Este trabajo fue apoyado en parte por (1) el Programa de Investigación del Cáncer (US Army Medical Investigación Adquisición Actividad) premio # W81XVVH-10-1-0781 a LAL y VE, y (2) la donación de la Fundación Susan G. Komen Breast Departamento de Defensa IR122224446 a Lal y VE. Apoyo Patología y taquillera tejido fue proporcionado amablemente por Inova Fairfax Departamento de Patología, el Dr. Hassan Nayer, el Dr. A. Gita Menezes, y el Dr. Charles Bechert. El consentimiento del paciente y la adquisición de la muestra fue hábilmente guiados por Inova Fairfax Hospital coordinadores de investigación clínica del acebo Gallimore, Heather Huryk, y Emil Kamar.

Tejido mamario humano se obtuvieron de pacientes que participaron en un estudio de investigación, con el consentimiento informado por escrito, siguiendo Departamento de Defensa, la Universidad George Mason, y los protocolos aprobados por Inova Health System Junta de Revisión Institucional. 1. Prepare rica en nutrientes medio con Factores de crecimiento y antibióticos <ol><li> Preparar soluciones madre de insulina, factor de crecimiento epidérmico (EGF), sulfato de estreptomicina y sulfato de gentamicina. <ol><li> Reconstituir la insulina en agua filtrada estéril hasta una concentración final de 10 mg / ml. Aspirar 10 ml de agua de tipo 1 grado reactivo en un 10 ml jeringa desechable estéril. Adjuntar un filtro de 0,22 micras polietersulfona a la jeringa. Dispensar el agua filtrada estéril en un tubo cónico de 15 ml estéril. </li><li> Añadir 10 ml de agua filtrada estéril a un vial de 100 mg de la insulina. Mezclar el contenido brevemente en un mezclador de vórtice. Conservar el vial de insulina en el hielo. Dispense 450 l de la insulina de la solución en la etiqueta, tubos de microcentrífuga estériles y almacenar a -20 ° C. Solución de insulina de valores es estable hasta la fecha de caducidad indicada en el vial. </li><li> Preparar la solución madre de factor de crecimiento epidérmico (EGF): Añadir 500 l de agua estéril y 500 mg de EGF (agotadas 1 mg / l). Mezclar el contenido brevemente en un mezclador de vórtice. Conservar el vial de EGF en el hielo. <ol><li> Preparar una solución madre de trabajo de EGF: Añadir 100 l de la solución madre FEAG a 900 l medio nutritivo (madre de trabajo será de 0,1 mg / l). Mezclar el contenido brevemente en un mezclador de vórtice. Dispensar 50 l de la solución de EGF trabajo stock en, tubos de microcentrífuga estériles etiquetados y almacenar a -80 ° C. NOTA: solución madre EGF (1 mg / l) es estable por 1 año a -80 ° C; trabajando solución madre (0,1 mg / l) es estable durante 2 meses a -80 ° C. </li></ol></li><li> Pesar 40 mg de sulfato de estreptomicina en una balanza analítica. Coloque el int sulfato de estreptomicinaOA estériles 15 ml tubo cónico. Añadir 4 ml de medio nutriente. Mezclar el contenido brevemente en un mezclador de vórtice. La solución debe ser ligeramente rosado. Almacenar a 4 ° C protegida de la luz. Solución de sulfato de estreptomicina es estable durante 7 días. </li><li> Pesar 20 mg de sulfato de gentamicina en una balanza analítica. Coloque el sulfato de gentamicina en un tubo cónico de 15 ml estéril. Añadir 2 ml de medio nutriente. Mezclar el contenido brevemente en un mezclador de vórtice. La solución debe ser de color amarillo. Almacenar a 4 ° C protegida de la luz. Solución de sulfato de gentamicina es estable durante 7 días. </li></ol></li><li> Preparar dos 200 ml lotes de medio nutriente suplementado con EGF recombinante humano (10 ng / ml concentración final.), Insulina (10 mg / ml conc.), Sulfato de estreptomicina (100 mg / ml conc.) Y sulfato de gentamicina (20 g / ml concentración final.). <ol><li> Añadir 200 ml medio nutriente a un matraz de filtro de vacío equipado con un matraz de filtro de 0,2 micras de polietersulfona y una botella de recibir 250 ml. Añadir 20 y# 181; l madre de trabajo EGF, 200 l de stock de insulina, 2 ml de stock de sulfato de estreptomicina, y 400 l de stock de sulfato de gentamicina a la 200 ml de medio nutriente. Conecte el matraz de filtración al vacío. Filtra el medio. Deseche el filtro y la etiqueta del frasco como &quot;medio rico en nutrientes&quot;. Almacenar a 4 ° C durante un máximo de 14 días. </li></ol></li></ol> 2. Tejido Adquisición y Ventas <ol><li> En la sala de operaciones, mantener una técnica estéril después de la adquisición de los tejidos del seno. Coloque el tejido mamario en una bandeja