 weeks of age, the endogenous epithelium will never populate the mammary fat pad and the fat pad is described as "cleared". At this time, mammary epithelium from another source can be transplanted in the cleared fat pad where it has the potential to extend mammary ductal trees through out the fat pad. This procedure has been utilized in many experimental models including the examination of tumor phenotype in transgenic mammary epithelial tissue without the confounding effects of genotype on the entire animal3, in the identification of mammary stem cells by transplanting cells in limited dilution4,5, determining if hyperplastic nodules proceed to mammary tumors6, and to assess the effect of prior hormone exposure on the behavior of the mammary epithelium7,8.
Three week old host mice are anesthetized, cleaned and restrained on a surgical stage. A mid-sagittal incision is made through the skin, but not the peritoneum, extending from the pubis to the sternum. Oblique cuts are made through the skin from the mid-sagittal incision across the pelvis toward each leg. The skin is pulled away from the peritoneum to expose the 4th inguinal mammary gland. The fat pad is cleared by removing the fat pad tissue anterior to the lymph node. Epithelium fragments or epithelial cells are transplanted into the remaining cleared fat pad and the mouse is closed.

This article demonstrates the procedure developed by DeOme KB et al. (1959) and the sparing procedure developed by Brill B et al. (2008) for clearing the 4th inguinal mammary fat pad of a pubescent mouse in preparation for transplantation of mammary fragments, mammary epithelial cells, or mammary tumor cells.

Procedimiento de mama epiteliales de Trasplantes

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

Aislamiento y cultivo de células madre adultas epiteliales de la piel humana

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Aislamiento de la cromatina mediante la purificación de ARN (chirp)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Inducción y Análisis de epitelial a mesenquimal de transición

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Aislamiento y cultivo de células madre epiteliales dental del adulto Ratón Incisivo

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Evaluating the Angiogenetic Properties of Ovarian Cancer Stem-Like Cells using the Three-Dimensional Co-Culture System, NICO-1

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular matrix. The ability of CSCs to participate in the development of endothelium constitutes an important characteristic that directly contributes to the general understanding of the mechanisms of tumorigenesis and tumor metastasis. The purpose of this work is to establish a reproducible methodology to investigate the tumor-initiation capability of ovarian cancer stem cells (OCSCs). Herein, we examined the neovascularization mechanism between endothelial cells and OCSCs along with the morphological changes of endothelial cells using the in vitro co-culture model NICO-1. This protocol allows visualization of the neovascularization step surrounding the OCSCs in a time course manner. The technique can provide insight regarding the angiogenetic properties of OCSCs in tumor metastasis.

Ovarian cancer stem cells (OCSC) are responsible for cancer initiation, recurrence, therapeutic resistance, and metastasis. The OCSC vascular niche is considered to promote self-renewal of OCSCs, leading to chemoresistance. This protocol provides the basis for establishing a reproducible OCSC vascular niche model in vitro.

Evaluación de las propiedades angiogenéticas de las células madre similares al cáncer de ovario utilizando el sistema de cocultivo tridimensional, NICO-1

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular ...

Staining and High-Resolution Imaging of Three-Dimensional Organoid and Spheroid Models

In vitro three-dimensional (3D) cell culture models, such as organoids and spheroids, are valuable tools for many applications including development and disease modeling, drug discovery, and regenerative medicine. To fully exploit these models, it is crucial to study them at cellular and subcellular levels. However, characterizing such in vitro 3D cell culture models can be technically challenging and requires specific expertise to perform effective analyses. Here, this paper provides detailed, robust, and complementary protocols to perform staining and subcellular resolution imaging of fixed in vitro 3D cell culture models ranging from 100 &#181;m to several millimeters. These protocols are applicable to a wide variety of organoids and spheroids that differ in their cell-of-origin, morphology, and culture conditions. From 3D structure harvesting to image analysis, these protocols can be completed within 4-5 days. Briefly, 3D structures are collected, fixed, and can then be processed either through paraffin-embedding and histological/immunohistochemical staining, or directly immunolabeled and prepared for optical clearing and 3D reconstruction (200 &#181;m depth) by confocal microscopy.

Here, we provide detailed, robust, and complementary protocols to perform staining and subcellular resolution imaging of fixed three-dimensional cell culture models ranging from 100 &#181;m to several millimeters, thus enabling the visualization of their morphology, cell-type composition, and interactions.

Tinción e imágenes de alta resolución de modelos organoides y esferoides tridimensionales

In vitro three-dimensional (3D) cell culture models, such as organoids and spheroids, are valuable tools for many applications including development and ...

Isolation of Epithelial Cells from Human Dental Follicle

The dental follicle (DF) was harvested during the removal of an impacted third molar by an oral maxillofacial surgeon. Epithelial cell isolation was performed on the day of DF harvest. The DF was washed three times with DPBS and then dissected with tissue scissors until the tissue had a pulpy or squishy consistency. Single-cell populations were pelleted by centrifugation and washed with keratinocyte serum-free medium. Heterogeneous cell populations were distributed in a culture dish. Keratinocyte serum-free medium was used to select the epithelial cells. The culture medium was changed daily until no floating debris or dead cells were observed. Epithelial cells appeared within 7-10 days after cell population distribution. Epithelial cells survived in serum-free medium, while &#945;-modification minimal essential medium supplemented with 10% fetal bovine serum allowed the proliferation of mesenchymal-type cells. The DF is a tissue source for the isolation of dental epithelial cells.
The purpose of this study was to establish a method for the isolation of epithelial cells from human DF. Periodontal ligament (PDL) was used for the isolation of human dental epithelial cells. Procuring epithelial cells from human PDL is not always successful due to the small tissue volume, leading to low numbers of epithelial cells. DF has a larger volume than PDL and contains more cells. DF can be a tissue source for the primary culture of human dental epithelial cells. This protocol is easier and more efficient than the isolation method using PDL. Procuring human dental epithelial cells may facilitate further studies of dental epithelial-mesenchymal interactions.

The dental follicle contains an epithelial population and mesenchymal cells. The epithelial population was selected from the heterogeneous dental follicle cell population by providing a distinct culture medium. Epithelial cells survived and formed colonies in a serum-free medium.

Aislamiento de células epiteliales del folículo dental humano

The dental follicle (DF) was harvested during the removal of an impacted third molar by an oral maxillofacial surgeon. Epithelial cell isolation was ...