Este trabajo fue apoyado por fondos del Departamento de Defensa de la próstata Programa de Investigación del Cáncer (W81XWH-12-1-0124), U54CA143876 del Instituto Nacional del Cáncer y el Fondo de Investigación Robert McCooey Oncología Genitourinaria. Nos gustaría dar las gracias al Dr. Annarita Lorenzo (Departamento de Patología) para proporcionar anticuerpos VE-cadherina, y el Dr. Marco Seandel (Departamento de Cirugía) para proporcionar HUVECs.

1. El cultivo de HUVEC microportaobjetos para Interacciones de Observación del CTC-endoteliales <ol><li> Bajo la campana de cultivo de tejidos, primero enjuague el microportaobjetos, ancho de canal de 1 mm con PBS. Suavemente la capa MicroSlide con 200 l de 50 mg / ml de fibronectina (disuelto en PBS) usando una jeringa de cierre Luer de 1 ml. </li><li> Cubrir el MicroSlide con la tapa y mantenerlo dentro de la campana de cultivo de tejido durante 30 min. De dispensación lenta del líquido en el MicroSlide impide la formación de burbujas en el canal. </li><li> Perfundir 200 l de agua tibia (37 ° C) de HUVEC medio de crecimiento (medio M199, HEPES 1 M, 20% de FBS, 5 mg / ml de heparina, factor de crecimiento celular endotelial 100 g / ml, y L-glutamina) sobre el MicroSlide e incubar 20 min a temperatura ambiente. Durante la perfusión, preparar la suspensión de células HUVEC. </li><li> Enjuague HUVECs con PBS y añadir 0,05% de tripsina-EDTA durante 1-2 min a TA. Centrífuga HUVECs en 2 ml de medio de crecimiento a 180 xg durante 5 min. </li><li> Medir la concentración de células usando un Neubahemocitómetro uer y preparar 10 7 HUVEC células/100 l medio de cultivo. A continuación, retire con cuidado el medio de la entrada de la microportaobjetos usando una punta de pipeta de 200 l. </li><li> Llevar el MicroSlide a nivel de los ojos y el uso de una jeringa de 1 ml Luer-Lock, perfundir suavemente 200 l de concentración de preparado de HUVECs en el canal. Se debe tener precaución en este paso para evitar la formación de burbujas. Si aparecen burbujas, mantenga la perfusión durante un tiempo un poco más largo hasta que las burbujas entren en el canal de salida. </li><li> Ponga un volumen igual (~ 80 l) de los medios de comunicación HUVEC tanto en la entrada y salida de la MicroSlide. Esto evita que el flujo de células en cualquier dirección. </li><li> Cubrir el portaobjetos y mantenerlo en la incubadora (37 ° C) durante 1,5 horas. </li></ol> 2. Preparación de Flujo Asamblea Cámara para pasar la noche HUVEC Cultura en un MicroSlide <ol><li> Coloque una jeringa estéril de 20 ml, conectores Luer hembra y macho, los tubos y una bomba de jeringa en la incubadora durante 15 millasn. </li><li> Para un volumen muerto mínimo, utilizar el tubo con diámetro interior de 0,04 pulgadas. Menor diámetro tubo impide la formación de burbujas. </li><li> Llene la jeringa de 20 ml con agua caliente (37 ° C) HUVEC medios de comunicación (12 ml). Una el tubo con los conectores en la jeringa. Quite las burbujas. Conecte este conjunto a la microportaobjetos. </li><li> Llenar completamente la entrada de la MicroSlide con medios HUVEC. Traiga la jeringa de 20 ml conectada al conector junto al microportaobjetos. Conecte con cuidado el conector a la microportaobjetos. </li><li> Traiga la puesta a punto en la incubadora y conectarlo a la bomba de jeringa, puesta a régimen de cizalladura 10 l / min. Deje las células O / N en la incubadora a 37 ° C. </li><li> Al día siguiente, desmontar la puesta a punto mediante la eliminación de el conector unido a la entrada de la MicroSlide. </li><li> Para regular positivamente la expresión de E-selectina sobre las CE, preparar el medio de cultivo fresco que contenía IL-1β a 10 ng / ml en 4 ml de medio. Aspirar los medios de comunicación en una jeringa de 10 ml. Retire tque los medios de comunicación a partir de la entrada de la microportaobjetos y conectar la jeringa a la diapositiva. </li><li> Ajuste la bomba de jeringa a 10 la velocidad de cizalla l / min durante 4 horas en la incubadora. </li></ol> 3. Elaboración de Anti-PSMA (J591-488) con etiqueta células del cáncer de próstata <ol><li> Durante la incubación IL-1β de HUVECs, preparar anti-PSMA J591-Alexa488 etiquetados células de CaP. Añadir 0,05% de tripsina-EDTA para células MDA durante 1 min. No exponer las células a tripsina durante tiempos más largos, ya que puede afectar a los glicopéptidos presentes en la superficie celular. Reactivo de disociación celular libre de enzimas también se puede utilizar en lugar de la tripsina. Centrifugar a 200 xg durante 5 min. </li><li> Resuspender sedimento celular MDA in 1 ml de H / H tampón (sal de Hanks equilibrada solution/0.1% HSA/10 HEPES / CaCl2 1 mM). Añadir anticuerpo-J591 Alexa488 anti-PSMA a 20 mg / mL durante 30 min a TA en un lugar oscuro. Resuspender las células durante la incubación. </li><li> Después de 30 min, se centrifuga la solución de células a 800 xg durante 5 min. Aspitasa y resuspender el precipitado en 1 ml de tampón H / H. Contar las células MDA etiquetados y llevar la concentración final de 1x10 6 células / ml. Llene una jeringa de 5 ml con J591-488 células MDA etiquetados y eliminar las burbujas. </li></ol> 4. Preparación de anti-PSMA (J591-488) Etiquetada CTCs Enriquecido de CaP pacientes <ol><li> Recoger 7,5 ml de sangre de pacientes con cáncer de próstata en un tubo de tapa azul (que contiene citrato de sodio). Suavemente diluir 1:01 sangre en 0,1% BSA / 1 mM EDTA / PBS. </li><li> Añadir 5,3 ml de Ficoll-Paque Plus en un tubo cónico de 50 ml. Capa diluyó la sangre en la parte superior de la Ficoll-Paque. Centrifugar a 400 xg durante 30 min. </li><li> Pre-coat todos los tubos o consejos de entrar en contacto con las CTC con un 2% FBS/RPMI-1640 / 1 mM CaCl 2/4 mM MgCl 2 (tampón R / S) para evitar la unión no específica de los CTC a las superficies. Mantener 4 ° C de temperatura de los medios y tampones que entran en contacto con los CTC. </li><li> Recoger el de células mononucleares de sangre periférica (PBMC) fraccióncontiene CTCs de la interfaz en otro tubo cónico de 50 ml que contiene 30 ml de tampón de R / S. Se centrifuga a 400 g durante 8 min. </li><li> Resuspender el precipitado y lavar de nuevo en tampón de R / S a 400 xg durante 8 min. Después de la centrifugación, se resuspende el sedimento en tampón H / h. Añadir anti-PSMA J591-488 anticuerpo a 20 mg / ml durante 30 min a TA en un lugar oscuro. </li><li> Después de 30 minutos, centrifugar los PBMCs que contengan J591-488 CTCs etiquetados a 800 xg durante 5 min. Volver a suspender en tampón H / H. Llene una jeringa de 1 ml (pre-recubiertos con tampón de R / S) con la muestra. </li></ol> 5. Preparación del microscopio, la bomba de jeringa y flujo Asamblea Cámara <ol><li> Encienda el microscopio invertido y ajuste la iluminación Kohler a 10X objetivo. Traiga la bomba de jeringa