estéril y cubrir la bandeja con papel de plástico estéril. <ol><li> Transporte el tejido en la bandeja cubierta de Radiología / Patología como es requerido por su institución. No abra la bandeja. Los plazos de transporte varían dentro de las instituciones. El tejido puede permanecer viable en esta bandeja cubierto a temperatura ambiente durante la escisión posterior de hasta 45 min. Sin embargo, el procesamiento oportuno de los tejidos en un medio rico en nutrientes proporciona las condiciones óptimas para el cultivo organoid posterior. </li></ol></li><li> Disección de tejido bruto a identificar las áreas de carcinoma ductal in situ dentro del tejido del seno: Usar guantes estériles, cuchillas, bisturís, tejido tinturas para marcar, y vinagre durante la disección de tejidos para mantener la esterilidad de los tejidos. <ol><li> Limpie el área de trabajo con etanol al 70% o el 1% de lejía. Abra los guantes estériles y colocar la envoltura de los guantes en la superficie de trabajo. Coloque el interior de la envoltura estéril guante hacia arriba. Póngase los guantes estériles. <ol><li> Limpiar la superficie de la recipiente de la muestra con etanol al 70%. Quite la envoltura de plástico del contenedor de muestras de tejido y colocar la muestra en la envoltura del guante. </li></ol></li><li> Sumerja dos hisopos con punta de algodón en el tinte marcado tejido. Aplicar el colorante a la superficie del tejido haciendo rodar los hisopos a través de la superficie del tejido. NOTA: Cada departamento de patología tiene un protocolo de orientación color del tinte / tejido estandarizado. Tissue está marcado con colorante para orientar el tejido en relación con su posición en el paciente. El colorante se borró opintada sobre el tejido. No vierta el colorante sobre el tejido. Verter el medio de contraste puede provocar que el medio de contraste para ejecutar / goteo en las grietas de tejido confundiendo así la orientación de los márgenes quirúrgicos. De orientación del tejido proporciona al cirujano y el patólogo con puntos de referencia anatómicos a a) describir la muestra de tejido, y b) determinar la ubicación de los márgenes quirúrgicos en relación con el tumor. </li><li> Vierta vinagre blanco destilado (5% de ácido acético) sobre gasas estériles. Seque el tejido teñido con el vinagre empapado gasa. Deseche la gasa. El vinagre se utiliza para fijar la marca de tintes de tejidos. </li><li> Cortar el tejido mamario en rebanadas verticales de aproximadamente 5 mm de espesor. No corte todo el camino a través del tejido (Figura 2). Observar / palpar el tejido para las áreas de calcificación. Identificar lesiones de CDIS por su firma característica, pálida apariencia rojiza rodeada, bordes de goma que se sienten arenosos (debido a las espículas de calcio). NOTA: En algunos casos, comedo áreas (espinilla-como pueden ser)visto como puntos blancos, que representan el material necrótico de gran diámetro lesiones de CDIS. </li><li> Cortar áreas de DCIS / tejido mamario calcificado, incluyendo una pequeña cantidad de tejido circundante del pecho. Coloque el tejido del seno en un tubo de 50 ml estéril que contiene 20-30 ml de medio rico en nutrientes preparado en la etapa 1. <ol><li> Mezclar el tejido y por medio suavemente invirtiendo el tubo varias veces. Deseche el medio y añadir medio fresco. Coloque el tubo que contiene el tejido y medio en un contenedor aislado y transportar el tejido al laboratorio de cultivo de tejidos. </li></ol></li></ol></li></ol> 3. Tissue Culture <ol><li> Trabajar en un gabinete de seguridad biológica, verter el tejido y una pequeña cantidad del medio nutriente en una placa de Petri estéril. Con un bisturí estéril cortar y desechar el tejido fibroso. Cortar el tejido mamario en piezas (organoides) aproximadamente 3 mm 2 (Figura 1C-D). Trate de cortar el tejido de manera que cada órganooid contiene al menos un segmento de conducto discernible con estroma circundante. </li><li> Abrir la tapa del frasco de cultivo de tejidos. El uso de pinzas o fórceps estériles, colocar los organoides en el matraz. Cierre la tapa. Deseche la placa de Petri. </li><li> Añadir medio rico en nutrientes suero libre de 11 ml (preparado en el paso 1) al matraz de cultivo de tejidos. Cerrar el frasco y agitar el frasco de manera que los organoides y medio se distribuyen de manera uniforme en toda la superficie del matraz (Figura 1E-F). </li><li> Incubar el matraz a 37 ° C