Damos las gracias a los esfuerzos de colaboración de Dr. Thao Dang y los miembros de su laboratorio para el uso de su equipo de imagen y el microscopio. También agradecemos al biorepositorio y Tejidos del centro de investigación (BTRF) núcleo, Jeff Harper, y los residentes de la Universidad de Virginia para que nos proporciona el tejido uterino. Damos las gracias a Karol Szlachta por la ayuda con Esquema general.

Especímenes de histerectomía utilizados en este manuscrito se recogieron en concordancia con un protocolo de ética aprobado por la IRB Universidad numerada IRB-HSR # 14424. 1. Adquisición de la muestra de la Fuente Clínica <ol><li> Obtener el gobierno y las normas éticas basadas en instituciones y la documentación de aprobación antes de comenzar. </li><li> Llevar a cabo todos los pasos en condiciones estériles. </li><li> Preservar el tejido derivado del paciente en los medios de comunicación (RPMI o DMEM / de alta glucosa) en un tubo de 50 ml a 4 ° C si la muestra no se puede procesar en la cultura de inmediato. Las muestras pueden conservarse en este estado durante un máximo de 24 horas. </li><li> Lavar muestra de tejido tres veces con 1X solución salina tamponada con fosfato estéril (PBS) y desechar la solución entre los lavados. </li></ol> 2. Preparación de líneas celulares primaria mediante el método de raspado <ol><li> Añadir 4-10 ml de medio de crecimiento (medio RPMI o DMEM / de alta glucosa suplementado con 10% de suero fetal bovino y 1% de penicilina-streptomyCIN) al tejido y añadir Fungizone (/ ml de concentración final 0,25 g) durante 30 min. </li><li> Descartar el medio de crecimiento. </li><li> Lavar el tejido dos veces con 1X PBS. </li><li> Coloque el tejido en una placa de cultivo de 6 cm de células para distinguir entre miometrio y endometrio capas (refiérase a las Figuras 2A-2C para una descripción adicional). Separe el endometrio. </li><li> Uso de una hoja de bisturí o la maquinilla de afeitar, seccionar el tejido en trozos pequeños, mientras que rascarse en la placa de cultivo de 6 cm de células. Estos arañazos facilitar la fijación de las células endometriales primarios emergentes. En comparación con el método de tripsina, se necesita más tejido (véase la Figura 2D para una aproximación). </li><li> Suavemente, añadir 2 ml de medio de cultivo para el plato rayado (fragmentos de tejido serán visibles e idealmente inmovilizado a partir del movimiento de rasguño). </li><li> Transferencia de la placa (s) a una incubadora de cultivo celular designada (37 ° C y 5% de CO 2). Designe un lugar para que las líneas de células primarias, lejos de other placas de cultivo celular. Los cultivos primarios son más sensibles a la infección y la contaminación. </li><li> Examine las células bajo un microscopio de luz diaria. Pequeñas poblaciones de células deberían surgir por dos o tres días a partir de alrededor de los tejidos en rodajas (ver Figura 3B). </li><li> Para mantener los cultivos, lavar suavemente con 1X PBS y añadir medio de cultivo fresco (2 ml) cada tres días. Cuando aumenta la tasa de proliferación, el medio de crecimiento adicional (3-4 ml) se pueden añadir a la placa de cultivo de 6 cm (s). Nota: Durante el lavado y los medios de comunicación los cambios, es probable que se aspiraron piezas de tejido. Esto no va a afectar el crecimiento de células adherentes. Las muestras de tejido deben ser aspirados por el paso subsiguiente (2,10), como es probable que surjan después de una semana de nuevas colonias. </li><li> Cuando la placa de 6 cm es aproximadamente el 75 - 80% confluentes (véase la Figura 3B), el paso usando 0,05% de tripsina. Las células primarias no pueden ser pasados ​​por tiempo indefinido - congelar por una placa de células tan pronto como dos o tres placas son maintained. Si se requieren más pasos, siga un protocolo de inmortalización (revisado en Ref. 3). </li></ol> 3. Preparación de líneas celulares primaria mediante el método de tripsina <ol><li> Coloque un pedazo pequeño de tejido endometrial (ver Figura 2D para una aproximación) en 2 ml de tripsina al 0,25% suplementado con kanamicina (0,03 mg / ml de concentración final). </li><li> Incubar el tejido a 