Agradecemos enormemente las contribuciones de redacción por Cheryl Schaeberle. Esta investigación fue apoyada por subvenciones de Avon # 02-2009-093 y 02-2011-095, y NIEHS / NIH ES 08314 a AMS. El contenido es responsabilidad exclusiva de los autores y no representa necesariamente las opiniones oficiales del Instituto Nacional de Ciencias de Salud Ambiental o de los Institutos Nacionales de Salud.

1. Preparación de los reactivos <ol><li> Se disuelve el promegestona sintético progestágeno (R5020) y 17-β-estradiol (E2) en etanol para hacer 10 -3 soluciones madre M. Disolver la prolactina en agua desionizada destilada para hacer una solución stock 1 mg / ml. Disolver el antiestrógeno ICI 182780 en DMSO para preparar una solución de stock 10 -2 M. Almacenar estas soluciones a -20 ° C durante un máximo de 6 meses. </li><li> dextrano carbón (CD) despojado suero y medio CDFBS: <ol><li> Heat-inactivar suero fetal bovino (FBS) en un baño de agua a 57 ° durante 30 min. Nota: Utilice sólo material de vidrio lavado con ácido durante el resto de este protocolo. </li><li> Calcular la cantidad de carbón necesaria. El uso de 10% más de volumen que la cantidad de suero para compensar el desplazamiento causado por el carbón de leña, pesar 5% en peso / vol carbón activo en polvo. </li><li> Lavar carbón dos veces con agua destilada desionizada fría usando el mismo volumen que la cantidad de suero. Para cada lavado centrifuge a 50 xg durante 2 min para sedimentar el carbón vegetal. Eliminar el agua después de cada lavado. Añadir 0,5% peso / vol de dextrano T-70 a la última pastilla de carbón. Vuelva a suspender con agua destilada, desionizada y se centrifuga a 100 xg durante 5 min. Aspirar el agua. </li><li> Añadir el volumen apropiado de suero al sedimento y volver a suspender el sedimento de carbón dextrano en el suero. Incubar, mientras que rueda (6 rpm), a 37 ° C durante 60 minutos. Centrifugar a 2000 xg durante 20 min. Si sobrenadante aparece nublado, repetir la centrifugación; Sin embargo, el suero puede no ser del todo claro en este momento, pero será limpiado durante la filtración. </li><li> Filtrar a través de un filtro de 0,80 micras y luego de nuevo a través de un filtro de 0,45 micrómetros para eliminar las partículas más grandes. Finalmente, filtro de esterilizar con un filtro de 0,20 micras. Tienda filtra CDFBS en tubos de vidrio o T-25 frascos de cultivo a -20 ºC. </li><li> Carbón de leña con dextrano-fetal bovino que contiene los medios de comunicación (medios CDFBS): mezclar 375 ml de fenol DMEM libre de rojo y 125 ml de DMEM/ F-12. Retire 37,5 ml y reemplazarlo con el mismo volumen de suero fetal bovino de carbón dextrano. Añadir 10 6 U / ml de penicilina y L-glutamina a una concentración final de 2 mM. Los medios de comunicación resultante es 75% de DMEM, 25% de DMEM / F-12, 7,5% de dextrano con carbón vegetal-FBS, 2 mM de L-glutamina, y 10 6 U / ml de penicilina. </li></ol></li><li> Preparar 10X PBS, NaOH 1 N y agua desionizada destilada. Preparar suficiente volumen de cada uno de acuerdo con el volumen de solución de colágeno sea necesario. Esterilizar todas las soluciones de filtrado. Almacenar el 10x PBS y agua destilada desionizada estéril a 4 ° C por hasta 1 mes. No almacene la solución de NaOH 1N. </li><li> Preparar la solución de colágeno a una 1 mg / ml de concentración final de acuerdo con el protocolo para el &quot;Procedimiento de gelificación alternativo para el colágeno I, de cola de rata&quot; proporcionado por el fabricante. Cada gel se hace de 1,5 ml de solución de colágeno. Por ejemplo, preparar 18,5 ml (0,5 ml para dar cuenta de la pérdida durante el pipeteado) de solución de colágeno para preparar12 geles. Utilice la solución de colágeno inmediatamente o mantener en hielo durante un máximo de 2-3 horas. </li><li> Carmín alumbre tinte: <ol><li> Combinar 1 g Carmine y 2,5 g de sulfato de aluminio y potasio en 500 ml de agua destilada. Hervir mientras se agita durante 20 min. Observe cuidadosamente para evitar una ebullición más. Ajustar el volumen final de nuevo a 500 ml con agua destilada adicional. </li><li> Añadir un cristal de timol para la actividad antimicrobiana y cubrir botella con papel de aluminio para protegerlo de la luz. Filtrar a través de un filtro de papel de grado de laboratorio antes de cada uso. Reutilizar la solución de carmín para un máximo de tres meses. Almacenar a temperatura ambiente, protegido de la luz. </li></ol></li><li> solución de colagenasa: disolver la colagenasa en medio DMEM / F12 en una concentración de 0,125% w / v. No guarde esta solución. </li></ol> 2. Cultura 3D de T47D células en la cola de la rata colágeno tipo 1 Geles y Tratamiento Hormonal Nota: mantener las pipetas serológicas estériles y puntas de pipeta a 4 ° C. <ol><li> AEEre una suspensión de una sola célula y realizar un recuento de células. cultivos de células T47D de división antes de que alcancen el 70% de confluencia. Se centrifuga el volumen que contiene la cantidad de células necesarias. Tenga en cuenta que un gel debe contener aproximadamente 75.000 células T47D. </li><li> Usando una pipeta de frío, vuelva a suspender el sedimento celular en el volumen apropiado de colágeno. Tenga