en un 5,0% de CO 2 humidificado ambiente durante 2 días. No mueva el frasco durante este tiempo. </li><li> El día 2 de post incubación, retirar el frasco de la incubadora para comprobar la posible contaminación bacteriana / por hongos. Evite movimientos repentinos y agudos, o remolino del matraz. Colocar el matraz en un escenario microscopio invertido. <ol><li> Observar el medio para las bacterias, levadura, y / o hongos. Si no se observa contaminación, devuelva el frasco a la incubadora durante un complementodía itional. Si se observa contaminación, deseche el matraz y su contenido en un recipiente apropiado. </li></ol></li><li> En el día 3 después de la incubación, sustituir el medio con medio fresco acondicionado. <ol><li> Colocar 11 ml de medio en un tubo estéril a 37 ° C durante 20-30 min. Retirar el matraz de cultivo de tejidos de la incubadora. Sin molestar a los organoides, retire y deseche el medio en el matraz con una pipeta serológica estéril. </li><li> Utilizando una nueva pipeta serológica estéril, añadir 11 ml de medio fresco precalentado. Girar muy suavemente el frasco para distribuir el medio a través de la superficie del matraz. </li><li> Incubar el matraz a 37 ° C en un 5,0% humidificado atmósfera de CO 2. </li></ol></li></ol> 4. Mantenimiento de la empresa organoide / colonias de células epiteliales <ol><li> Preparar medio fresco cada 2 semanas y reemplazar los medios de comunicación en el matraz de cultivo de tejido 3 veces por semana. <ol><li> Colocar 11 ml de medio en un recipiente estéril a 37° C durante 20-30 minutos. Retirar el matraz de la incubadora. Con una pipeta serológica estéril, retire y deseche el medio condicionado del frasco, teniendo cuidado de no molestar a los organoides. </li><li> Utilizando una nueva pipeta serológica estéril, añadir 11 ml de medio fresco precalentado. Girar muy suavemente el frasco para distribuir el medio a través de la superficie del matraz. Incubar el matraz a 37 ° C en un 5,0% humidificado atmósfera de CO 2. </li></ol></li><li> Después de 10 - 14 días en cultivo, retire todos los trozos de tejido en el frasco de cultivo que no son adherentes. <ol><li> Células y / o organoides del matraz para la propagación en nuevos matraces de cultivo periódicamente cosecha, para transplante de xenoinjerto, o para fenotípica y / o análisis molecular. </li><li> Retirar el matraz de cultivo de tejidos de la incubadora. Pulverizar el matraz con etanol al 70%. Limpie el exceso de etanol en el matraz con una toalla de papel limpia que ha sido rociado con etanol al 70%. </li><li> Propagación organoid: &lt;ol&gt; <li> Abra la tapa del frasco y coloque la tapa boca arriba en la cabina de seguridad biológica. Bajo la visualización microscópica, localizar el organoide (s) para ser cosechado. Utilice pinzas o fórceps estériles para recoger un organoide. </li><li> Para propagar la organoide en un nuevo frasco de cultivo, coloque el organoide en el nuevo frasco. Añadir 11 ml de medio fresco, cálido. Incubar a 37 ° C, en una atmósfera de 5% CO 2 humidificado tal como se describe en los pasos 3.3 - 3.6.3. Sustituir el medio de cultivo celular en el cultivo de tejidos matraz de tres veces por semana. </li></ol></li><li> La recolección de células: <ol><li> Bajo vixualization microscópico directo, raspan suavemente y las células de aspirado y mammospheres utilizando una pipeta de 1.000 l con puntas de pipeta desechables estériles. Aspirar las células y el medio circundante. Prescindir de la células / medio en un tubo de microcentrífuga estéril. </li></ol></li><li> Haga girar las células brevemente en un mini-centrifugar a 12.100 xg durante 5 seg. Retire y deseche el medio. &lt;ol&gt; <li> Para el análisis de ADN, congelar inmediatamente el sedimento de células en hielo seco, en un pequeño volumen de medio (10 l), con almacenamiento a largo plazo a -80 ° C. </li><li> Para el análisis proteómico, la lisis de las células en 10 l de urea 8 M para espectrometría de masas o 2x SDS tampón tris-glicina para el Western Blot / inversa microarrays de proteínas de fase. Alternativamente, las células girar en una citocentrífuga para hacer frotis de células para el análisis inmunohistoquímico. </li></ol></li><li> Después de cosechar las células de un frasco, retire y deseche el medio restante. Añadir 11 ml de medio rico caliente, nutriente fresco. Incubar el matraz a 37 ° C, en un 5% humidificado atmósfera de CO 2. </li></ol></li></ol>