al mismo nivel que la etapa de la muestra del microscopio y lo fijó en 1 dyn / cm 2 esfuerzo de corte (~ 10 l / min). </li><li> Abra el software Zeiss Axiovision. Seleccione el objeto en la pantalla de ordenador. Crear un nuevocarpeta en la opción de herramientas en el software. </li><li> Abra la opción experimentos inteligentes en Axiovision y ajuste la configuración para grabar vídeos de corta duración 30 seg durante 30 min. </li><li> Para el vídeo fluorescente en vivo, establezca las opciones-12 de imagen ms de exposición, 2 x 2 bandejas, 5 Gain. Estos parámetros ayudan en la consecución de los vídeos cercanas a la velocidad de fotogramas de vídeo (~ 23 cuadros por segundo) con la cámara Zeiss MRM. </li><li> Para visualizar ancho del canal de flujo a 10 X objetivo en la pantalla del ordenador, utilice un adaptador de montura C (0,63 mm xf/60 Interface). </li><li> Encienda la lámpara de mercurio. </li><li> Coloque la jeringa y el conector que contiene las células en la bomba de jeringa. </li><li> Llevar la IL-1β estimulada HUVEC de la incubadora. Enchufe el conector en el canal de entrada de lleno de la microportaobjetos contiene HUVECs. </li><li> Conectar el canal de salida con el conector unido a la tubería. Ponga el tubo en un plato o 15 ml tubo cónico para recoger el flujo a través. </li><li> Inicie la infusia través de la MicroSlide a 10 l / min. Tenga en cuenta las interacciones entre las células endoteliales y las células MDA marcados o etiquetados CTC derivados de pacientes menores de filtro de 488 nm en el microscopio de epifluorescencia. </li><li> Comienza la grabación del experimento como 30 seg videos cortos. Durante el análisis de la reproducción, medir la velocidad de rotación. La velocidad de rotación se mide dividiendo la distancia recorrida por las células con el tiempo. </li></ol> 6. La inmunotinción de la MicroSlide <ol><li> Retire el papel de la entrada y salida del microportaobjetos después de la perfusión de las células MDA en IL-1β estimulada HUVECs durante 10 min. </li><li> Con una punta de 200 l, poner caliente (37 ° C) de PBS que contiene calcio y magnesio en la entrada de la microportaobjetos durante 5 min. Incline la microportaobjetos usando su tapa como un apoyo en el banquillo. Mantenga el MicroSlide en una posición inclinada para todas las incubaciones durante inmunotinción. </li><li> Fijar HUVECs perfundiendo cálida formaldehído al 2% en la entrada </li><li&gt; incubar durante 20 minutos a temperatura ambiente. Enjuague dos veces con PBS durante 5 min cada uno. </li><li> Añadir Triton X-100 (0,1%) en 5% de BSA en PBS durante 10 min a TA. </li><li> Enjuague dos veces con PBS durante 5 min cada uno. Se bloquea con 5% de BSA en PBS durante 30 min. </li><li> Se incuba con anticuerpo de cabra anti-primaria VE-cadherina (1:100) en 2,5% BSA O / N a 4 ° C. </li><li> Al día siguiente, lavar dos veces con PBS durante 5 min cada uno. Añadir burro secundario de cabra anti-647 de anticuerpos durante 45 minutos. Lavar dos veces con PBS durante 5 min cada uno. </li><li> Incubar a temperatura ambiente con anticuerpo humanizado conjugado-J591 Alexa488 durante 1 hora. </li><li> Lavar dos veces con PBS durante 5 min cada uno. Añadir DAPI durante 5 min. Lavar con agua destilada durante 5 min. </li><li> Añadir PBS (o Mowiol para el almacenamiento a largo plazo). Visualice el microportaobjetos bajo el microscopio confocal. </li></ol>

