Este trabajo fue apoyado por subvenciones del NIH R01 CA107469, R01 CA125577 y U01CA154224 a CG Kleer, la Universidad del Centro del Cáncer de Apoyo de Michigan.

1. Preparación de los materiales y medios de cultivo <ol><li> Factor de crecimiento Descongele reducida (TFG) Matrigel a 4 ° CO / N. </li><li> Calentar medio completo para la línea celular requerida a 37 ° C. MCF10A-ATCC especifica los medios de comunicación; Medio F-12 de HME-Ham suplementado con suero bovino fetal al 5%, ml de hidrocortisona, 5 mg / ml de insulina, 10 ng de factor de 1 mg / / ml de crecimiento epidérmico, y 100 ng de toxina / ml de cólera; F-12 de los medios de comunicación SUM149-Ham suplementado con FBS al 5%, 2 mg / ml de hidrocortisona y 5 mg / ml de insulina; y MDA-MB-231-FBS al 10%-DMEM </li><li> PreCool un porta con cámara de 8 pocillos en hielo. </li><li> Prepare el tejido capucha y suministros cultura. </li></ol> 2. Cultivo tridimensional en 8-así Slide Cámara <ol><li> Escudo cada pocillo del portaobjetos de 8 pocillos con 40 l de Matrigel TFG. Añadir el Matrigel en el centro de la diapositiva y luego se extendió el uso de una punta de pipeta P200. Trata de difundir lo más uniformemente posible, evitando las burbujas y la muchedumbre que puede llevar aformación de menisco. </li><li> Incubar los portaobjetos a 37 ° C en 5% de CO 2. Mientras que el Matrigel está solidificando, trypsinize las células, recoger y girar a 150 xg en cultivo de tejido se centrifuga durante 4 min. </li><li> Contar las células y diluir a 12.500 células / ml en medio completo de la línea celular respectivo. Añadir TFG Matrigel a 2% y añadir 400 l a cada pocillo de un portaobjetos de cámara de Matrigel con capa preliminar. </li><li> Incubar los portaobjetos en la incubadora de CO 2. Dar los cambios de medios completos frescas cada 4 º día hasta 15 días. </li><li> El tratamiento con inhibidores farmacológicos: Para estudios de inhibidores añaden inhibidores de moléculas pequeñas a los medios de comunicación completa en el momento de la siembra, seguido de cambios de medio fresco suplementado con los inhibidores de cada tres días, mientras que el crecimiento de las células en Matrigel TFG. </li><li> El tratamiento con proteína purificada: <ol><li> Pasos Placa las células después de 02.01 a 02.03. Permita que los cultivos crezcan durante 4 días seguidos de starvati suerodurante 16 horas. </li><li> En el día 5, el tratamiento de las células con una concentración apropiada de proteína purificada en el 0,1% de SFB suplementado medios de comunicación para las células de hambre suero. Dale cambio de medio fresco con proteína purificada después de 2 días. </li><li> En el día 10 reemplazar el medio condicionado con medio completo HME. Permita que los cultivos crezcan por otros 5 días. </li></ol></li><li> La tinción de inmunofluorescencia de las estructuras acinares cultivadas en Matrigel: <ol><li> Preparar 2% de paraformaldehído, 0.5% de Triton X-100 y glicina 100 mM en 1x PBS, pH 7,4. Preparar tampón de inmunofluorescencia (IF tampón) que comprende de 1x PBS que contenía 0,1% de albúmina de suero bovino, 0,2% de Triton X-100, 0,05% de Tween-20. </li><li> Utilice una punta de pipeta P200 para aspirar cuidadosamente el medio condicionado. </li><li> Fijar las estructuras acinares con 2% de paraformaldehído recién preparado a temperatura ambiente (TA) durante 20 min. </li><li> Permeabilizar las células con 0,5% de Triton X-100 durante 10 min a 4 ° C. </li><li> Lave la culturas de tres veces con glicina 100 mM, 10-15 minutos para cada lavado. </li><li> Bloquee las células con 10% de suero de cabra en tampón IF, llamado bloque de primaria, durante 1 hora a temperatura ambiente. </li><li> Incubar los cultivos con un 20 ug / ml de anticuerpo de cabra anti F (ab &#39;) 2 fragmento en tampón de bloqueo primario durante 30 min ratón. Este paso es importante para bloquear la especie de IgG de ratón inmunorreactivas en el Matrigel. </li><li> Incubar con el anticuerpo primario en la segunda solución de bloqueo (bloque principal 20 mg / ml de cabra anti-ratón F (ab &#39;) 2) O / N a 4 ° C. </li><li> Lavar tres veces con tampón IF durante 10-15 minutos cada uno con suave balanceo. </li><li> Se incuba con anticuerpos secundarios conjugados fluorescentes en el tampón IF de 45 min. </li><li> Lave 3 veces durante 10 minutos cada uno con tampón IF con suave balanceo. </li><li> Monte los portaobjetos con el reactivo anti-fade contiene DAPI para visualizar los núcleos. </li><li> Deje que los portaobjetos se sequen O / N a temperatura ambiente. </li><li> La adquisición de imágenes: detección confocal de imágenes en las millas de coloniasdsection de las células cultivadas en la parte superior de Matrigel. Para obtener más información adquirir una serie de secciones ópticas a lo largo de toda la longitud de la estructura acinar por Z apilamiento. </li></ol></li></ol> 3. Cultivo tridimensional en 2 pocillos Cámara Diapositivas por inmunotransferencia <ol><li> PreCool una diapositiva cámara de 2 pozos en el hielo. Siga los pasos 2.1 a 2.4 la ampliación de los reactivos para la 2-well cámara de diapositivas por ejemplo, cubrir con 160 l de Matrigel y agregar 1,6 ml de células / Matrigel mezcla a cada pocillo de la diapositiva 2 pocillos. </li><li> Recoger las estructuras acinares de la Matrigel utilizando el kit de recolección de células de cultivo 3D disponible comercialmente siguiendo las instrucciones del fabricante. </li><li> Diluir los tampones de lavado celular y recolección de células a 1x y preenfriar a 4 ° C. Lavado y desprendimiento de Matrigel deben llevarse a cabo en hielo. </li><li> Lávese las estructuras acinares 3x utilizando el tampón de lavado celular. </li><li> Recoger los acinos en 15 ml tubos de centrífuga utilizando bu recolección de célulasffer seguido de 30 minutos de incubación en un agitador a 4 ° C. Mezclar bien la suspensión con una pipeta de 1 ml. Esto romperá el Matrigel en trozos pequeños y liberar la mayor parte de las células en el tampón de la recolección de células. </li><li> Sedimentar las células a 200 xg en una centrífuga de cultura. Retire la capa flotante de hidrogel de las células sedimentadas con una punta de pipeta P200. </li><li> Resuspender los lisados ​​en tampón de muestra de SDS se suministran con el kit. Tampón de lisis RIPA también se puede utilizar. </li></ol> 4. Cuantificación de vs maligno de mama benigna Fenotipo <ol><li> Se cultivan las células por triplicado para cada conjunto de tratamiento siguiendo los pasos 2.1 a 2.4. </li><li> Tomar imágenes de contraste de fase de las estructuras acinares de cada pocillo a 5X o resolución 10 veces utilizando un microscopio de contraste de fase unida a una palanca de cambios de diapositivas y un ordenador. Tome las imágenes moviendo el portaobjetos de un cuadro a otro y por lo tanto cubre todo el pozo de la diapositiva cámaras. </li><li> Cuente el número total de las estructuras acinares,el número de acinos sola plana redonda y el número de estructuras multiacinar o estructuras que crecen sin orden ni concierto que carecen de organización y con los procesos de ramificación que emanan de ella. </li><li> Calcular el porcentaje de estructuras acinares plana redondos individuales frente a las estructuras malignas y la trama como gráfico de columnas. Calcular la importancia de la prueba t de Student. </li></ol>

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Los autores declaran no tener intereses financieros en competencia.

Hemos descrito aquí el cultivo de tres dimensiones de las células epiteliales de mama sobre una membrana basal reconstituida y la utilidad de este sistema para diferenciar entre vs maligno fenotipo benigna de la mama. Este sistema también puede ser usado para cultivar diferentes tipos de células de otros tejidos glandulares y se ha informado que se han utilizado con éxito para la cultura epitelial de próstata, del epitelio bronquial, y las células epiteliales de tiroides, para nombrar unos pocos 12-14....

Cultivo tridimensional (3D) de las células epiteliales de mama en la membrana basal reconstituida es un importante sistema modelo para estudiar el complejo fenotipo y la señalización asociada de mama normal y cáncer de mama 2,3. La unidad funcional de mama es la unidad lobular del conducto terminal (TDLU) que consiste en un pequeño conducto que se ramifica en los acinos. Los acinos son estructuras altamente organizadas, compuestas de dos capas de células, células epiteliales y mioepiteliales, que están rodeados por una membrana basal 5. Las características más distintivas de acinos normales son la polarización celular, unión a la membrana basal subyacente y contactos especializados célula-célula. Esta organización intrincada se interrumpe en los carcinomas invasivos. Las culturas 2D ampliamente utilizados, donde las células se cultivan como monocapas, no permiten la formación de acinos. Por lo tanto, el cultivo en monocapa se carece en proporcionar un sistema para capturar la relación compleja entre las células epitelialesen condiciones normales de mama y su desregulación en el cáncer de mama. Culturas epiteliales monotípicas funcionales de las células del seno se han desarrollado en varios laboratorios 2,3. Cultura 3D implica el crecimiento de células de mama en Matrigel, que es un extracto solubilizado derivado de Engelbreth-Holm-Swarm células de sarcoma de ratón y está disponible comercialmente. Otros sustratos 3D como colágeno I también se utilizan. Las células de la mama pueden ser cultivadas en 3D mediante ensayo incorporado 3D, donde se cultivan las células incrustado en Matrigel 2. Alternativamente, las células pueden ser cultivadas por 3D en ensayo superior, también llamado ensayo de superposición 3D, que es rentable, ya que requiere menor volumen de Matrigel, y facilita el seguimiento de lapso de tiempo de la formación de colonias por imágenes de contraste de fase y es ideal para la proyección de imagen en situ 2 -3. El ensayo de 3D ​​en la parte superior se ha utilizado para definir la correlación entre el fenotipo acinar y perfil de expresión génica 7. Cultura 3D se puede effectively utiliza para distinguir las células normales y malignas de carcinoma invasor. Las células normales y malignas de mama forman crecimiento arrestado estructuras polarizadas con una luz bien definidos en el centro de Matrigel mientras que las células de carcinoma invasivo crecen prolíficamente y sin orden ni concierto, sin compensación de la luz. E6/E7 inmortalizado células epiteliales mamarias humanas y de la línea de células MCF10A, que es una línea celular inmortalizada espontáneamente aislada de un paciente de 36 años con los cambios fibroquísticos, se han utilizado con éxito para recuperar el fenotipo benigno de mama en 3D 3,8 y han servido como un sistema modelo para estudiar la función de supresor de oncogénica y tumoral de varias moléculas de 8,9. Estas células benignas pueden ser diseñados para manipular gen (s) de interés por la introducción de los lentivirus / retrovirus. Si el gen (s) de interés es un gen supresor tumoral, desmontables shRNA mediada conducirá a una transformación maligna mientras que si el gen de interés es una sobreexpresión de oncogenes en células benignasdebe mostrar un fenotipo maligno. En ambos casos las estructuras acinares formados en 3D pueden capturar la transformación maligna. Células individuales benignos chapados en 3D comienzan a proliferar y formar ronda estructuras esféricas. Por días 5-8 este agregado esférico de células se diferencien en una capa que rodea polarizada de células que está en contacto con el Matrigel, y una masa interior de células menos polarizadas, que carecen de contacto con el Matrigel. En los próximos 10 a 15 días las células internas empezarán a morir por apoptosis formando un lumen clara. Las células malignas crecen desordenadamente perder su polaridad. Células altamente malignos generalmente se forman estructuras de ramificación. Estas diferencias en el fenotipo puede ser capturado por imágenes de contraste de fase de lapso de tiempo y por in situ inmunotinción con marcadores moleculares relevantes. Los marcadores más comúnmente utilizados son alfa 6 integrina, un marcador de polaridad basales, en combinación con el marcador de proliferación fosfo-histona 3 y el apoptotic marcador escinde la caspasa-3. Esto último es importante para determinar si existe la formación del lumen en el centro de los acinos, una característica de las células normales y benignos de mama. Otros marcadores de polaridad y de contacto célula-célula incluyen GM130, laminina, ERM, E-cadherina. Alternativamente las líneas celulares de cáncer de mama pueden ser diseñados para manipular el gen de interés. En este caso, la reversión a un fenotipo más se detiene el crecimiento benigno se puede utilizar como una lectura de distinguir de la fenotipo del cáncer. Cultura 3D también puede usarse con cuidado para delinear las vías de señalización implicadas en la transformación maligna 10-11. Usando la anteriormente mencionada-outs leído, inhibidores farmacológicos, anticuerpos de bloqueo o proteínas recombinantes puede ser utilizado ser identificar en la señalización que conducen a la transformación maligna. Con la continuación de los esfuerzos de diversos laboratorios es posible extraer las células de 3D para aislar ARN y también preparar lisados ​​para inmunotransferencia y inmunidadnoprecipitation. Estas estrategias se describen en un paso a paso y de manera detallada en el protocolo.

<table border="0" cellpadding="0" cellspacing="0" width="585">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Growth Factor Reduced matrigel</td>
			<td style="width:83px;">BD biosciences</td>
			<td style="width:119px;">354230</td>
			<td style="width:167px;">Avoid repeat freeze thaw&#160;</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Lab-Tek glass hamber slides 8-well</td>
			<td style="width:83px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">177402</td>
			<td>Precool on ice&#160;</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Lab-Tek glass chamber slides 2- well&#160;</td>
			<td style="width:83px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">177380</td>
			<td>Precool on ice&#160;</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">goat anti mouse F(ab&#180;)2 fragment</td>
			<td style="width:83px;">Jackson Immunoresearch</td>
			<td style="width:119px;">115-006-006</td>
			<td></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Normal goat serum</td>
			<td style="width:83px;">Jackson Immunoresearch</td>
			<td style="width:119px;">005-000-121</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Fluorescent conjugated Alexa antibodies</td>
			<td style="width:83px;">Molecular probes</td>
			<td style="width:119px;"></td>
			<td>Please look at molecular probes website</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Prolong gold antifade reagent with DAPI</td>
			<td style="width:83px;">Molecular Probes</td>
			<td style="width:119px;">P36935</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">3D Culture Cell Harvesting kit</td>
			<td style="width:83px;">Trevigen</td>
			<td style="width:119px;">3448-020-k</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-Apha-6-integrin</td>
			<td style="width:83px;">Fisher Scientifics&#160;</td>
			<td style="width:119px;">MAB1378</td>
			<td>1:00 dilution for IF</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-GM130</td>
			<td style="width:83px;">BD Biosciences</td>
			<td style="width:119px;">610822</td>
			<td>1:00 dilution for IF</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-phospho-histone 3 (Ser10)FITC Conjugated</td>
			<td style="width:83px;">Fisher Scientifics&#160;</td>
			<td style="width:119px;">16-222</td>
			<td>1:100 dilution for IF</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-Cleaved caspase 3 (Asp 175)</td>
			<td style="width:83px;">Cell Signaling</td>
			<td style="width:119px;">9661s</td>
			<td>1:50 dilution for IF</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-E-cadherin</td>
			<td style="width:83px;">BD Biosciences</td>
			<td style="width:119px;">610181</td>
			<td>1:200 dilution for IF</td>
		</tr>
	</tbody>
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three dimensional cultures: a tool to study normal acinar architecture vs. malignant transformation of breast cells

Carcinomas de mama invasivo son un grupo de tumores epiteliales malignos caracterizados por la invasión de tejidos adyacentes y la propensión a la metástasis. La interacción de señales entre las células cancerosas y su microentorno ejerce una poderosa influencia sobre el crecimiento del cáncer de mama y el comportamiento biológico 1. Sin embargo, la mayoría de estas señales de la matriz extracelular se pierden o su relevancia está poco estudiada cuando las células se cultivan en dos culturas dimensiones (2D) como una monocapa. En los últimos años, cultivo tridimensional (3D) sobre una membrana basal reconstituida ha surgido como un método de elección para recapitular la arquitectura del tejido de las células mamarias benignas y malignas. Las células cultivadas en 3D conservan las señales importantes de la matriz extracelular y proporcionan un sistema ex vivo 2,3 fisiológicamente relevante. Es de destacar que existe una creciente evidencia que sugiere que las células se comportan de manera diferente cuando se cultiva en 3D en comparación con 2D 4. Cultura 3Dpuede ser utilizado eficazmente como un medio para diferenciar el fenotipo maligno de la fenotipo benigna de la mama y para sustentar la señalización celular y molecular implicado 3. Una de las características distintivas de las células epiteliales benignos es que están polarizados de manera que el citoplasma apical es hacia el lumen y el citoplasma basal descansa sobre la membrana basal. Esta polaridad apico basal se pierde en los carcinomas de mama invasivos, que se caracterizan por la desorganización celular y la formación de anastomosis y ramificación de túbulos que se infiltra al azar el estroma circundante. Estas diferencias histopatológicas entre las glándulas benignas y el carcinoma invasor se pueden reproducir en 3D 6,7. Usando las lecturas de espera apropiadas como la cuantificación de las estructuras acinares individuales redondos, o la expresión diferencial de marcadores moleculares validados para la proliferación celular, la polaridad y la apoptosis en combinación con otras técnicas de biología molecular y celular, cultur 3De puede proporcionar una herramienta importante para entender mejor los cambios celulares durante la transformación maligna y para delinear la señalización responsable.

Cultura 3D se puede utilizar con eficacia para diferenciar fenotipo maligno de las células epiteliales de mama humanos de la benigna de la mama. Células benignas individuales chapada en Matrigel crecen como estructuras esféricas organizadas que pueden ser fácilmente incluidas en la imagen como única acinos redonda plana en un plano utilizando imágenes de contraste de fase. Las células malignas individuales chapada en Matrigel crecen sin orden ni concierto, muestran muy alto índice de refracción y son difíciles...

Watch this Scientific Journal Video about Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells at JoVE.com

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

Cultivo tridimensional de células epiteliales mamarias sobre una membrana basal reconstituida es un método útil para recapitular la arquitectura in vivo de la benigna de la mama, y para diferenciar el fenotipo maligno de la fenotipo benigna de la mama. Es importante destacar que este sistema se puede aplicar para estudiar los carcinomas invasivos en otros tejidos.

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 ...

three-dimensional-cultures-tool-to-study-normal-acinar-architecture

Research

JoVE Journal

Medicine

19.8K vistas.  University of Michigan Comprehensive Cancer Center.  Cultivo tridimensional de células epiteliales mamarias sobre una membrana basal reconstituida es un método útil para recapitular la arquitectura in vivo de la benigna de la mama, y para diferenciar el fenotipo maligno de la fenotipo benigna de la mama. Es importante destacar que este sistema se puede aplicar para estudiar los carcinomas invasivos en otros tejidos. 

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

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Humanos neuroendocrinos líneas de células tumorales como un modelo tridimensional para el estudio de la terapia humana tumor neuroendocrino

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

Investigación

Medicina

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 ...

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 ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

Inyección ortotópico de células de cáncer de mama en el Mamaria Fat Pad de ratones para estudiar el crecimiento del tumor.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

Utilizando adeno-asociado virus como una herramienta para estudiar las barreras de retina en la enfermedad

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Tumor Ensayo tridimensional (3D) esferoide Invasion

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

Utilización del ensayo Soft Agar formación de colonias para identificar inhibidores de tumorigenicidad en células de cáncer de mama

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

Tridimensional de alginato-grano Cultura de células humanas del adenoma pituitario

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including Parkinson's and Huntington's can disrupt it. People with Parkinson's disease, for example, tend to have increased saccadic latencies. Consequently, the quantitative measurement of saccadic eye movements has received considerable attention as a potential biomarker for neurodegenerative conditions. A lot more can be learned about the brain in both health and disease by observing what happens to eye movements when the function of specific brain areas is perturbed. Deep brain stimulation is a surgical intervention used for the management of a range of neurological conditions including Parkinson's disease, in which stimulating electrodes are placed in specific brain areas including several sites in the basal ganglia. Eye movement measurements can then be made with the stimulator systems both off and on and the results compared. With suitable experimental design, this approach can be used to study the pathophysiology of the disease being treated, the mechanism by which DBS exerts it beneficial effects, and even aspects of normal neurophysiology.

This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

Usando Saccadometry con estimulación cerebral profunda para estudiar la función cerebral normal y patológica

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...

A Clinical Trial Assessing the Safety, Efficacy, and Delivery of Olive-Oil-Based Three-Chamber Bags for Parenteral Nutrition

Limited evidence exists to precisely estimate efficacy and safety differences between parenteral nutrition (PN) prepared using olive-oil-based three-chamber bags (3CBs) and soybean-oil-based compounded bags (CoBs) in hospitalized adult patients. We designed a multicenter, randomized, prospective, open-label, noninferiority protocol to compare the efficacy, safety, and distribution of olive-oil-based 3CBs and soybean-oil-based CoB formulations in adult Chinese patients requiring PN during surgical intervention. Subjects were randomized to receive either one of the study treatments using an interactive voice or web-based recognition system in accordance with the randomization code. Randomization was further stratified based on the study site and surgical category. Both treatment groups received similar amounts of calories and protein. In addition, the two study treatments contained a similar composition of the amino-acid component. The only difference between the two PN formulations was the lipid constitution. The duration of administration of study treatments was a minimum of 5 days up to a maximum of 14 days after the surgical procedure. The primary efficacy endpoint was serum prealbumin levels on day 5 of the study. Noninferiority was proved if the anti-log of the lower bound of the 95% confidence interval (CI) of the treatment difference was at least 0.80. Other efficacy measures included treatment preparation time; duration to achieve tolerability of oral nutrition; associated infectious complications; length of hospitalization; and laboratory assessment of markers of nutrition, inflammation, metabolism, and oxidative stress. A total of 458 patients were enrolled in the study. The results showed that olive-oil-based 3CBs were non-inferior to soybean-based CoBs, besides being well tolerated. The infection rate was found to be significantly lower in the olive-oil-based 3CB group. Thus, this study may be used as a reference for future research on lipid emulsion and 3CBs.

Here, we present a protocol for a study comparing the efficacy, safety, and delivery of olive-oil-based 3CB and soybean-oil-based CoB formulations in adults requiring parenteral nutrition. The results revealed that olive-oil-based 3CBs is non-inferior and well tolerated compared to soybean formulations.