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El sistema de cultivo se describe en el presente documento constituye un nuevo modelo para la generación que viven las células mamarias tumorales pre-invasivas para estudios básicos y traslacionales de investigación. En el pasado, la progresión del cáncer de mama pre-maligna típicamente se ha estudiado utilizando tres métodos diferentes. El primer método es histopatológico y el análisis genético de microdissected especímenes humanos congelados o fijos 12-14. El segundo método utiliza modelos de ...

Proliferación de las células epiteliales dentro de los confines de los conductos mamarios y los alvéolos (carcinoma ductal in situ) es reconocido como un precursor obligado a ductal infiltrante y el carcinoma de mama lobular. Sin embargo, los mecanismos moleculares y dinámica de la población de células que permiten la progresión a cáncer invasor, son poco conocidos. Dilucidar los mecanismos de supervivencia utilizados por las células de carcinoma de mama pre-invasoras, o cualquier tumor pre-invasivo, puede revelar las estrategias terapéuticas para matar, o incluso prevenir, neoplasias pre-invasoras 1. Sin embargo, los métodos simples de bajo costo para estudiar funcionalmente lesiones pre-invasoras humanos han faltado. Aunque el cultivo monocapa in vitro de líneas celulares transformadas es un método de laboratorio establecida, el fenotipo y el genotipo de estas líneas celulares inmortalizadas falla para recapitular el estado molecular de las células tumorales humanas primarias 2. Además, incluso la no tumorigénicos línea celular MCF-10A, que recapitulates 3-D arquitectura de la glándula mamaria, falla para representar adecuadamente el fenotipo funcional y características moleculares de pre-invasiva lesión de la mama de un paciente individual 3,4. Para determinar si las células neoplásicas-madre como capaces de invasión existían en el carcinoma ductal in situ (CDIS) población de células, hemos desarrollado una metodología para la recolección y el cultivo de tejidos de mama humana estéril en el momento de la cirugía (Figura 1) 5. Nuestro sistema de cultivo ex vivo organoide de mama no se basa en la interrupción enzimática de tejido, extracto de matriz de membrana basal, o el agotamiento de fibroblastos, para aislar y propagar células formadoras de mammosphere fresco ductal de mama humano tejido de carcinoma de 6-8. Nuestro nuevo sistema se basa en el principio de transmisión de célula / migración 5. Los conductos mamarios discernibles, y estroma circundante se sumergen en un volumen mínimo de medio nutriente libre de suero (sólo correonough para cubrir los fragmentos de cajón) para maximizar el intercambio de gas, con la superficie de corte del conducto expuesta al medio de cultivo, pero en ninguna orientación específica en el matraz (Figura 1E-F). Este sistema de cultivo permite que las células migran fuera del conducto y en / sobre el matraz de estroma y de cultivo autólogo. El medio nutriente, suplementado sólo con el Factor de Crecimiento Epidérmico (EGF), insulina y antibióticos, apoya el crecimiento de las poblaciones de células mixtas que emanan de la organoide. El matraz de cultivo tisular tiene un re-sellable tapa extraíble, que permite que los organoides y / o células que se recogerán sin interrumpir la totalidad del frasco o vecinos organoides, mientras que el mantenimiento de un entorno humidificado estéril.