37 ° C en la plataforma giratoria durante 30 min. </li><li> Brevemente agite el tejido. </li><li> Centrifugar la muestra a 200-400 xg durante 2 min y descartar el sobrenadante. </li><li> Añadir tripsina fresca y kanamicina. </li><li> Incubar el tejido a 37 ° C mientras se hace girar por una hora. </li><li> Agite el tejido durante 5-10 segundos. </li><li> Añadir 2 ml de medio de crecimiento (suplementado con 10% de suero fetal bovino y 1% de penicilina-estreptomicina) para desactivar la tripsina. </li><li> Centrifugar las células a 200-400 xg durante 2-3 min. </li><li> Desechar el sobrenadante y añadir 2 ml de cultivoª medios a las células sedimentadas. </li><li> Placa sobre una placa de cultivo de 6 cm de células. Raspar la placa como en el Paso 2.5 Preparación de líneas celulares primaria mediante el método de raspado no es necesario, pero facilita la unión y el crecimiento de los cultivos primarios. </li><li> Monitorear las células bajo un microscopio de luz diaria. Las células son generalmente visibles con un microscopio óptico después de 24 a 48 horas (ver Figura 3 C). </li><li> Para mantener los cultivos, monitorear las células y cambiar el medio cada 2-3 días como en el paso 2.9. </li><li> Para el paso de las células, utilizan 0,05% o 0,25% de tripsina cuando las células alcanzan 75-80% de confluencia (véase la Figura 3C). </li></ol> 4. Ahorro de tejido extra for Analysis (Snap Congelación y Fijación de formalina) <ol><li> Lavar la muestra dos veces con 1X PBS. </li><li> Aspirar la mayor cantidad de líquido posible. </li><li> Para Snap congelación, coloque la muestra de tejido endometrial en un tubo de centrífuga de 1,7 (ver Figuras 2F y 2G). Coloque el 1,7tubo de centrífuga en nitrógeno líquido durante 10 segundos o hasta que se puede ver que el tejido ha congelado hacia abajo. Nota: Tenga cuidado de no tocar directamente el nitrógeno líquido en la preparación de muestras. Las muestras se pueden almacenar a -80 ° C hasta su posterior procesamiento o análisis. </li><li> Para la fijación en formol, cortar un pequeño trozo de tejido endometrial (ver Figura 2 H) y sumergirlo en el 10% formalina tamponada zinc. Después de 24 horas, deseche formalina y añadir etanol al 70% para muestras de tejido hasta su posterior procesamiento. </li></ol> 5. Inmunofluorescencia (IF) <ol><li> La fijación de la célula <ol><li> Preparar las células en un portaobjetos de la cultura o de la placa. </li><li> Suavemente, lavar las células con PBS 1X. </li><li> Añadir pre-enfriada (4 º C) metanol y acetona (mezclado en una proporción de 1:1) durante 5 min. </li><li> Seque el portaobjetos antes de proceder. Slide se puede almacenar a 4 ° C durante 1-2 semanas si es necesario. </li></ol></li><li> Procesamiento SI <ol><li> Rehidrate células con 1X PBS durante 10 min. Para la following los pasos 1-6, llevar a cabo todos los lavados e incubaciones en una plataforma giratoria. </li><li> Bloquear usando 1X PBS suplementado con 1% albúmina de suero bovino (BSA) y 1% de especies de suero específico (fuente de anticuerpo 2 º) durante 30 min a temperatura ambiente (RT). </li><li> Incubar en anticuerpo primario (ver las recomendaciones del fabricante para las diluciones de anticuerpos). Ver Sustancias de anticuerpos específicos. Diluir el anticuerpo primario en tampón de bloqueo fresco como en el Paso 5.2.2. Esta incubación puede llevarse a cabo durante al menos 2 horas a RT o durante la noche a 4 ° C. </li><li> Lavar 3 veces con 1X PBS durante 5 min cada uno. </li><li> Incubar en anticuerpo secundario (ver las recomendaciones del fabricante para las diluciones de anticuerpos). Diluir el anticuerpo secundario en tampón de bloqueo fresco como en el Paso 5.2.2. Para evitar la foto-blanqueo, es importante llevar a cabo esto y los pasos subsiguientes en la ausencia de luz. </li><li> Lavar 3 veces con 1X PBS durante 5 min cada uno. </li><li> Thoroughly secar las diapositivas. </li><li> Añadir un medio de montaje y solución de contratinción DAPI. </li><li> Aplicar cubreobjetos y sellar los bordes usando sellante (s). Los portaobjetos se pueden almacenar a 4 ° C en la oscuridad durante un máximo de 2 semanas. </li></ol></li></ol> 6. Extracción