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Dr. Bander es el inventor de las patentes que se asignan a la Fundación de Investigación de Cornell (&quot;IRC&quot;) para el anticuerpo J591 utilizada en este artículo. Dr. Bander es consultor y posee acciones en BZL Biologics, la empresa a la que las patentes han sido autorizados por el CRF de una mayor investigación y desarrollo.

Debido al bajo número de CTC entre las células de la sangre, es difícil aislar los CTC como una población pura de células. Con el fin de estudiar las interacciones CTC / CE, la población rara e impuro de los CTC plantea dos grandes retos: a) Identificación de los CTC entre las células de la sangre; b) Observación de las interacciones CTC / CE. Para superar la primera limitación de la identificación de los CTC de próstata entre los glóbulos, tomamos ve...

La metástasis es un proceso de múltiples pasos compleja que sigue siendo poco conocida. El eje ligando E-selectin/selectin se ha demostrado que desempeñan un papel importante en la metástasis tumoral mediante la promoción de interacciones adhesivas primarias entre el endotelio vascular y células de cáncer de 1,2. Endotelial (E)-selectina es una proteína transmembrana expresada por las células endoteliales activadas, mientras diferente ligando (s) de E-selectina se expresan por las células tumorales 3. Numerosos enfoques in vitro se han empleado con éxito para modelar las interacciones de ligando E-selectin/selectin entre las células tumorales y las células endoteliales (EC) 1. Para el estudio de estas interacciones, se están empleando diferentes sistemas de cámara de flujo para simular el sistema vascular arterial. Entre conjuntos de cámara de flujo, la cámara de flujo de placas paralelas (PPFC) en conjunción con las EC se utiliza rutinariamente como un modelo in vitro simulando in vivo en condiciones de estrés de cizallamiento. En estemétodo, ECs se cultivan en un plato de 35 mm y después de la consecución de una monocapa, las EC se adjuntan a la PPFC y experimentos basados ​​en la tensión de cizallamiento se realizan. Sin embargo, PPFC y otros sistemas actuales presentan muchas limitaciones para el estudio de las interacciones adhesivas entre las células tumorales (CTC) derivadas de pacientes y EC, que circula principalmente, porque CTC son una población rara de células, arrojar desde el tumor primario, que circula entre los millones de células de la sangre (1 CTC por 10 9 células de la sangre) 4. Por lo tanto, a diferencia de suministro ilimitado de líneas de células cultivadas, bajo recuento de CTC conducen a muy pocas y raras interacciones CTC / CE, requiriendo adecuado ancho de canal de flujo para registrar las interacciones de análisis de la reproducción. Además, dado que los pacientes CTC derivados son una población impura, por lo tanto, se requiere un marcador de identificación para rastrear las CTC en específico. Para solucionar este problema, hemos desarrollado un nuevo método para identificar el cáncer de próstata (CaP) CTCs tomando ventaja del hecho de que prácticamente todos estos CTC expresan antígeno prostático específico de membrana (PSMA) en su superficie celular 5,6. En este informe, se utilizó la línea celular de cáncer de próstata, MDA PCa2b (MDA), para demostrar la utilidad potencial de nuestro nuevo sistema para el estudio de la próstata interacciones CTC con EC, con el tiempo para comprender el mecanismo de la metástasis. Nuestra metodología se puede aplicar para varios experimentos de cizalla basada en la simulación de sistema vascular in vivo en 7-9. Además de examinar las interacciones CaP CTC / CE, el sistema de cámara de flujo de corriente podría adaptarse fácilmente para el análisis de células mononucleares de sangre periférica o de las interacciones de células tumorales con EC. La facilidad de montaje y desmontaje de la cámara de flujo, un microportaobjetos III (0,1) (en lo sucesivo denominada como microportaobjetos), permite el cultivo de EC bajo perfusión y la estimulación de los EC con diferentes citocinas para inducir prOtein expresión. Además, cultivos de ECS, proteínas recombinantes, tales como E-y P-selectina se pueden cubrir en el microportaobjetos y las interacciones con las células tumorales pueden ser observados bajo condiciones de flujo laminar 10.

<table border="0" cellpadding="0" cellspacing="0" width="744">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Microslide</td>
			<td style="width:192px;">Ibidi</td>
			<td style="width:119px;">80331</td>
			<td style="width:189px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Fibronectin</td>
			<td style="width:192px;">Millipore</td>
			<td style="width:119px;">FC010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Plastic tubing</td>
			<td style="width:192px;">Tygon</td>
			<td style="width:119px;">AAQ04103</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Male luer adapter</td>
			<td style="width:192px;">GlycoTech</td>
			<td style="width:119px;">31-001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Female luer adapter</td>
			<td style="width:192px;">GlycoTech</td>
			<td style="width:119px;">31-001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Syringe pump</td>
			<td style="width:192px;">Chemyx Inc</td>
			<td style="width:119px;">Fusion 100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Luer-lock syringe</td>
			<td style="width:192px;">BD Biosciences</td>
			<td style="width:119px;">309628</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">M199 medium</td>
			<td style="width:192px;">Sigma</td>
			<td style="width:119px;">M7653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Endothelial Mitogen</td>
			<td style="width:192px;">Biomedical Technologies</td>
			<td style="width:119px;">BT-203</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">HBSS</td>
			<td style="width:192px;">Sigma</td>
			<td style="width:119px;">H9269</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:244px;">Anti-PSMA J591-488</td>
			<td style="width:192px;">Weill Cornell Medical College-Lab of Urologic Oncology</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Interleukin-1 beta</td>
			<td style="width:192px;">Peprotech</td>
			<td style="width:119px;">200-01B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Trypsin</td>
			<td style="width:192px;">Millipore</td>
			<td style="width:119px;">SM-2002-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Heparin</td>
			<td style="width:192px;">Sigma</td>
			<td style="width:119px;">H-3149</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:244px;">HUVECs</td>
			<td style="width:192px;">Weill Cornell Medical College-Department of Surgery</td>
			<td style="width:119px;"></td>
			<td>provided by Marco Seandel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">VE-Cadherin</td>
			<td style="width:192px;">Santa Cruz</td>
			<td style="width:119px;">sc-5648</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">10x objective</td>
			<td style="width:192px;">Zeiss Plan Neofluar</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:244px;">Enzyme free cell dissociation reagent</td>
			<td style="width:192px;">Millipore</td>
			<td style="width:119px;">S-004-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">RPMI-1640</td>
			<td style="width:192px;">Lonza</td>
			<td style="width:119px;">12-702-F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Ficoll-paque plus</td>
			<td style="width:192px;">GE healthcare</td>
			<td style="width:119px;">17-1440-02</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro method to observe e-selectin-mediated interactions between prostate circulating tumor cells derived from patients and human endothelial cells

La metástasis es un proceso en el que las células tumorales se desprenden de tumor intravasate vascular de la sangre primaria y el sistema linfático, de ese modo, obtener el acceso a la extravasación y formar un nicho secundaria. La extravasación de las células tumorales desde el sistema vascular de la sangre puede ser estudiada usando células endoteliales (ECs) y las células tumorales obtenidas a partir de diferentes líneas celulares. Se llevaron a cabo estudios iniciales usando condiciones estáticas, pero se ha documentado bien que los EC se comportan de manera diferente bajo condiciones de flujo fisiológico. Por lo tanto, los diferentes conjuntos de cámara de flujo actualmente se utilizan para el estudio de las interacciones celulares de cáncer con EC. Ensambles de cámara de flujo actuales ofrecen resultados reproducibles, ya sea utilizando diferentes líneas celulares o de líquido a diferentes condiciones de estrés de cizalla. Sin embargo, para observar y estudiar las interacciones con las células raras, como las células del tumor (CTCs) en circulación, se requieren ciertos cambios que deben introducirse en el conjunto de la cámara de flujo convencional. CTCsson una población de células raras entre millones de células sanguíneas. En consecuencia, es difícil obtener una población pura de CTC. La contaminación de las CTC con diferentes tipos de células que se encuentran normalmente en la circulación es inevitable el uso de presente de enriquecimiento o técnicas de agotamiento. En el presente informe se describe un método único para la etiqueta circular las células de cáncer de próstata con fluorescencia y estudiar sus interacciones con EC en un sistema de cámara de flujo auto-ensamblado. Esta técnica se puede aplicar además a observar las interacciones entre CTC próstata y cualquier proteína de interés.

La figura 1 muestra un cultivo O / N de una monocapa de EC en la MicroSlide. Los batiduras en la Figura 1A muestra que el 100% de la MicroSlide es visible usando objetivo 5X mientras que el 70% es visible utilizando un objetivo de 10 aumentos (figura 1B). Para E-selectina mediada por las interacciones, las células de rodadura en los bordes no se consideran que hace más de 70% de la MicroSlide disponible para la grabación en vídeo y análisis de la reproducción. En ...

Watch this Scientific Journal Video about In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells at JoVE

In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

Nuestro informe se describe un método único para visualizar y analizar las interacciones CTC / CE en el cáncer de próstata en condiciones de flujo fisiológico.

Método in vitro para observar interacciones mediadas por E-selectina Entre próstata células tumorales circulantes derivadas de pacientes y las células endoteliales humanas

Metastasis is a process in which tumor cells shed from the primary tumor intravasate blood vascular and lymphatic system, thereby, gaining access to ...

in-vitro-method-to-observe-e-selectin-mediated-interactions-between

Research

JoVE Journal

Medicine

In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

9.0K vistas.  Weill Cornell Medical College.  Nuestro informe se describe un método único para visualizar y analizar las interacciones CTC / CE en el cáncer de próstata en condiciones de flujo fisiológico. 

Video: In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Diferenciación de aislamiento, caracterización y comparativo de las células madre derivadas de la Pulpa Dental de los dientes permanentes mediante dos métodos diferentes

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Investigación

Medicina

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Paciente celulares derivadas Cultura y aislamiento de CD133<sup&gt; +</sup&gt; Putativos células madre del cáncer de melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

El aislamiento de las células madre del cáncer de muestras de cáncer de próstata humano

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with muscular dystrophy is an invaluable cellular source for in&#160;vitro disease model systems and can be used for drug screening studies. Patient-derived urine cells have been used in successful reprogramming into induced pluripotent stem cells in order to model dystrophic cardiomyopathy1. Addressing the safety concerns of integrating vector systems, we present a protocol using a non-integrating Sendai virus vector for transduction of Yamanaka factors into urine cells collected from patients with muscular dystrophy. This protocol generates fully reprogrammed clones within 2&#8211;3 weeks. The pluripotent cells are vector-free by passage-13. These dystrophic iPSCs can be differentiated into cardiomyocytes and used either to study disease mechanisms or for drug screening.

This protocol entails detailed procedures for isolation of urine derived cells from muscular dystrophy patients; their efficient and rapid reprogramming through Sendai virus transduction.

Generación de pluripotentes inducidas Células Madre de la distrofia muscular pacientes: Eficiente Reprogramación-Integración libre de orina células derivadas

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with ...

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent &#8216;floater&#8217; cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

En Enriquecimiento vitro de células iniciadoras del tumor del cáncer de ovario

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

El estudio de cáncer de páncreas Tallo Características de las células para el desarrollo de nuevas estrategias de tratamiento

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

El aislamiento y la propagación de células tumorales circulantes de un Cáncer de ratón

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

Aislamiento y cultivo de expansión de las células endoteliales-tumorales específicas

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

Generación del paciente con cáncer de próstata Derivado modelos de xenoinjertos de células tumorales circulantes

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

El agotamiento de las células de ratón a partir de xenoinjertos de tumores humanos mejora significativamente análisis de aguas abajo de las células diana

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...