<table border="0" cellpadding="0" cellspacing="0" width="1024">
	<tbody>
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			<td height="21" style="height:21px;width:353px;">Name of Reagent/ Equipment</td>
			<td style="width:187px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:365px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Ethanol</td>
			<td style="width:187px;">Fisher</td>
			<td style="width:119px;">A405-P</td>
			<td>prepare a 70% solution in Type 1 reagent grade water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">18 M&#937;-cm water, sterile filtered</td>
			<td style="width:187px;"></td>
			<td style="width:119px;"></td>
			<td>sterile filtered, Type 1 reagent grade water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10cc plastic disposable syringe, sterile</td>
			<td style="width:187px;">BD</td>
			<td style="width:119px;">305482</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.2&#181;m polyethersulfone (PES) syringe filter, sterile</td>
			<td style="width:187px;">Thermo Scientific</td>
			<td style="width:119px;">194-2520</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">15ml polypropylene conical tubes, sterile</td>
			<td style="width:187px;">Fisher</td>
			<td>14-959-49B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">50ml polypropylene conical tubes, sterile</td>
			<td style="width:187px;">Fisher</td>
			<td>05-539-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">1.5ml low retention microcentrifuge tubes,sterile</td>
			<td style="width:187px;">Fisher</td>
			<td style="width:119px;">02-681-331</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">nutrient medium, DMEM-F12/HEPES</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:119px;">11330-032</td>
			<td>with L-glutamine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insulin, human recombinant</td>
			<td style="width:187px;">Roche</td>
			<td style="width:119px;">11376497001</td>
			<td>10mg/ml stock</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Epidermal Growth Factor (EGF), human recombinant</td>
			<td style="width:187px;">Millipore</td>
			<td style="width:119px;">GF144</td>
			<td>100&#181;g/ml stock</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptomycin sulfate</td>
			<td style="width:187px;">Sigma-Aldrich</td>
			<td style="width:119px;">S1567</td>
			<td>10mg/ml stock</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gentamicin sulfate</td>
			<td style="width:187px;">Sigma-Aldrich</td>
			<td style="width:119px;">G19114</td>
			<td>10mg/ml stock</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Filtration flask and filter top, sterile</td>
			<td style="width:187px;">Millipore</td>
			<td>SCGPU02RE</td>
			<td>0.22&#181;m PES membrane</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">25ml sterile, disposable pipettes</td>
			<td style="width:187px;">Fisher</td>
			<td>4489</td>
			<td>paper-plastic wrapped</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10ml sterile, disposable pipettes</td>
			<td style="width:187px;">Fisher</td>
			<td style="width:119px;">4488</td>
			<td>paper-plastic wrapped</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:353px;">Tissue marking dyes (black, blue, red, green, yellow and orange)</td>
			<td style="width:187px;">CDI</td>
			<td style="width:119px;">MD2000</td>
			<td style="width:365px;">after opening use only with single-use, sterile cotton tipped applicators, or use once and discard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Cotton tipped applicators, sterile</td>
			<td style="width:187px;">Fisher</td>
			<td>23-400-115</td>
			<td>single use only</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Gauze pads, 10x10cm, sterile</td>
			<td style="width:187px;">Fisher</td>
			<td style="width:119px;">2187</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plastic transfer pipettes, sterile, disposable</td>
			<td style="width:187px;">Samco</td>
			<td>202-20S</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;">Vinegar, white distilled</td>
			<td style="width:187px;">household use</td>
			<td></td>
			<td style="width:365px;">5% acetic acid; after opening use only with sterile pipettes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">#10 scalpels, sterile, disposable&#160;</td>
			<td style="width:187px;">Thermo Scientific</td>
			<td>31-200-32&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">petri dish, sterile</td>
			<td style="width:187px;">Fisher</td>
			<td>FB0875713A</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">TPP 115cm2 flask, with removable lid</td>
			<td style="width:187px;">MidSci</td>
			<td style="width:119px;">90652</td>
			<td>screw cap with filter</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:353px;">CO2 incubator</td>
			<td style="width:187px;">Fisher</td>
			<td>13-998-074</td>
			<td>5% CO2, 37 oC, humidified chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">inverted light microscope</td>
			<td style="width:187px;">Olypmus</td>
			<td>IX51</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">8M urea</td>
			<td style="width:187px;">Fisher</td>
			<td style="width:119px;">BP169-500</td>
			<td>optional, for mass spectrophotometric analysis of cultured cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">2X SDS tris-glycine buffer</td>
			<td style="width:187px;">Life Technologies</td>
			<td>LC2676</td>
			<td>optional, for proteomic analysis of cultured cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Cytocentrifuge</td>
			<td style="width:187px;">Thermo Scientific</td>
			<td>A78300003</td>
			<td>optional, for preparing cell smears</td>
		</tr>
	</tbody>
</table>

non-enzymatic, serum-free tissue culture of pre-invasive breast lesions for spontaneous generation of mammospheres

Ductal carcinoma de mama in situ (DCIS), por definición, es la proliferación de células epiteliales neoplásicas dentro de los confines del conducto de la mama, sin violar la membrana basal de colágeno. Mientras DCIS es un precursor no obliga a los cánceres de mama invasivos, los mecanismos moleculares y las poblaciones de células que permiten la progresión a cáncer invasivo no se conocen totalmente. Para determinar si las células progenitoras capaces de invasión existían dentro de la población de células DCIS, se desarrolló una metodología para la recogida y el cultivo de tejido de mama humano estéril en el momento de la cirugía, sin interrupción enzimática de tejido. Tejido del seno estéril que contiene segmentos ductales se cosecha a partir de tejido de mama extirpada quirúrgicamente tras el examen patológico de rutina. Tejido que contiene DCIS se coloca en un medio rico en nutrientes, que contiene antibiótico, sin suero, y se transporta al laboratorio de cultivo de tejidos. El tejido mamario es más dissected para aislar las áreas calcificadas. Múltiples piezas de tejido de mama (organoides) se colocan en un volumen mínimo de medio libre de suero en un matraz con una tapa extraíble y se cultivaron en un incubador de CO2 humidificado. Poblaciones de células epiteliales y fibroblastos emergen de la organoide después de 10 - 14 días. Mammospheres formar espontáneamente en y alrededor de la monocapa de células epiteliales. Poblaciones específicas de células pueden ser cosechadas directamente desde el matraz sin interrumpir las células vecinas. Nuestro sistema de cultivo de tejidos no enzimática revela de forma fiable, células progenitoras invasivos citogenéticamente anormales de lesiones de CDIS humanos frescos.