de ARN <ol><li> Pellet 1 x 10 7 células por centrifugación. Todos los materiales y reactivos de este protocolo deben RNasa libre. </li><li> Lisar células en 1 ml de reactivo de TRIZOL, e incubar las muestras se homogeneizaron durante 10 min a temperatura ambiente para completar la disociación de los complejos de nucleoproteínas. </li><li> Añadir 200 l de cloroformo por 1 ml de TRIZOL. Agitar enérgicamente a mano durante 15 segundos y se incuba durante 2-3 minutos a temperatura ambiente. </li><li> Se centrifuga a la velocidad máxima (15.000 xg) durante 15 min a 4 ° C. Darán como resultado tres capas. </li><li> Pasar la fase acuosa superior a un tubo de microcentrífuga nuevo. Añadir 1 l de glucógeno para aumentar el rendimiento de ARN; Sin embargo, este paso sólo es necesario cuando un pequeñoSe prevé cantidad de ARN. </li><li> Añadir 0,5 ml de alcohol isopropílico a la capa acuosa, y invertir las muestras 3-5 veces. Incubar las muestras a temperatura ambiente durante 10 min. Para aumentar el rendimiento, las muestras se pueden colocar en -20 ° C o -80 ° C durante 30 min. </li><li> Centrifugar a máxima velocidad durante 10 min a 4 ° C. </li><li> En este punto, un pequeño sedimento de ARN debe ser visible. Eliminar el sobrenadante. </li><li> Lavar el ARN con 1 ml de etanol al 75%. </li><li> Centrifugar a máxima velocidad durante 5 min y descartar el sobrenadante. Las muestras se pueden lavar una vez más para eliminar contaminantes inorgánicos, pero este paso no es necesario. </li><li> En pocas palabras, se seca el precipitado de ARN hasta el ARN de pellets está seca (por lo general toma 7-10 min). Nota: Un paso adicional puede ser utilizado para disminuir la materia inorgánica y disminuir el tiempo de secado. Después de desechar el sobrenadante del lavado con etanol, las muestras de centrifugado a la velocidad máxima durante un minuto adicional y aspirar líquido residual. Tenga cuidado de no aspirar pellet. </li><li> Disolver ARN en agua libre de RNasa, dependiendo del tamaño del sedimento de ARN. Los volúmenes varían típicamente desde 10 hasta 50 l. </li><li> Incubar las muestras a 55 ° C durante 10 min, y medir la concentración de ARN. </li><li> Almacene las muestras a -20 ° C hasta su posterior procesamiento. </li></ol> 7. Transcripción inversa <ol><li> Llevar el ARN de la muestra (1-3 mg) hasta un volumen de 9 l con H 2 O. </li><li> ARN de calor a 70 ° C durante 10 min. </li><li> Para generar una mezcla maestra de AMV, combinar los siguientes reactivos: 5 l de dNTP (concentración de trabajo de 50 mM), 5 l de oligos de ADN N6 (concentración de trabajo de 80 mM), 5 l de tampón 5X de AMV, y 1 l de AMV . Esta solución de mezcla maestra está diseñado por una muestra. </li><li> Añadir 16 l de la mezcla maestra a la ARN climatizada. El volumen final de reacción será de 25 l de reacción. </li><li> Utilice las siguientes condiciones de PCR para la transcripción inversa: (Etapa 1) 42 ° C durante 90 min, (StaGE 2) 95 ° C durante 5 min, y (Etapa 3) 4 ° C hasta su posterior procesamiento. </li></ol> 8. Tiempo real PCR <ol><li> Llevar a cabo las reacciones de PCR usando los cebadores, temperaturas de recocido, los números de ciclo, y los reactivos se encuentran en la Tabla 1. Nota: Es ideal para seguir las instrucciones del fabricante al usar reactivos de PCR (consulte los números de la empresa y catálogo de Materiales). </li></ol> 9. In vitro Protocolo decidualization (Derivado de Ref. 4 y 5) <ol><li> Placa células endometriales. </li><li> Haga crecer hasta una confluencia del 75-85%. </li><li> Después las células se vuelven confluentes, lavar una vez con PBS 1X. </li><li> Rápidamente, agregar elementos multimedia sin hormonas (fenol libre de RPMI suplementado con 5% de carbón tira de FBS y 1% de penicilina-estreptomicina). </li><li> Después de 24 horas, agregue los medios libres de hormonas complementadas con acetato de medroxiprogesterona (MPA) a una concentración final de 1 mM y 8-bromoadenosine 3 &#39;, 5&#39;Monofosfato cíclico (AMPc) a una concentración final de 0,5 mM. </li><li> Después de al menos 48 horas, detener la reacción y salvar a la placa (s) para su posterior procesamiento. </li></ol>