en cuenta que cada gel se compone de 1,5 ml de colágeno. Mezclar las células y el colágeno suavemente para evitar la introducción de burbujas. </li><li> Para evitar el efecto de la electricidad estática en la distribución de células, cepillo de la frontera, la parte superior e inferior de la placa de 12 pocillos con un cepillo de reducción estática después de abrir el plato. Para evitar la acumulación de carga estática durante el contacto con la superficie de trabajo de la campana, colocar esta placa en la parte superior de la otra placa de 12 pocillos (placa en blanco) que también ha sido aplicado con brocha con la brush.Gently reductores estática, verter 1,5 ml de la mezcla en cada pocillo de la placa de 12 pocillos. </li><li> Colocar la placa en una incubadora a 37 ° C durante 30 minutos. Retire THe placa de la incubadora y colocarlo de nuevo en la campana. </li><li> Utilizando una punta estéril p200, separar cuidadosamente el gel en un suave movimiento circular de la frontera del pozo. El gel también se debe separar de la parte inferior del pozo. </li><li> Diluir hormonas en los medios de comunicación CDFBS que se añaden a los pocillos. Use E2 y R5020 a una concentración final 10 -10 M y prolactina en un en 10 -7 M concentración final. Como control, utilizar medios CDFBS sin hormonas, pero que todavía contiene el mayor volumen de DMSO iguales a las reservas de hormonas utilizadas. Asegúrese de que cada pocillo contiene 1,5 ml de medio. Coloque las culturas 3D dentro de una 37 ° C, 5% de CO 2, el 100% tejido humedad cultura incubadora. Mantener los cultivos durante 1 o 2 semanas, el cambio de los medios de comunicación cada 2 días. </li></ol> 3. Tratamiento Gel para toda Monturas <ol><li> Retire los medios de cultivo y añadir 1,5 ml de PBS a cada pocillo. </li><li> Transferir el gel a una diapositiva. Cortar el gel en un medio utilizando una curvacuchilla quirúrgica d. Continuar con el procesamiento con una mitad y guardar la otra mitad para el análisis histológico. </li><li> Transferir el gel de la mitad de todo el montaje a una diapositiva. Deje secar gel durante 5 minutos y luego transferir la diapositiva a una jarra de Coplin que contiene un 10% de formalina. Nota: todas las incubaciones deben realizarse a temperatura ambiente. </li><li> Fijar geles en una jarra Coplin en un agitador O / N. </li><li> Lavar los portaobjetos dos veces con PBS durante 10 min cada uno en agitador. Transferencia desliza a 70% EtOH; incubar durante 1 hr en un agitador. transferencia de diapositivas a Carmine Alum O / N. </li><li> El día siguiente, deshidratar diapositivas en un agitador en 70% de EtOH, 95% de EtOH y 100% de EtOH durante 1 hora cada uno. Transferencia desliza a xileno dos veces durante 1 hora cada uno y mantener en un agitador. Utilice medio de montaje a base de tolueno para montar los geles y 1,5 mm de espesor cubreobjetos. </li><li> Examine su conjunto se monta bajo luz normal (microscopio o estereoscopio). Para un análisis más detallado de la forma y el lumen utilizar microscopía confocal y visualizar colorante carmín con 633 nm láser de He-Ne. </li></ol>4. Tratamiento Gel para transformación para el análisis Histología <ol><li> Transferir el medio restante de la gel a un vial de vidrio de 20 ml que contiene 10% de formalina y fijar en un agitador O / N. </li><li> Lavar geles dos veces con PBS durante 10 min cada uno en agitador. Coloque los geles en casetes de procesamiento para la inclusión en parafina. </li><li> geles de transferencia de hasta 70% de EtOH, se incuban durante 1 hora y siguen con EtOH al 95% durante 1 hora, cambian al fresco EtOH al 95% durante 1 hora, EtOH al 100% durante 1 hora, y cambian a fresca EtOH al 100% durante 1 hora. En este punto, los geles se pueden almacenar en 100% de EtOH O / N a temperatura ambiente o se incubaron con xileno durante 1 hr y luego de nuevo en xileno fresco para 1 hr. </li><li> casetes de transferencia de parafina durante 1 hora a 60 ° C en una incubadora de vacío, repetir con parafina fresca dos veces más. </li><li> Rellenar los moldes de plástico con parafina limpia. Abra el casete, retire el gel y lo coloca en el molde que contiene parafina plástico. Llenar el depósito de parafina y añadir la parte superior de casete que contiene la etiqueta en la parte superior y dejar que el bloque frío. </li><li> Retire el plastic molde y la sección del bloque utilizando un microtomo. Obtener secciones de 5 um de espesor para la tinción HE e inmunocitoquímica. </li></ol> 5. La extracción de células a partir de células a partir de geles Uso de tratamiento con colagenasa <ol><li> Eliminar gel del pozo y la transfiere a una placa de Petri que contiene PBS. Enjuague el gel brevemente. </li><li> Transferir el gel a un tubo de 15 ml que contiene 2 ml de 0,125% de colagenasa. Pipetear arriba y abajo 4-5 veces con el fin de romper el gel en trozos pequeños. </li><li> Incubar a 37 ° C durante 25 min (hasta que el gel se haya disuelto y las células están en suspensión) </li><li> Añadir 2 ml de medio de cultivo suplementado con suero. Recoger las células por centrifugación (1.000 xg, 5 min), el sobrenadante de descarte, volver a suspender las células y contar. </li></ol>

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	<li>Barnes, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+single+cells+to+tissues:+interactions+between+the+matrix+and+human+breast+cells+in+real+time.">From single cells to tissues: interactions between the matrix and human breast cells in real time.</a> PLoS ONE. 9, e93325 (2014).</li><li>Berthois, Y., Katzenellenbogen, J. A., Katzenellenbogen, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenol+red+in+tissue+culture+is+a+weak+estrogen:+implications+concerning+the+study+of+estrogen+responsive+cells+in+culture.">Phenol red in tissue culture is a weak estrogen: implications concerning the study of estrogen responsive cells in culture.</a> Proc.Nat.Acad.Sci.USA. 83, 2496-2500 (1986).</li><li>Chalbos, D., Vignon, F., Keydar, I., Rochefort, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogens+stimulate+cell+proliferation+and+induce+secretory+proteins+in+a+human+breast+cancer+cell+line+(T47D).">Estrogens stimulate cell proliferation and induce secretory proteins in a human breast cancer cell line (T47D).</a> J.Clin.Endocrinol.Metab. 55, 276-283 (1982).</li><li>Dhimolea, E., Maffini, M. V., Soto, A. 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M., Sonnenschein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+epithelial+tissue+morphology+is+affected+in+3D+cultures+by+species-specific+collagen-based+extracellular+matrix.">Breast epithelial tissue morphology is affected in 3D cultures by species-specific collagen-based extracellular matrix.</a> J.Biomed.Mat.Res.A. 100, 2905-2912 (2012).</li><li>Krause, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+regulation+of+breast+tubulogenesis+using+extracellular+matrix+composition+and+stromal+cells.">Dual regulation of breast tubulogenesis using extracellular matrix composition and stromal cells.</a> Tissue Eng Part A. 18, 520-532 (2012).</li><li>Krause, S., Maffini, M. V., Soto, A. M., Sonnenschein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+3D+in+vitro.+culture+model+to+study+stromal-epithelial+interactions+in+the+mammary+gland.">A novel 3D in vitro. culture model to study stromal-epithelial interactions in the mammary gland.</a> Tissue Eng.Part C Methods. 14, 261-271 (2008).</li><li>Krause, S., Maffini, M. V., Soto, A. M., Sonnenschein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microenvironment+determines+the+breast+cancer+cells'+phenotype:+organization+of+MCF7+cells+in+3D+cultures.">The microenvironment determines the breast cancer cells' phenotype: organization of MCF7 cells in 3D cultures.</a> BMC Cancer. 10, 263-275 (2010).</li><li>Lee, G. Y., Kenny, P. A., Lee, E. H., Bissell, 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=Three-dimensional+culture+models+of+normal+and+malignant+breast+epithelial+cells.">Three-dimensional culture models of normal and malignant breast epithelial cells.</a> Nat.Methods. 4, 359-365 (2007).</li><li>Martinson, H. A., Jindal, S., Durand-Rougely, C., Borges, V. F., Schedin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing-like+immune+program+facilitates+postpartum+mammary+gland+involution+and+tumor+progression.">Wound healing-like immune program facilitates postpartum mammary gland involution and tumor progression.</a> Int.J.Cancer. , (2014).</li><li>Soto, A. M., Murai, J. T., Siiteri, P. K., Sonnenschein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+cell+proliferation:+evidence+for+negative+control+on+estrogen-sensitive+T47D+human+breast+cancer+cells.">Control of cell proliferation: evidence for negative control on estrogen-sensitive T47D human breast cancer cells.</a> Cancer Res. 46, 2271-2275 (1986).</li><li>Speroni, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormonal+regulation+of+epithelial+organization+in+a+3D+breast+tissue+culture+model.">Hormonal regulation of epithelial organization in a 3D breast tissue culture model.</a> Tissue Eng.Part C Methods. 20, 42-51 (2014).</li><li>Vignon, F., Bardon, S., Chalbos, D., Rochefort, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antiestrogenic+effect+of+R5020,+a+synthetic+progestin+in+human+breast+cancer+cells+in+culture.">Antiestrogenic effect of R5020, a synthetic progestin in human breast cancer cells in culture.</a> J.Clin.Endocrinol.Metab. 56, 1124-1130 (1983).</li></ol>

The authors have nothing to disclose.

Here, we describe a hormone-sensitive 3D culture model to test the action of hormones on breast epithelium. The response to hormones can be assessed at the tissue morphology, cell proliferation and gene expression levels 12. One limitation of this technique is that visualization during the culture period is restricted to light microscopy since the cultures are grown in a plastic bottom plate. The 3D culture system could be adapted to glass bottom plates to allow for live imaging of the cultures 1.</...

A diferencia de los cultivos 2D estándar, modelos sustitutos de cultivo de células 3D permiten el estudio del comportamiento de las células epiteliales en un contexto fisiológicamente relevante, uno parecido a un tejido. culturas 3D de la glándula mamaria han ayudado a aclarar muchos aspectos del desarrollo de la glándula mamaria y la neoplasia. Sin embargo, la mayoría de los modelos de cultivo 3D disponibles actualmente no son adecuados para estudiar la acción hormonal porque las líneas de células epiteliales humanos utilizados para la tarea carecen de expresión del receptor de la hormona 6,7,9. En este documento, se describe un modelo de cultivo 3D del epitelio de mama humano que es adecuado para estudiar la acción hormonal 12. Este modelo usa la línea disponible en el mercado sensible a hormonas humana epiteliales de mama celular, T47D 3,11,13, que se deriva originalmente de un derrame pleural obtenida de un paciente femenino de 54 años con un carcinoma ductal infiltrante de la mama. Utilizamos rata colágeno de cola de tipo 1 como una matriz. Este culto 3Dmodelo ure es apropiado para el estudio de la acción de las tres hormonas mammotropic principales (estradiol, promegestona (un análogo de progesterona), y prolactina) en células epiteliales de mama humanos. La morfología del epitelio inducida por la hormona se puede evaluar cuantitativamente a través del tiempo mediante análisis morfométricos 12. Una densidad de siembra apropiada permite a estos cultivos en 3D que se le mantenga durante 2 semanas. Por este tiempo, el desarrollo de estructuras es suficiente para una evaluación cuantitativa robusta de la acción hormonal en la morfología epitelial. Los geles también se pueden cosechar en puntos de tiempo anteriores para la proliferación celular y los análisis de la expresión génica. Además, este modelo es adecuado para probar los efectos de un tratamiento hormonal secuencial; por ejemplo, después del tratamiento con estradiol durante la primera semana y el reemplazo con otra hormona / combinación de hormonas durante la siguiente semana. El efecto de los compuestos y antiestrógenos estrogénicos, tales como ICI 182780, también se puede Stumurió el uso de este modelo de cultivo 3D 12.

<table><tbody><tr><td>12-well Tissue Culture Plates (Falcon)</td><td>Fisher Scientific</td><td>08-772-29</td><td></td></tr><tr><td>15 ml polystyrene conical Tubes</td><td>Fisher Scientific</td><td>14-959-49D</td><td></td></tr><tr><td>Activated Charcoal</td><td>Sigma</td><td>C-5510</td><td></td></tr><tr><td>Carmine Alum</td><td>Sigma</td><td>C1022-100G</td><td></td></tr><tr><td>Collagenase, Type 3</td><td>Worthington</td><td>S0C11784</td><td></td></tr><tr><td>Confocal Microscope</td><td>Zeiss</td><td>LSM510</td><td>Equiped with HeNe 633nm laser</td></tr><tr><td>Dextran T-70</td><td>Abersham/Pharmacia</td><td>17-0280-01</td><td></td></tr><tr><td>DMEM/F-12, HEPES, no phenol red&amp;nbsp;</td><td>Life Technologies</td><td>11339-021</td><td>Phenol red-free media for hormone use</td></tr><tr><td>DMEM, low glucose, pyruvate, no glutamine, no phenol red</td><td>Life Technologies</td><td>11054-020</td><td>Phenol red-free media for hormone use</td></tr><tr><td>17-&amp;beta;-Estradiol</td><td>EMD Millipore</td><td>3301</td><td>Dissolved in Ethanol</td></tr><tr><td>Ethanol</td><td>Koptec</td><td>V1001</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Hyclone</td><td>SH30070.03</td><td>For use with hormones, must be Charcoal Dextran stripped&amp;nbsp;</td></tr><tr><td>Filters (115 ml)</td><td>Nalgene</td><td>380-0080, 245-0045, 120-0020</td><td>0.88, 0.45, 0.20 micron, respectively&amp;nbsp;</td></tr><tr><td>Formalin, 10%</td><td>Fisher Scientific</td><td>SF93-20</td><td></td></tr><tr><td>L-Glutamine (200 mM)</td><td>Life Technologies</td><td>25030-081</td><td></td></tr><tr><td>ICI 182,780 (fulvestrant)</td><td>Sigma Aldrich</td><td>I4409-25MG</td><td>Dissolved in DMSO</td></tr><tr><td>Microtome&amp;nbsp;</td><td>Leica</td><td>RM2155</td><td></td></tr><tr><td>Tissue embedding media</td><td>McCormick Scientific</td><td>39502004</td><td></td></tr><tr><td>Penicillin</td><td>Sigma</td><td>7794-10MU</td><td>Dissolved in 10 ml of distilled deionized water</td></tr><tr><td>Permount</td><td>Fisher Scientific</td><td>SP15-500</td><td></td></tr><tr><td>Phosphate Buffered Saline pH 7.4</td><td>Sigma Aldrich</td><td>P3813-10PAK</td><td></td></tr><tr><td>Prolactin</td><td>Sigma Aldrich</td><td>L4021-50UG</td><td>Dissolved in distilled deionized water</td></tr><tr><td>Promegestone</td><td>Perkin Elmer</td><td>NLP004005MG</td><td>Dissolved in Ethanol</td></tr><tr><td>Rat-Tail Collagen&amp;nbsp;</td><td>Corning&amp;nbsp;</td><td>354236</td><td>Lots may contain varying concentrations, note accordingly</td></tr><tr><td>Scalpel</td><td>Miltex</td><td>4311</td><td></td></tr><tr><td>Semi-enclosed Benchtop Tissue Processor</td><td>Leica</td><td>TP1020</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>Sigma Aldrich</td><td>S5881</td><td>Prepare 1N NaOH stock</td></tr><tr><td>StaticMaster Anti-static brush&amp;nbsp;</td><td>Amstat</td><td>C3500</td><td></td></tr><tr><td>Stripette Serological Pipettes&amp;nbsp;</td><td>Corning&amp;nbsp;</td><td>4101</td><td></td></tr><tr><td>T-25 flasks</td><td>Corning&amp;nbsp;</td><td>430168</td><td></td></tr><tr><td>Tissue Cassettes</td><td>Fisher Scientific</td><td>15-200-403E</td><td></td></tr><tr><td>Wheaton Vials, Glass, 20 ml</td><td>Fisher Scientific</td><td>03-341-25D&amp;nbsp;</td><td></td></tr><tr><td>Xylene&amp;nbsp;</td><td>VWR</td><td>95057-822</td><td></td></tr></tbody></table>

a hormone-responsive 3d culture model of the human mammary gland epithelium

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is limited to cell proliferation and gene expression endpoints. However, in the organism, mammary morphogenesis occurs in a 3D environment. 3D culture systems help bridge the gap between monolayer cell culture (2D) and the complexity of the organism. Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action. It uses the commercially available hormone-responsive human breast epithelial cell line, T47D, and rat tail collagen type 1 as a matrix. This 3D culture model responds to the main mammotropic hormones: estradiol, progestins and prolactin. The influence of these hormones on epithelial morphogenesis can be observed after 1- or 2-week treatment according to the endpoint. The 3D cultures can be harvested for analysis of epithelial morphogenesis, cell proliferation and gene expression.

La Figura 1 resume el procedimiento para la preparación de las culturas 3D sensibles a las hormonas. estructuras epiteliales se observan en preparaciones completas de los geles se cultivaron durante 2 semanas en presencia de E2 solo y en combinación con otras hormonas. Sólo las células individuales o grupos de 2-3 células están presentes cuando se añaden ninguna hormona al medio de cultivo (medio CDFBS) (Figura 2). Esta condición sirve como un control negativo. <p class="jov...

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A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

We describe a 3D culture model of the human breast epithelium that is suitable to study hormone action.

Un 3D Cultura Modelo sensibles a hormonas de la glándula mamaria humana epitelio

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is ...

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8.8K vistas.  Tufts University School of Medicine. We describe a 3D culture model of the human breast epithelium that is suitable to study hormone action.

Video: A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction of differentiation of two mouse breast epithelial cell lines, HC11 and EpH4, and their use to study complementary stages of mammary gland development and neoplastic transformation.
The HC11 mouse breast epithelial cell line originated from the mammary gland of a pregnant Balb/c mouse. It differentiates when grown to confluence attached to a plastic Petri dish surface in medium containing fetal calf serum and Hydrocortisone, Insulin and Prolactin (HIP medium). Under these conditions, HC11 cells produce the milk proteins &#946;-casein and whey acidic protein (WAP), similar to lactating mammary epithelial cells, and form rudimentary mammary gland-like structures termed &#34;domes&#34;.
The EpH4 cell line was derived from spontaneously immortalized mouse mammary gland epithelial cells isolated from a pregnant Balb/c mouse. Unlike HC11, EpH4 cells can fully differentiate into spheroids (also called mammospheres) when cultured under three-dimensional (3D) growth conditions in HIP medium. Cells are trypsinized, suspended in a 20% matrix consisting of a mixture of extracellular matrix proteins produced by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, plated on top of a layer of concentrated matrix coating a plastic Petri dish or multiwell plate, and covered with a layer of 10% matrix-containing HIP medium. Under these conditions, EpH4 cells form hollow spheroids that exhibit apical-basal polarity, a hollow lumen, and produce &#946;-casein and WAP.
Using these techniques, our results demonstrated that the intensity of the cadherin/Rac signal is critical for the differentiation of HC11 cells. While Rac1 is necessary for differentiation and low levels of activated RacV12 increase differentiation, high RacV12 levels block differentiation while inducing neoplasia. In contrast, EpH4 cells represent an earlier stage in mammary epithelial differentiation, which is inhibited by even low levels of RacV12.

We describe techniques for differentiation induction of two breast epithelial lines, HC11 and EpH4. While both require fetal calf serum, insulin, and prolactin to produce milk proteins, EpH4 cells can fully differentiate into mammospheres in three-dimensional culture. These complementary models are useful for signal transduction studies of differentiation and neoplasia.

Diferenciación de células epiteliales de mama de ratón HC11 y EpH4

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction ...

Investigación

Investigación sobre el cáncer

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

Un tejido tridimensional Cultura modelo para estudiar primaria médula ósea humana y sus malignidades

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Medicina

Phospholipid Mediator Induced Transformation in Three-Dimensional Cultures

Several models have been developed to study cancer, such as rodent models and established cell lines. Valuable insights into carcinogenesis have been provided by studies using these models. Cell lines have provided an understanding of the deregulation of molecular signaling associated with breast tumorigenesis, while rodent models are widely used to study cellular and molecular characteristics of breast cancer in vivo. The establishment of 3D cultures of breast epithelial and cancerous cells aids in bridging the gap between in vivo and in vitro models by mimicking the in vivo conditions in vitro. This model can be used to understand the deregulation of complex molecular signaling events and the cellular characteristics during breast carcinogenesis. Here, a 3D culture system is modified to study a phospholipid mediator-induced (Platelet Activating Factor, PAF) transformation. Immunomodulators and other secreted molecules play a major role in tumor initiation and progression in the breast. In the present study, 3D acinar cultures of breast epithelial cells are exposed to PAF exhibited transformation characteristics such as loss of polarity and altered cellular characteristics. This 3D culture system will assist in shedding light on genetic and/or epigenetic perturbations induced by various small molecule entities in the tumor microenvironment. Additionally, this system will also provide a platform for the identification of novel as well as known genes that may be involved in the process of transformation.

The present protocol describes the setting up of 3D 'on top' cultures of a non-transformed breast epithelial cell line, MCF10A, that has been modified to study Platelet Activating Factor (PAF) induced transformation. Immuno-fluorescence has been used to assess the transformation and is discussed in detail.

Transformación inducida por mediadores fosfolípidos en cultivos tridimensionales

Several models have been developed to study cancer, such as rodent models and established cell lines. Valuable insights into carcinogenesis have been ...

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

Tres Culturas dimensionales: una herramienta para estudiar normal Acinar Arquitectura vs transformación maligna de las células de la mama

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

La cuantificación de la invasividad de las células del cáncer de mama El uso de un modelo (3D) Tres Dimensiones

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after propagation from primary tissue or stem cells. This method of organoid generation forgoes single-cell differentiation through multiple passages and instead uses differential centrifugation to isolate mammary epithelial organoids from mechanically and enzymatically dissociated tissues. This protocol provides a streamlined technique for rapidly producing small and large epithelial organoids from both mouse and human mammary tissue in addition to techniques for organoid embedding in collagen and basement extracellular matrix. Furthermore, instructions for in-gel fixation and immunofluorescent staining are provided for the purpose of visualizing organoid morphology and density. These methodologies are suitable for myriad downstream analyses, such as co-culturing with immune cells and ex vivo metastasis modeling via collagen invasion assay. These analyses serve to better elucidate cell-cell behavior and create a more complete understanding of interactions within the tumor microenvironment.

This protocol discusses an approach for generating epithelial organoids from primary normal and tumor mammary tissue through differential centrifugation. Furthermore, instructions are included for three-dimensional culturing as well as immunofluorescent imaging of embedded organoids.

Generación y obtención de imágenes de organoides epiteliales humanos y de ratón a partir de tejido mamario normal y tumoral sin pasar

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

Ingeniería tridimensionales epiteliales tejidos embebidos dentro de la matriz extracelular

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Bioingeniería

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

Generación de organoides mamarios de mosaico por trippsinización diferencial

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

Biología del Desarrollo

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

Una<em&gt; Ex vivo</em&gt; Modelo para estudiar la acción hormonal en el pecho humano

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

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

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

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 ...