Flujo de trabajo para la adquisición y el cultivo de carcinoma ductal de mama estéril en el tejido in situ Esterilidad El tejido mamario se mantiene desde el quirófano al laboratorio de cultivo celular a través de pequeños cambios en el flujo de trabajo típico de patología del hospital (Figura 1). El tejido se transporta en un recipiente estéril con una cubierta de película de plástico que permite la evaluación radiológica, mientras que se mantiene...

Watch this Scientific Journal Video about Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres at JoVE.com

Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

Cultivo de células primarias de tejido intacto utilizando organoides proporciona un sistema modelo que imita el microambiente multi-celular in vivo. Desarrollamos un modelo de cultivo libre de suero primaria tejido epitelio de mama que perpetúa los linajes de cultivo de células mixtas y exposiciones morfología diferenciados, sin interrupción tejido enzimática. Organoides mamarios permanecen viables durante&gt; 6 meses.

No enzimática, libre de suero Cultivo de Tejidos de lesiones de mama pre-invasivas para la generación espontánea de mammospheres

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without ...

non-enzymatic-serum-free-tissue-culture-pre-invasive-breast-lesions

Research

JoVE Journal

Biology

12.5K vistas.  George Mason University.  Cultivo de células primarias de tejido intacto utilizando organoides proporciona un sistema modelo que imita el microambiente multi-celular in vivo. Desarrollamos un modelo de cultivo libre de suero primaria tejido epitelio de mama que perpetúa los linajes de cultivo de células mixtas y exposiciones morfología diferenciados, sin interrupción tejido enzimática. Organoides mamarios permanecen viables durante> 6 meses. 

Video: Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

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

Generation of Single-Cell Suspensions from Mouse Neural Tissue

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a repertoire of cell surface molecules, or molecular determinants, through which it can be identified and characterized. Currently, robust cell identification and separation technologies require single-cell preparations to be generated while simultaneously limiting cell death and destruction of characteristic surface protein. The gentleMACS Dissociator, when used in combination with trypsin or papain-based dissociation kits, can effectively and gently dissociate brain tissue while preserving antigen epitopes and limiting cell loss. Standardized preparation of single-cell suspensions is achieved using C Tubes and optimized, preset gentleMACS Programs. Once generated, single-cell suspensions can be treated with monoclonal conjugates like Anti-Prominin-1 MicroBeads, which identify neural progenitors, or purified further using Myelin Removal Beads.

Dissociating cells from specific tissue types requires specific parameters for tissue aggitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS Dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on nerual tissue is explained.

Generación de un solo suspensiones de células de tejido de ratón Neural

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a ...

Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for mesenchymal stromal cells (MSCs), fibroblasts and endothelial colony-forming cells with platelets activated by thrombin3-5 or lysed by freeze/thaw cycles6-14 before the platelet releasate is added to the cell culture medium. The trophic effect of platelet derived growth factors has already been tested in several trials for tissue engineering and regenerative therapy.1,15-17 Varying efficiency is considered to be at least in part due to individually divergent concentrations of growth factors18,19 and a current lack of standardized protocols for platelet preparation.15,16 This protocol presents a practicable procedure to generate a pool of human platelet lysate (pHPL) derived from routinely produced platelet rich plasma (PRP) of forty to fifty single blood donations. By several freeze/thaw cycles the platelet membranes are damaged and growth factors are efficiently released into the plasma. Finally, the platelet fragments are removed by centrifugation to avoid extensive aggregate formation and deplete potential antigens. The implementation of pHPL into standard culture protocols represents a promising tool for further development of cell therapeutics propagated in an animal protein-free system.

Preparation of Pooled Human Platelet Lysate pHPL as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Human platelet lysate is a rich source of growth factors and a potent supplement in cell culture. This protocol presents the process of preparing a large pool of human platelet lysate by starting from platelet rich plasma, performing several freeze-thaw cycles and depleting the platelet fragments.

Preparación de combinados lisado plaquetario humano (pHPL) como un suplemento eficaz de los animales libres de suero humano culturas de células madre

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for ...