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Otros grupos han descrito y adaptado metodología para la preparación de cultivos estromales endometriales, la mayoría de los cuales utilizan colagenasa 4,12,13,15-18. En este manuscrito, hemos proporcionado la metodología y la evidencia de dos métodos de cultivo estromal simplificados primarias endometrial, los cuales son utilizados por nuestro laboratorio por razones económicas y de la disponibilidad adecuada de tripsina y / o una hoja de afeitar. Al comparar nuestros dos m...

El corpus útero humano se compone de tres capas, la perimetrio (o serosa), el miometrio, y el endometrio. Distinguir cada una de estas capas es un paso importante para establecer líneas celulares del endometrio. El perimetrio es la capa más externa del útero y compone de células delgadas y serosas. El miometrio es la capa gruesa, medio del útero y compuesta de células del músculo liso. El endometrio se identifica como la capa interior del útero e incluye células epiteliales y estromales poblaciones. El endometrio se subdivide a su vez en la capa basal cuyo tallo población celular es la hipótesis de la repoblación de la capa functionalis aproximadamente cada 28 días 1. La capa functionalis del endometrio humano sufre cambios bioquímicos y morfológicos significativas en la respuesta a las hormonas circulantes. Estas hormonas se derivan de la glándula pituitaria y los ovarios. Lacoordinó la producción y liberación de hormonas resulta en un ciclo reproductivo. El ciclo reproductivo está diseñado para preparar el endometrio para eventos potenciales de implantación del embrión. En los humanos, el ciclo reproductivo se conoce como &quot;ciclo menstrual&quot; y se divide en tres fases - proliferativa, secretora y menstrual. La fase proliferativa implica la proliferación de la capa de endometrio functionalis mientras que la fase secretora se caracteriza por functionalis maduración. En concreto, las alteraciones extracelulares, secreciones y la diferenciación celular señalan un potencial de implantación. Si la implantación no se produce antes de que el final de la fase secretora, la capa de endometrio functionalis se desprende durante la fase menstrual. La importancia de la menstruación y los acontecimientos que desencadenan el desprendimiento de la capa functionalis todavía se está debatiendo. En los seres humanos, se ha planteado que la menstruación es el resultado de un evento específico mediados de secretora diferenciación fase conocidacomo &quot;decidualization espontánea&quot; 2. En este manuscrito, proporcionamos metodología detallada para ambos métodos de aislamiento de células estromales endometriales, y utilizamos una combinación de inmunofluorescencia e imágenes digitales para demostrar la eficacia de estos enfoques. Además, se aplica una de uso común en modelo in vitro de decidualización espontánea para confirmar el aislamiento de células del estroma endometrial.

<table border="0" cellpadding="0" cellspacing="0" width="713">
	<tbody>
		<tr height="24">
			<td dir="LTR" height="24" style="height:24px;width:235px;">0.25 Trypsin or 0.05% Trypsin&#160;</td>
			<td dir="LTR" style="width:201px;">Hyclone&#160;</td>
			<td dir="LTR" style="width:216px;">SH3023602 or SH30004202&#160;&#160;</td>
			<td style="width:63px;"></td>
		</tr>
		<tr height="27">
			<td dir="LTR" height="27" style="height:27px;width:235px;">1.7 micro Centrifuge Tube&#160;&#160;</td>
			<td dir="LTR" style="width:201px;">Genesee Scientific&#160;</td>
			<td dir="LTR" style="width:216px;">22-272A&#160;</td>
			<td></td>
		</tr>
		<tr height="25">
			<td dir="LTR" height="25" style="height:25px;width:235px;">1&#181;l,20&#181;l, 200ml and 1000&#181;l Pipette&#160;&#160;</td>
			<td dir="LTR" style="width:201px;">Genesee Scientific&#160;</td>
			<td dir="LTR" style="width:216px;">24-401,24-402, 24-412, 24-430&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td dir="LTR" height="22" style="height:22px;width:235px;">15ml Conical Tube&#160;</td>
			<td dir="LTR" style="width:201px;">Hyclone&#160;</td>
			<td dir="LTR" style="width:216px;">339650</td>
			<td></td>
		</tr>
		<tr height="23">
			<td dir="LTR" height="23" style="height:23px;width:235px;">50ml Conical Tube&#160;</td>
			<td dir="LTR" style="width:201px;">Hyclone&#160;</td>
			<td dir="LTR" style="width:216px;">339652</td>
			<td></td>
		</tr>
		<tr height="24">
			<td dir="LTR" height="24" style="height:24px;width:235px;">6cm Cell Culture Dish&#160;</td>
			<td dir="LTR" style="width:201px;">Thermo scientific&#160;&#160;</td>
			<td dir="LTR" style="width:216px;">12-556-002&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td dir="LTR" height="22" style="height:22px;width:235px;">8 well Chambers&#160;</td>
			<td dir="LTR" style="width:201px;">Thermo Scientific&#160;</td>
			<td dir="LTR" style="width:216px;">AB-4162&#160;</td>
			<td></td>
		</tr>
		<tr height="23">
			<td dir="LTR" height="23" style="height:23px;width:235px;">Acetate&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher scientific&#160;</td>
			<td dir="LTR" style="width:216px;">C4-100&#160;</td>
			<td></td>
		</tr>
		<tr height="24">
			<td dir="LTR" height="24" style="height:24px;width:235px;">AMV RT Enzyme/Buffer&#160;</td>
			<td dir="LTR" style="width:201px;">Bio Labs&#160;</td>
			<td dir="LTR" style="width:216px;">M077L&#160;</td>
			<td></td>
		</tr>
		<tr height="24">
			<td dir="LTR" height="24" style="height:24px;width:235px;">Bovine Serum Albumin (BSA)&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher Scientific&#160;</td>
			<td dir="LTR" style="width:216px;">BP-1605-100&#160;</td>
			<td></td>
		</tr>
		<tr height="23">
			<td dir="LTR" height="23" style="height:23px;width:235px;">Buffered Zinc Formalin&#160;</td>
			<td dir="LTR" style="width:201px;">Thermo&#160;</td>
			<td dir="LTR" style="width:216px;">59201ZF&#160;</td>
			<td></td>
		</tr>
		<tr height="24">
			<td dir="LTR" height="24" style="height:24px;width:235px;">Charcoal strip FBS&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher&#160;</td>
			<td dir="LTR" style="width:216px;">NC9019735&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="24">
			<td dir="LTR" height="24" style="height:24px;width:235px;">Chloroform&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher Scientific&#160;</td>
			<td dir="LTR" style="width:216px;">BP1145-1</td>
			<td></td>
		</tr>
		<tr height="24">
			<td dir="LTR" height="24" style="height:24px;width:235px;">Cover slip&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher Brand&#160;&#160;</td>
			<td dir="LTR" style="width:216px;">12-544D&#160;</td>
			<td></td>
		</tr>
		<tr height="24">
			<td dir="LTR" height="24" style="height:24px;width:235px;">Cyclic AMP (cAMP)&#160;</td>
			<td dir="LTR" style="width:201px;">Sigma&#160;</td>
			<td dir="LTR" style="width:216px;">B7880</td>
			<td></td>
		</tr>
		<tr height="24">
			<td dir="LTR" height="24" style="height:24px;width:235px;">DMEM/High Glucose&#160;</td>
			<td dir="LTR" style="width:201px;">Hyclone&#160;</td>
			<td dir="LTR" style="width:216px;">SH30243FS&#160;</td>
			<td></td>
		</tr>
		<tr height="25">
			<td dir="LTR" height="25" style="height:25px;width:235px;">dNTP&#160;</td>
			<td dir="LTR" style="width:201px;">Bioline&#160;</td>
			<td dir="LTR" style="width:216px;">BIO-39025&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Donkey Anti Goat -TRITC&#160;</td>
			<td dir="LTR" style="width:201px;">Santa Cruz&#160;</td>
			<td dir="LTR" style="width:216px;">SC-3855&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Donkey Serum&#160;</td>
			<td dir="LTR" style="width:201px;">Jackson&#8217;s lab&#160;</td>
			<td dir="LTR" style="width:216px;">&#160;017-000-002&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">E Cadherin Antibody&#160;&#160;</td>
			<td dir="LTR" style="width:201px;">Epitomics&#160;</td>
			<td dir="LTR" style="width:216px;">1702-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Ethanol&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher Scientific&#160;&#160;</td>
			<td dir="LTR" style="width:216px;">BP2818-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Fetal Bovine Serum (FBS)&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher Scientific</td>
			<td dir="LTR" style="width:216px;">03-600-511&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Fungizone Amphotericin B&#160;</td>
			<td dir="LTR" style="width:201px;">Gibco&#160;</td>
			<td dir="LTR" style="width:216px;">15290-018&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">GAPDH Probe&#160;</td>
			<td dir="LTR" style="width:201px;">Life Technologies&#160;</td>
			<td dir="LTR" style="width:216px;">HS99999905&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Glycogen&#160;</td>
			<td dir="LTR" style="width:201px;">5Prime&#160;</td>
			<td dir="LTR" style="width:216px;">2301440</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Goat Anti Mouse -FITC&#160;</td>
			<td dir="LTR" style="width:201px;">Jackson&#8217;s Lab&#160;</td>
			<td dir="LTR" style="width:216px;">115-096-003&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Isopropanol&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher Scientific&#160;</td>
			<td dir="LTR" style="width:216px;">BP2618-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Kanamycin&#160;&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher Scientific&#160;</td>
			<td dir="LTR" style="width:216px;">BP906-5&#160;</td>
			<td></td>
		</tr>
		<tr height="41">
			<td dir="LTR" height="41" style="height:41px;width:235px;">Medroxyprogesterone acetate (MPA)&#160;</td>
			<td dir="LTR" style="width:201px;">Sigma&#160;</td>
			<td dir="LTR" style="width:216px;">M1629</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">MeOH (Methanol)&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher Scientific&#160;</td>
			<td dir="LTR" style="width:216px;">A4-08-1&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Mounting Media (w/DAPI)&#160;</td>
			<td dir="LTR" style="width:201px;">Vector Labratories&#160;</td>
			<td dir="LTR" style="width:216px;">H-1500&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">N6 DNA Oligos&#160;</td>
			<td dir="LTR" style="width:201px;">Invitrogen&#160;</td>
			<td dir="LTR" style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Number 15 Scraper&#160;&#160;</td>
			<td dir="LTR" style="width:201px;">BD&#160;</td>
			<td dir="LTR" style="width:216px;">371615</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Pan Cytokeratin&#160; Mouse mAB&#160;</td>
			<td dir="LTR" style="width:201px;">Cell Signaling&#160;&#160;</td>
			<td dir="LTR" style="width:216px;">4545</td>
			<td></td>
		</tr>
		<tr height="29">
			<td dir="LTR" height="29" style="height:29px;width:235px;">PBS (phosphate buffered saline)&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher Scientific&#160;</td>
			<td dir="LTR" style="width:216px;">BP-399-4&#160;</td>
			<td></td>
		</tr>
		<tr height="41">
			<td dir="LTR" height="41" style="height:41px;width:235px;">Penicillin-Streptomycin Glutamine Solution 100X&#160;&#160;</td>
			<td dir="LTR" style="width:201px;">Hyclone&#160;&#160;</td>
			<td dir="LTR" style="width:216px;">SV30082.01&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">PML Anti Goat Anti body&#160;</td>
			<td dir="LTR" style="width:201px;">Santa Cruz&#160;</td>
			<td dir="LTR" style="width:216px;">SC-9862&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Primer(s)&#160;</td>
			<td dir="LTR" style="width:201px;">Eurofins&#160;</td>
			<td dir="LTR" style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">RPMI&#160;</td>
			<td dir="LTR" style="width:201px;">Hyclone&#160;&#160;</td>
			<td dir="LTR" style="width:216px;">SH30027FS&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">RPMI (Phenol free)&#160;</td>
			<td dir="LTR" style="width:201px;">Gibco&#160;</td>
			<td dir="LTR" style="width:216px;">11835</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Sybr Green&#160;&#160;</td>
			<td dir="LTR" style="width:201px;">Thermo Scientific&#160;</td>
			<td dir="LTR" style="width:216px;">AB-4162&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Taqman&#160;</td>
			<td dir="LTR" style="width:201px;">Thermo&#160;</td>
			<td dir="LTR" style="width:216px;">AB-4138</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Trizol&#160;</td>
			<td dir="LTR" style="width:201px;">Life Technologies&#160;</td>
			<td dir="LTR" style="width:216px;">15596018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Vimentin Antibody&#160;</td>
			<td dir="LTR" style="width:201px;">Epitomics&#160;</td>
			<td dir="LTR" style="width:216px;">4211-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

two methods for establishing primary human endometrial stromal cells from hysterectomy specimens

Muchos esfuerzos se han dedicado a establecer en sistemas de cultivo celular in vitro. Estos sistemas están diseñados para modelar un gran número de procesos in vivo. Sistemas de cultivos celulares derivados de muestras endometriales humanas no son una excepción. Las aplicaciones varían desde procesos fisiológicos normales cíclicos a patologías endometriales, tales como los cánceres ginecológicos, enfermedades infecciosas, y fallos del aparato reproductor. Aquí, le ofrecemos dos métodos para establecer células estromales endometriales primarios a partir de especímenes de histerectomía endometrio resecado quirúrgicamente. El primer método se conoce como &quot;el método de raspado&quot; e incorpora raspado mecánico utilizando cuchillas quirúrgicas o de afeitar mientras que el segundo método se denomina &quot;el método de tripsina.&quot; Este último método utiliza la actividad enzimática de tripsina para promover la separación de células y primaria excrecencia celular. Nos ilustran la metodología paso a paso a través de imágenes digitales y la microscopía. También provide ejemplos de validación de líneas celulares del estroma endometrial por medio de reacciones cuantitativas en tiempo real de la cadena de la polimerasa (qPCR) e inmunofluorescencia (IF).

Como se destaca en la sección Protocolo, asegúrese de llevar a cabo todos los métodos en el gobierno, institucionales y directrices éticas al manipular y preparar los tejidos humanos. Se incluyen en este manuscrito es una ilustración del flujo de trabajo general de &quot;el método de raspado&quot; (Figura 1A) y &quot;el método de tripsina&quot; (Figura 1B) se utiliza para establecer cultivos endometriales primarios. Estos métodos se ...

Watch this Scientific Journal Video about Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens at JoVE.com

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

El establecimiento de los sistemas primarios de células endometriales estromales de cultivo a partir de especímenes de histerectomía es una técnica biológica valiosa y un paso crucial antes de continuar con una amplia gama de objetivos de investigación. Aquí se describen dos métodos utilizados para establecer cultivos de estroma de los tejidos del endometrio resecado quirúrgicamente de pacientes humanos.

Dos métodos para fijar las células humanas primarias endometrial estromales de la histerectomía especímenes

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell ...

two-methods-for-establishing-primary-human-endometrial-stromal-cells

Research

JoVE Journal

Medicine

22.5K vistas.  University of Virginia.  El establecimiento de los sistemas primarios de células endometriales estromales de cultivo a partir de especímenes de histerectomía es una técnica biológica valiosa y un paso crucial antes de continuar con una amplia gama de objetivos de investigación. Aquí se describen dos métodos utilizados para establecer cultivos de estroma de los tejidos del endometrio resecado quirúrgicamente de pacientes humanos. 

Video: Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

Generación experimental de carcinoma asociada a los fibroblastos (CAF) a partir de fibroblastos humanos mamarios

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

Investigación

Medicina

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 ...

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

Protocolo mejorado para microdisección láser de islotes pancreáticos humanos a partir de muestras quirúrgicas

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl

Retinal detachment (RD) describes a separation of the neurosensory retina from the retinal pigmented epithelium (RPE). The RPE is essential for normal function of the light sensitive neurons, the photoreceptors. Detachment of the retina from the RPE creates a physical gap that is filled with extracellular fluid. RD initiates cellular and molecular adverse events that affect both the neurosensory retina and the RPE since the physiological exchange of ions and metabolites is severely perturbed. The consequence for vision is related to the duration of the detachment since a rapid reapposition of the two tissues results in the restoration of vision 1. The treatment of RD is exclusively surgical. Removal of vitreous gel (vitrectomy) is followed by the removal non essential part of the retina around the detached area to favor retinal detachment. The removed retinal specimens are res nullius (nothing) and consequently normally discarded. To recover RNA from these surgical specimens, we developed the procedure jouRNAl that allows RNA conservation during the transfer from the surgical block to the laboratory. We also standardized a protocol to purify RNA by cesium chloride ultracentrifugation to assure that the purified RNAs are suitable for global gene expression analysis. The quality of the RNA was validated both by RT-PCR and microarray analysis. Analysis of the data shows a simultaneous involvement of inflammation and photoreceptor degeneration during RD.

We used retinal samples from retinectomy for a transcriptomic analysis of retinal detachment. We developed a procedure that allows RNA conservation between the surgical blocks and the laboratory. We standardized a protocol to purify RNA by cesium chloride ultracentrifugation to assure that the purified RNAs are suitable for microarray analysis.

Análisis Transcriptomic de especímenes quirúrgicos retina humana usando Diario

Retinal detachment (RD) describes a separation of the neurosensory retina  from the retinal pigmented epithelium (RPE). The RPE is essential for normal  ...

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging.&#160;Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

Método para la obtención de las células del cáncer de ovario primarios a partir de muestras sólidas

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

El enriquecimiento de las células madre quimiorresistente cáncer ovárico de líneas celulares humanas

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

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 ...

A Novel Clinical Grade Isolation Method for Human Kidney Perivascular Stromal Cells

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and transplantation. Perivascular Stromal Cells (PSCs) share characteristics with bone marrow MSCs (bmMSCs). However, they also possess, most likely due to local imprinting, tissue-specific properties and play a role in local tissue homeostasis. This tissue specificity may result in tissue specific repair, also within the human kidney. We previously showed that human kidney PSCs (kPSCs) have enhanced kidney epithelial wound healing whereas bmMSCs did not have this potential. Moreover, kPSCs can ameliorate kidney injury in vivo. Therefore, kPSCs constitute an interesting source for cell therapy, particularly for kidney diseases and renal transplantation. Here we show the detailed isolation and culture method for kPSCs from transplant-grade human kidneys based on whole-organ perfusion of digestive enzymes via the renal artery and enrichment for the perivascular marker NG2. In this way, large cell quantities can be obtained that are suitable for cellular therapy.

Here we present a novel clinical grade isolation and culture method for kidney Perivascular Stromal Cells (kPSCs) based on whole organ perfusion with digestive enzymes and NG2-cell enrichment. With this method, it is possible to acquire sufficient cell numbers for cellular therapy.

Un método de aislamiento de grado clínico novedoso para Stromal células de riñón humano perivasculares

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and ...

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized stromal cells and immune cells, are responsible for the secretion of hormonal and inflammatory factors which are critical for successful blastocyst implantation, placental development and play a role in the initiation of labor at term and preterm. Many pregnancy complications can arise from perturbations of a fine balance of different cell populations comprising decidua. Alterations in the proportion of specific decidual cell types may disrupt these crucial processes and increase the risk of developing serious complications of pregnancy, such as embryo implantation failure, intrauterine growth restriction, preeclampsia and preterm labor. The protocol outlined here demonstrates a cost and time effective method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae. By combining enzymatic digestion and gentle mechanical disruption of the decidual tissue, a high yield of decidual cells was obtained with virtually no chorion contamination. Importantly, isolated decidual cells were characterized (stromal cells (55-60%), leukocytes (35%), epithelial (1%) or trophoblast (0.01%) cells) and maintained high viability (80%) which was confirmed by multicolor imaging flow cytometry assay. This protocol is specific to the decidua parietalis and can be adapted to first and second trimester placentae. Once isolated, decidual cells can be used for a multitude of experimental applications aiming to understand the role of different decidual cell sub-populations in pregnancy complications.

This protocol demonstrates a method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae which can be used for a variety of applications (i.e. immunocytochemistry, flow cytometry, etc.) aiming to study the role of different cell populations in pregnancy complications.

Aislamiento de células Decidual humanas primarias de las membranas fetales de la placenta de término

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...

Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release different bioactive molecules that control metabolic, hormonal, and immune processes; currently, it is unclear how these processes are regulated within the adipose tissue. Therefore, the development of methods evaluating the contribution of each cell population to the pathophysiology of adipose tissue is crucial. This protocol describes the isolation steps and provides the necessary troubleshooting guidelines for efficient isolation of viable mature adipocytes and SVF from human VAT biopsies in a single process, using a collagenase enzymatic digestion technique. Moreover, the protocol is also optimized to identify macrophage subsets and perform mature adipocyte RNA isolation for gene expression studies, which allows performing studies dissecting the interaction between these cell populations. Briefly, VAT biopsies are washed, minced mechanically, and digested to generate a single-cell suspension. After centrifugation, mature adipocytes are isolated by flotation from the SVF pellet. The RNA extraction protocol ensures a high yield of total RNA (including miRNAs) from adipocytes for downstream expression assays. Simultaneously, SVF cells are used to characterize macrophage subsets (pro- and anti-inflammatory phenotype) through flow cytometry analysis.

This protocol provides an efficient collagenase digestion method for isolation of viable adipocytes and stromal vascular fraction-SVF cells from human visceral fat in a single process, including methodology to obtain high-quality RNA from adipocytes and phenotypification of SVF-macrophages through staining of multiple membrane-bound markers for analysis by flow cytometry.

Aislamiento de adipocitos viables y fracción vascular estromal a partir de tejido adiposo visceral humano adecuado para análisis de ARN y fenotipado de macrófagos

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release ...