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address the requirements (e.g., mechanics and vascularity) of more intricate organs and tissues. Electrospinning is a popular technique to create fibrous scaffolds that mimic the architecture and size scale of the native extracellular matrix. These fibrous scaffolds are also useful as cell culture substrates since the fibers can be used to direct cellular behavior, including stem cell differentiation (see extensive reviews by Mauck et al. and Sill et al. for more information). In this article, we describe the general process of electrospinning polymers and as an example, electrospin a reactive hyaluronic acid capable of crosslinking with light exposure (see Ifkovits et al. for a review on photocrosslinkable materials). We also introduce further processing capabilities such as photopatterning and multi-polymer scaffold formation. Photopatterning can be used to create scaffolds with channels and multi-scale porosity to increase cellular infiltration and tissue distribution. Multi-polymer scaffolds are useful to better tune the properties (mechanics and degradation) of a scaffold, including tailored porosity for cellular infiltration. Furthermore, these techniques can be extended to include a wide array of polymers and reactive macromers to create complex scaffolds that provide the cues necessary for the development of successful tissue engineered constructs.

The process of electrospinning polymers for tissue engineering and cell culture is addressed in this article. Specifically, the electrospinning of photoreactive macromers with additional processing capabilities of photopatterning and multi-polymer electrospinning is described.

Electrospinning andamios fibroso polímero para ingeniería de tejidos y cultivos celulares

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address ...

Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later distally, is a major factor in patient prognosis. Experimental systems of breast cancer rely on cell lines usually derived from primary tumours or pleural effusions. Two major obstacles hinder this research: (i) some known sub-types of breast cancers (notably poor prognosis luminal B tumours) are not represented within current line collections; (ii) the influence of the tumour microenvironment is not usually taken into account.
We demonstrate a technique to culture primary breast cancer specimens of all sub-types. This is achieved by using three-dimensional (3D) culture system in which small pieces of tumour are embedded in soft rat collagen I cushions. Within 2-3 weeks, the tumour cells spread into the collagen and form various structures similar to those observed in human tumours1. Viable adipocytes, epithelial cells and fibroblasts within the original core were evident on histology. Malignant epithelial cells with squamoid morphology were demonstrated invading into the surrounding collagen. Nuclear pleomorphism was evident within these cells, along with mitotic figures and apoptotic bodies.
We have employed Optical Projection Tomography (OPT), a 3D imaging technology, in order to quantify the extent of tumour spread in culture. We have used OPT to measure the bulk volume of the tumour culture, a parameter routinely measured during the neo-adjuvant treatment of breast cancer patients to assess response to drug therapy. 
Here, we present an opportunity to culture human breast tumours without sub-type bias and quantify the spread of those ex vivo. This method could be used in the future to quantify drug sensitivity in original tumour. This may provide a more predictive model than currently used cell lines.

We have developed a collagen-based in vitro assay which promotes proliferation and invasion from samples of all breast cancer subtypes. Optical Projection Tomography, a three dimensional microscopy technique was utilised to visualise and quantify tumour expansion. This assay may be used to quantify drug response of individual tumour samples.

A largo plazo la cultura de muestras humanas del cáncer de pecho y su análisis mediante tomografía óptica de proyección

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

Embriones de ratón con el desarrollo en todo un sistema de cultivo de embriones sin suero

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

A Tissue Culture Model of Estrogen-producing Primary Bovine Granulosa Cells

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca and, in particular, of the granulosa cell layer profoundly change their morphological, physiological, and molecular characteristics and form the progesterone-producing corpus luteum that is responsible for maintaining pregnancy. Cell culture models are essential tools to study the underlying regulatory mechanisms involved in the folliculo-luteal transformation. The presented protocol focuses on the isolation procedure and cryopreservation of bovine GC from small- to medium-sized follicles (&#60; 6 mm). With this technique, a nearly pure population of GC can be obtained. The cryopreservation procedure greatly facilitates time management of the cell culture work independent of a direct primary tissue (ovaries) supply. This protocol describes a serum-free cell culture model that mimics the estradiol-active status of bovine GC. Important conditions that are essential for a successful steroid-active cell culture are discussed throughout the protocol. It is demonstrated that increasing the plating density of the cells induces a specific response as indicated by an altered gene expression profile and hormone production. Furthermore, this model provides a basis for further studies on GC differentiation and other applications.

A long-term culture model of bovine granulosa cells under serum-free conditions is described. This model allows researchers to study the effects of diverse factors and conditions as different plating densities on the characteristics of estrogen-producing bovine granulosa cells.

Un modelo de cultivo de tejidos de Granulosa bovino primario producción de estrógeno de las células

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca ...

Tissue Collection of Bats for -Omics Analyses and Primary Cell Culture

As high-throughput sequencing technologies advance, standardized methods for high quality tissue acquisition and preservation allow for the extension of these methods to non-model organisms. A series of protocols to optimize tissue collection from bats has been developed for a series of high-throughput sequencing approaches. Outlined here are protocols for the capture of bats, desired demographics to be collected for each bat, and optimized methods to minimize stress on a bat during tissue collection. Specifically outlined are methods for collecting and treating tissue to obtain (i) DNA for high molecular weight genomic analyses, (ii) RNA for tissue-specific transcriptomes, and (iii) proteins for proteomic-level analyses. Lastly, also outlined is a method to avoid lethal sampling by creating viable primary cell cultures from wing clips. A central motivation of these methods is to maximize the amount of potential molecular and morphological data for each bat and suggest optimal ways to preserve tissues so they retain their value as new methods develop in the future. This standardization has become particularly important as initiatives to sequence chromosome-level, error-free genomes of species across the world have emerged, in which multiple scientific parties are spearheading the sequencing of different taxonomic groups. The protocols outlined herein define the ideal tissue collection and tissue preservation methods for Bat1K, the consortium that is sequencing the genomes of every species of bat.

This is a protocol for the optimal tissue preparation for genomic, transcriptomic, and proteomic analyses of bats caught in the wild. It includes protocols for bat capture and dissection, tissue preservation, and cell culturing of bat tissue.

Colección de tejidos de murciélagos para análisis de émica y cultivo celular primario

As high-throughput sequencing technologies advance, standardized methods for high quality tissue acquisition and preservation allow for the extension of ...

Mucociliary Epithelial Organoids from Xenopus Embryonic Cells: Generation, Culture and High-Resolution Live Imaging

Mucociliary epithelium provides the first line of defense by removing foreign particles through the action of mucus production and cilia-mediated clearance. Many clinically relevant defects in the mucociliary epithelium are inferred as they occur deep within the body. Here, we introduce a tractable 3D model for mucociliary epithelium generated from multipotent progenitors that were microsurgically isolated from Xenopus laevis embryos. The mucociliary epithelial organoids are covered with newly generated epithelium from deep ectoderm cells and later decorated with distinct patterned multiciliated cells, secretory cells, and mucus-producing goblet cells that are indistinguishable from the native epidermis within 24 h. The full sequences of dynamic cell transitions from mesenchymal to epithelial that emerge on the apical surface of organoids can be tracked by high-resolution live imaging. These in vitro cultured, self-organizing mucociliary epithelial organoids offer distinct advantages in studying the biology of mucociliary epithelium with high-efficiency in generation, defined culture conditions, control over number and size, and direct access for live imaging during the regeneration of the differentiated epithelium.

We describe a simple protocol to develop mucociliary epithelial organoids from deep ectoderm cells isolated from Xenopus laevis embryos. The multipotent progenitors regenerate epithelial goblet cell precursors and allow live tracking of the initiation and progression of the cell transitions on the surface of organoids.

Organoides epiteliales mucocilios de células embrionarias de Xenopus: generación, cultura e imágenes en vivo de alta resolución

Mucociliary epithelium provides the first line of defense by removing foreign particles through the action of mucus production and cilia-mediated ...

Bovine Ovarian Cortex Tissue Culture

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors from somatic cells and cumulus cell-oocyte communication. Little is known about the ovarian microenvironment and how the cytokines and steroids produced in the surrounding milieu affect follicle progression or arrest. In vitro culture of ovarian cortex enables follicles to develop in a normalized environment that remains supported by adjacent stroma. Our objective was to determine the effect of nutritional Stair-Step diet on the ovarian microenvironment (follicle development, steroid, and cytokine production) through in vitro culture of bovine ovarian cortex. To accomplish this, ovarian cortical pieces were removed from heifers undergoing two different nutritionally developed schemes prior to puberty: Control (traditional nutrition development) and Stair-Step (feeding and restriction during development) that were cut into approximately 0.5-1 mm3 pieces. These pieces were subsequently passed through a series of washes and positioned on a tissue culture insert that is set into a well containing Waymouth&#39;s culture medium. Ovarian cortex was cultured for 7 days with daily culture media changes. Histological sectioning was performed to determine follicle stage changes before and after the culture to determine effects of nutrition and impact of culture without additional treatment. Cortex culture medium was pooled over days to measure steroids, steroid metabolites, and cytokines. There were tendencies for increased steroid hormones in ovarian microenvironment that allowed for follicle progression in the Stair-Step versus Control ovarian cortex cultures. The ovarian cortex culture technique allows for a better understanding of the ovarian microenvironment, and how alterations in endocrine secretion may affect follicle progression and growth from both in vivo and in vitro treatments. This culture method may also prove beneficial for testing potential therapeutics that may improve follicle progression in women to promote fertility.

In vitro culture of bovine ovarian cortex and the effect of nutritional Stair-step diet on ovarian microenvironment is presented. Ovarian cortex pieces were cultured for seven days and steroids, cytokines, and follicle stages were evaluated. The Stair-Step diet treatment had increased steroidogenesis resulting in follicle progression in culture.

Cultivo de tejido de corteza ovárica bovina

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors ...