Este estudio fue apoyado por becas SR1 CA 135561, R01 CA151494, R01 CA174432 y P3013330 de los Institutos Nacionales de Salud. Nos gustaría dar las gracias a Michele Houston, Elena Dhima y la doctora Anna Velcich por sus valiosos comentarios en el desarrollo del protocolo de aislamiento cripta. También agradecemos a la Formación de la Diabetes y el Centro de Investigación del Colegio Albert Einstein de Medicina apoyado por el NIH P60DK20541, y el Dr. Michael Brownlee y el Dr. Xue Liang-Du, que dirigen y operan las instalaciones de Seahorse, respectivamente.

Este estudio se realizó de acuerdo con las recomendaciones de la Guía para el Cuidado y Uso de Animales de Laboratorio de los Institutos Nacionales de Salud. El protocolo fue aprobado por el Comité de Ética de Experimentación Animal de la Einstein College de Medicina Albert. 1. Cripta Aislamiento y Cultura <ol><li> El aislamiento de las criptas del intestino delgado: <ol><li> Aislar criptas intestinales de cualquier modelo de ratones de interés. La eutanasia a los ratones con CO 2 seguido por dislocación cervical. </li><li> Abrir el abdomen longitudinalmente y llenar el intestino delgado (SI) con solución salina tamponada con fosfato enfriada en hielo, PBS (-Ca 2+; -Mg 2+) con 2x antibiótico-antimicótico (anti-anti). Rápidamente aislar intestino delgado. </li><li> Completamente diseccionar libre de grasa mesentérica utilizando un bisturí. Tenga cuidado de no perforar el tejido - en todo momento mantener el tejido húmedo con PBS enfriado con hielo. Abre inteStine longitudinalmente y lavar a fondo con helado de PBS. </li><li> Cortar el intestino delgado en dos secciones y aplanar con una punta de algodón húmedo, suavemente raspar las vellosidades utilizando una diapositiva pre-enfriado. Lave vigorosamente en PBS enfriado con hielo varias veces. </li><li> Incubar 3 min en 20 ml de 1x PBS por 3 mM de ácido etilendiaminotetraacético (EDTA) /0.5 mM ditiotreitol (DTT) para la disociación no enzimática del tejido. </li><li> Cortar el tejido en trozos pequeños (2-4 mm) utilizando una cuchilla de afeitar en un portaobjetos pre-enfriado. Transferir las piezas en un tubo de 50 ml que contiene 20 ml de PBS helado. </li><li> Pipeta hacia arriba y abajo suavemente 10 veces con una pipeta desechable estéril de 10 ml. Deje que los fragmentos de tejido sedimentan por gravedad. Eliminar el sobrenadante con una pipeta. Repita tres veces más; asegúrese de sobrenadante es claro. </li><li> Añadir 20 ml de PBS por EDTA 2 mM. Remolino e incubar a 4 ° C durante 30 minutos con agitación suave. </li><li> Deje que los sedimentos del tejido y desechar el sobrenadante. Añadir 15 ml de hielo frío PBS y pipetaarriba y abajo 5x con una pipeta 10 ml. Dejar que los fragmentos de tejido de sedimentos y recoger el sobrenadante como la fracción 1 (F1). Repita la recogida de F2-F5, cada fracción mantenido separado. </li><li> Añadir 15 ml de hielo PBS frío y este batido tiempo vigorosamente a mano durante 15 segundos. Recoger F6 y F7 para repetir, F8. Si las piezas de tejido comienzan a flotar, toque para ayudarles a establecerse. </li><li> Inspeccionar una parte alícuota de cada fracción bajo el microscopio. Piscina aquellas fracciones que contienen criptas. </li><li> Pasar las fracciones reunidas a través de un filtro de células de 70 micras de nylon, la recogida de las criptas en un tubo de 50 ml. </li><li> Centrifugar a 100 xg durante 5 min a 4 ° C, se descarta el sobrenadante y resuspender el pellet en 10 ml PBS enfriado con hielo con criptas anti-anti y transferencia 2X a un tubo de 15 ml. Repita el lavado una vez más. </li><li> Lavar las criptas una vez con 10 ml enfriado en hielo ADF, Advanced DMEM / F-12 (Dulbecco &quot;s Medio Eagle Modificado / jamón&quot; s F-12) - 2x anti-anti. </li><li> Lavar las criptas una vez con 10 ml ADF - 1x anti-anti. </li><li> Cuente las criptas utilizando un hemocitómetro. Ajustar el volumen de la solución cripta y transferir a un tubo de 1,5 ml para que la concentración final cuando cripta resuspendió será 100-500 criptas por 50 l de mezcla de proteína gelatinosa (Matrigel). Centrifugar a 100 xg durante 5 min a 4 ° C y volver a suspender las criptas en la mezcla de proteína gelatinosa. </li><li> Placa de 50 l de cripta - suspensión gelatinosa mezcla de proteína por pocillo en una placa de 24 pocillos (área de crecimiento: 2 cm 2), colocando cuidadosamente la caída de suspensión en el centro de cada pocillo. Mantener la placa en una CO 2, 37 ° C incubadora durante ~ 30 min hasta que la suspensión se solidifica. </li><li> Añadir 500 medios de comunicación helada l de cultivo completo (Advanced DMEM / F12 con 1x anti - anti, 10 mM ácido 4- (2-hidroxietil) -1-piperazinetanosulfónico (HEPES), 1x GlutaMAX, Suplemento B27 1x, 1x N2 Suplemento, 1 mM N-acetil-L-cisteína (NAC), 50 ng / ml de factor de crecimiento epidérmico (EGF), 100 ng / mlNoggin, 500 ng / ml R-Spondin). <ol><li> Reconstituir factores de crecimiento en 0,1% albúmina de suero bovino (BSA) en PBS. </li></ol></li></ol></li><li> Cripta organoide cultura y Pasaje: <ol><li> Passage organoides cripta 14-21 días después de la siembra (o cuando sea necesario) como sigue: <ol><li> Cambio de los medios de comunicación todos los viernes y lunes. Los miércoles, reemplace la mitad de los medios de comunicación con los medios de comunicación fresca. </li></ol></li><li> Retire el medio de cultivo. Lavar la muestra dos veces con 500 l de PBS a intervalos de 5 min. </li><li> Retire PBS y romper suavemente la mezcla de proteína gelatinosa usando una punta micro pipeta P1000 estéril. </li><li> Añadir 1 ml de medios de cultivo completa a un solo pozo y resuspender las organoides en 1 ml de medio. </li><li> Interrumpir suavemente los organoides utilizando un P1000 pipeteando arriba y abajo 20-30x (comprobar bajo el microscopio para la disociación organoid adecuada con un buen rendimiento cripta hasta que una técnica consistente se desarrolla). </li><li> Transfiera las criptas a un tubo de microcentrífuga de 1,5 ml;centrifugar a 100 xg a 4 ° C, 5 min. </li><li> Lavar el sedimento dos veces con 1 ml de medio de cultivo completo. </li><li> Split en una proporción de 1: 3 o 1: 6, según sea necesario, criptas resuspender en 50 l de Matrigel por pocillo. Proceda como se describe anteriormente (sección 1.1.16-18). </li></ol></li><li> Cripta de congelación organoide: <ol><li> Retire el medio de cultivo. Lavar la muestra en 500 l de PBS. </li><li> Dispersar la mezcla de proteína gelatinosa usando una punta de pipeta P1000. </li><li> Añadir 1 ml de medio a un solo pozo y resuspender las organoides en 1 ml de medio. </li><li> Romper suavemente hasta los organoides utilizando un P1000 pipeteando arriba y abajo 20-30x. </li><li> Transfiera los organoides a un tubo de 1,5 ml. Centrifugar a 100 xg a 4 ° C, 5 min. </li><li> Lavar el sedimento dos veces con 1 ml de medio de cultivo completo. </li><li> Criptas Resuspender en 500 l de congelación medios de comunicación. </li><li> Criptas transferencia a un criotubo, coloque el tubo en un recipiente de congelación y almacenar en un congelador a -80ºC overnilucha. </li><li> Transferencia de criptas a líquido del tanque N 2. <ol><li> Recuperar organoides cripta congelados descongelando rápidamente en un baño a 37 ° C. Lavar con 500 l medio completo de cultivo una vez, centrifugar a 100 xg, 5 min y resuspender en mezcla de proteína gelatinosa, y la cultura como se describe anteriormente. Para una mejor recuperación, añada inhibidor ROCA (Y-27632) a los medios de congelación. </li></ol></li></ol></li></ol> 2. Cripta organoide Metabolismo Ensayo <ol><li> 24 y Preparación de la placa: <ol><li> Aislar y contar criptas de acuerdo con el protocolo (ver sección 1.1 para el aislamiento de las criptas y la sección 1.2 para el paso cripta). </li><li> Criptas Resuspender en mezcla de proteína gelatinosa (100-200 criptas por 20 l). </li><li> Placa-cripta gelatinosa suspensión mezcla de proteínas en una placa de 24 pocillos de ensayo (asegúrese de que hay al menos por triplicado para cada muestra) y permita que la suspensión se solidifique a 37 ° C en un CO 2incubadora; a continuación, añadir 500 l de los medios de cultivo completo. </li><li> Criptas Cultura (como se describe en la sección 1.1) y observar al microscopio hasta criptas crecen en organoides plenamente desarrollados. </li></ol></li><li> Extracelular Ensayo de flujo: <ol><li> Cartucho Hidratación durante la noche en un 2 no-CO (0% de CO 2), 37 ° C incubadora. </li><li> Retire el medio de cultivo y se lava dos veces con 500 organoides l DMEM (sin: glucosa, L-glutamina, piruvato de sodio y bicarbonato de sodio; y con: rojo de fenol). Espere 5 min. </li><li> Añadir 675 l por pocillo de medio de ensayo (DMEM con 2 mM L-glutamina y 5 mM de D-glucosa) a cada pocillo. </li><li> Compruebe la morfología microscópica cripta organoides para asegurar que organoides y la mezcla de proteína gelatinosa están intactas después de los lavados. Incubar 1 hora en un no-CO2, 37 ° C incubadora. </li><li> Preparar 10 mM compuestos inyectables (oligomicina, cianuro de carbonilo-p-trifluoro-metoxi-fenil-hidrazona (FCCP), rotenone) en medio de ensayo. </li><li> Puesta en marcha del cartucho mediante la carga de 75 l de 10 mM compuestos inyectables en los puertos de la secuencialmente cartucho: Port A - oligomicina; Puerto B - FCCP; y Port C - rotenona (Las concentraciones finales durante el ensayo será de 1 M). </li><li> Incubar cartucho de 30 min - 1 hora a 37 ° C en una incubadora de 2-CO no. </li><li> Al mismo tiempo, active el XF Analyzer y crear la plantilla de protocolo de ensayo. </li><li> Coloque el cartucho y la placa de utilidad en el Analizador XF y ejecutar &quot;calibrar&quot; cartucho. </li><li> Compruebe organoides cripta microscópicamente para asegurarse de que están unidos a la mezcla de proteína gelatinosa. </li><li> Placa de cultivo de células de carga en protocolo de ensayo de instrumentos y de ejecución. </li></ol></li></ol>

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No hay revelaciones.

Hemos probado la tasa de consumo de oxígeno (OCR) y el índice de acidificación extracelular (ECAR) de criptas aisladas a partir de 8 meses de edad los ratones y se hicieron crecer en organoides ex vivo. Después de la medición de la tasa basal, el metabolismo cripta se evaluó mediante la adición de oligomicina, cianuro de carbonilo -p-trifluoromethoxyphenylhydrazone (FCCP) y rotenona, secuencialmente. Basal OCR y ECAR basal se registraron 0-29 min (Figura 2A y...

El cáncer colorrectal (CCR) es la tercera causa principal de muertes relacionadas con el cáncer en los Estados Unidos. Cáncer de colon esporádico - es decir, que surge más tarde en la vida (&gt; 50 años de edad) y sin factores genéticos predisponentes claras - las cuentas de ~ 80% de todos los casos, con una incidencia muy influida por los patrones de la dieta a largo plazo 1,2. Estos tumores exhiben un cambio metabólico hacia la dependencia de la glucólisis oxidativo, conocido como el efecto Warburg, lo que puede hacer, en parte, concentraciones más altas de bloques de construcción celular y la energía disponibles (a través de glutaminolysis) para permitir y tal vez conducir altas tasas de proliferación de células tumorales 3-5 . Estudios de cáncer de colon, así como otros cánceres gastrointestinales que incluyen cánceres pequeños intestino proporcionan información importante sobre la causa de la formación de tumores. La investigación de las diferencias metabólicas entre estados normales, pro-tumorigénicos y tumorigénicos de sistemas de órganos gastrointestinales pueden ayudar determination de riesgo relativo para el desarrollo del tumor así como la detección precoz de la neoplasia. Por otra parte, la comprensión de metabolismo bioenergético que involucra la respiración mitocondrial y la glucólisis proporcionará información fundamental en la forma en la fisiología celular, el envejecimiento y la enfermedad estado perturba la homeostasis intestinal. La utilización de la tecnología de ensayo de la bioenergética para el análisis de flujo extracelular puede evaluar las tasas de respiración mitocondrial y la glucólisis simultáneamente en las células que crecen en cultivo en tiempo real 6,7. Hasta hace poco, los estudios in vitro de intestino delgado se limitan a las líneas celulares derivadas de tumores benignos o malignos 8,9 y no representan la fisiología del epitelio intestinal normal y la influencia del microambiente en el que residen. En 2009, Sato et al. 10 introdujo un sistema ex vivo la cultura a crecer en tres dimensiones del ratón (3D) organoides epiteliales intestinales, o epithElial &quot;mini-tripas&quot;, apto para experimentales, diagnósticos y terapéuticos investigaciones 10,11. Por otra parte, criptas aisladas de ratones restricción calórica mantienen sus propiedades de crecimiento alterados como organoides en tales culturas 12. En comparación con las líneas celulares transformadas, culturas organoides de las criptas se pueden utilizar para generar datos fisiológicamente relevantes que presentan un mejor modelo para entender el estado in vivo. Hemos adaptado la tecnología de análisis bioenergético para analizar el metabolismo energético de las criptas intestinales organoides. Ratón organoides criptas intestinales fueron cultivadas ex vivo para desarrollar los estudios de metabolismo energético organoides cripta presentados. La tasa de consumo de oxígeno (OCR) y el índice de acidificación extracelular (ECAR) de organoides de las criptas se midieron en ausencia y presencia de dos diferentes inhibidores metabólicos (oligomicina, rotenona) y un portador de iones (cianuro de carbonilo-p-trifluoromethoxyphenylhydrazone). La orga criptarespuesta metabólica noid a estos compuestos químicos se refleja con éxito a través de cambiar ECAR y valores de OCR. Estudios bioenergéticos celulares tendrán dilucidar las interacciones recíprocas entre el estado metabólico y riesgo de enfermedad y fenotipo en el cáncer, la obesidad, la diabetes, los trastornos metabólicos y las enfermedades mitocondriales y ayudar a los métodos de detección de avance con implicaciones directas para la medicina traslacional. A continuación, describimos un protocolo detallado para aislar pequeñas criptas intestinales y de la cultura organoides cripta. Por otra parte, se introduce un nuevo método de usar las culturas organoides cripta para ensayos metabólicos.

<table border="0" cellpadding="0" cellspacing="0" width="1633">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">BD Matrigel Basement Membrane Matrix, GFR, Phenol Red-free, LDEV-free</td>
			<td style="width:229px;">BD Biosciences</td>
			<td style="width:135px;">356231</td>
			<td style="width:456px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">PBS (phosphate buffered saline), no magnesium, no calcium, pH 7.2</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">20012-027</td>
			<td style="width:456px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Advanced DMEM/F-12 (1X)</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">12634-028</td>
			<td style="width:456px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Dulbecco&#8242;s Modified Eagle&#8242;s Medium without glucose, L-glutamine, Phenol Red, sodium pyruvate and sodium bicarbonate</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">D5030</td>
			<td style="width:456px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Phenol Red sodium salt&#160;</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">P4758</td>
			<td style="width:456px;">Final Concentration 15 mg / L in DMEM (D5030) - step 2.2.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Antibiotic-Antimycotic, 100X, 100ml</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">15240-062&#160;</td>
			<td style="width:456px;">Final Concentration 1 X or 2 X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Penicilin-Streptomycin, liquid</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">15140-122</td>
			<td style="width:456px;">Final Concentration 1 X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Gibco&#174; GlutaMAX&#8482; supplement</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">35050061</td>
			<td style="width:456px;">Final Concentration 1 X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Gibco&#174; HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid), 1 M</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">15630-080</td>
			<td style="width:456px;">Final Concentration 10 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">N-Acetyl-L-Cysteine, 25g</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">A9165-25G</td>
			<td style="width:456px;">Final Concentration 1 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">100X N-2 supplement, liquid</td>
			<td style="width:229px;">Invitrogen</td>
			<td style="width:135px;">17502-048</td>
			<td style="width:456px;">Final Concentration 1 X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">50X B-27&#174; supplement minus Vitamin A, liquid</td>
			<td style="width:229px;">Invitrogen</td>
			<td style="width:135px;">12587-010</td>
			<td style="width:456px;">Final Concentration 1 X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Recombinant Mouse R-Spondin 1, CF, 50ug</td>
			<td style="width:229px;">R&#38;D Systems</td>
			<td style="width:135px;">3474-RS-050&#160;</td>
			<td style="width:456px;">Final Concentration 500 ng / mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Recombinant Murine EGF, 100ug&#160;</td>
			<td style="width:229px;">Peprotech</td>
			<td style="width:135px;">315-09&#160;</td>
			<td style="width:456px;">Final Concentration 50 ng / mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Recombinant Murine Noggin, 20ug&#160;</td>
			<td style="width:229px;">Peprotech</td>
			<td style="width:135px;">250-38</td>
			<td style="width:456px;">Final Concentration 100 ng / mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Gibco&#174; L-glutamine, 200 mM</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">25030-081</td>
			<td style="width:456px;">Final Concentration 2 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Gibco&#174; Glucose powder</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">15023-021</td>
			<td style="width:456px;">Final Concentration 5 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Ambion&#174; 0.5 M EDTA (Ethylenediaminetetraacetic acid), pH 8.0</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">AM9260G</td>
			<td style="width:456px;">Final Concentration 3 mM for step 1.1.5; 2 mM for step 1.1.8&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">DTT (Dithiothreitol), 1M</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">P2325</td>
			<td style="width:456px;">Final Concentration 3 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Albumin from bovine serum (BSA)</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">A2058</td>
			<td style="width:456px;">0.1 % in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">16000-044</td>
			<td style="width:456px;">1 % in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Recovery&#8482; Cell Culture Freezing Medium</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">12648-010</td>
			<td style="width:456px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">ROCK inhibitor (Y-27632)&#160;</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">Y0503</td>
			<td style="width:456px;">Final Concentration 10 &#181;M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Oligomycin</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">O4876</td>
			<td style="width:456px;">Final Concentration 1 &#181;M&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Carbonyl cyanide-p-trifluoro-methoxy-phenyl-hydrazone (FCCP)</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">C2920</td>
			<td style="width:456px;">Final Concentration 1 &#181;M&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Rotenone</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">R8875</td>
			<td style="width:456px;">Final Concentration 1 &#181;M&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Sodium hydroxide</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">221465</td>
			<td style="width:456px;">Final Concentration 0.1 N in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">XF24 Extracellular Flux Analyzer (XF Analyzer)</td>
			<td style="width:229px;">Seahorse Bioscience</td>
			<td style="width:135px;"></td>
			<td style="width:456px;"></td>
		</tr>
	</tbody>
</table>

real time analysis of metabolic profile in ex vivo mouse intestinal crypt organoid cultures

La mucosa del intestino delgado exhibe una arquitectura repetitivo organizada en dos estructuras fundamentales: vellosidades, que sobresalen en el lumen intestinal y compuestas de los enterocitos maduros, células caliciformes y células enteroendocrinas; y criptas, que residen proximal a la submucosa y la capa muscular, la acogida madre adultas y células progenitoras y células de Paneth madura, así como del estroma y las células inmunes del microambiente cripta. Hasta los últimos años, los estudios in vitro de intestino delgado se limitó a las líneas celulares derivadas de tumores benignos o malignos, y no representaban la fisiología del epitelio intestinal normal y la influencia del microambiente en el que residen. Aquí, se demuestra un método adaptado de Sato et al. (2009) para el cultivo de organoides de las criptas intestinales del ratón primarios derivados de ratones C57BL / 6. Además, se presenta el uso de cultivos de organoides cripta para analizar el perfil metabólico cripta en tiempo real por medidación del consumo basal de oxígeno, tasa glucolítica, la producción de ATP y la capacidad respiratoria. Organoides mantienen propiedades definidas por su origen y conservan aspectos de su adaptación metabólica se refleja en el consumo de oxígeno y las tasas de acidificación extracelular. Estudios metabólicos en tiempo real en esta cripta organoid sistema de cultivo son una poderosa herramienta para estudiar el metabolismo energético organoid cripta, y la forma en que pueden ser modulados por factores nutricionales y farmacológicos.

Organoides cripta se establecieron a partir de 8 meses de C57BL / 6 ratones alimentados con la dieta purificada roedor Instituto Americano de Nutrición 76A (AIN76A). Organoides criptas intestinales pueden ser cultivadas en cultivo durante períodos extendidos de un solo cripta (Figura 1A, sola flecha roja). Organoides surgen estructuras de cripta en 18-20 días en cultivo (Figura 1B, flechas rojas). Las criptas se pasaron cada 3 semanas y organoides recuperaron de manera eficiente desp...

Watch this Scientific Journal Video about Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures at JoVE.com

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Pequeñas organoides las criptas intestinales cultivadas ex vivo proporcionan un sistema de cultivo de tejidos que recapitula el crecimiento de las criptas dependientes de células madre y su nicho. Hemos establecido un método para analizar el perfil metabólico en tiempo real en organoides cripta ratón primaria. Encontramos organoides mantienen propiedades fisiológicas definidas por su origen.

Bienes Análisis Tiempo de perfil metabólico en<em&gt; Ex Vivo</em&gt; Culturas ratón intestinal Cripta organoides

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and ...

real-time-analysis-metabolic-profile-ex-vivo-mouse-intestinal-crypt

Research

JoVE Journal

Biology

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

16.1K vistas.  Albert Einstein College of Medicine.  Pequeñas organoides las criptas intestinales cultivadas ex vivo proporcionan un sistema de cultivo de tejidos que recapitula el crecimiento de las criptas dependientes de células madre y su nicho. Hemos establecido un método para analizar el perfil metabólico en tiempo real en organoides cripta ratón primaria. Encontramos organoides mantienen propiedades fisiológicas definidas por su origen. 

Video: Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Eukaryotic Polyribosome Profile Analysis

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase or the elongation phase by a variety of cellular stresses such as amino acid starvation or growth factor withdrawal. Alternatively, translation of individual mRNAs can be regulated by mRNA localization or the presence of cognate microRNAs. Studies of protein synthesis frequently utilize polyribosome analysis to shed light on the mechanisms of translation regulation or defects in protein synthesis. In this assay, mRNA/ribosome complexes are isolated from eukaryotic cells.  A sucrose density gradient separates mRNAs bound to multiple ribosomes known as polyribosomes from mRNAs bound to a single ribosome or monosome. Fractionation of the gradients allows isolation and quantification of the different ribosomal populations and their associated mRNAs or proteins. Differences in the ratio of polyribosomes to monosomes under defined conditions can be indicative of defects in either translation initiation or elongation/termination. Examination of the mRNAs present in the polyribosome fractions can reveal whether the cohort of individual mRNAs being translated changes with experimental conditions. In addition, ribosome assembly can be monitored by analysis of the small and large ribosomal subunit peaks which are also separated by the gradient. In this video, we present a method for the preparation of crude ribosomal extracts from yeast cells, separation of the extract by sucrose gradient and interpretation of the results.  This procedure is readily adaptable to mammalian cells.

This article describes a protocol for the extraction of translating ribosomes from eukaryotic cells. Once extracted, ribosomes are separated into monosomes and polyribosomes by sucrose gradient fractionation to allow different ribosomal populations to be analyzed. As such, this method is the gold standard for examining the regulation of translation.

Análisis eucariotas perfil Polyribosome

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase ...

Investigación

Biología

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

En vivo de imágenes y tratamientos terapéuticos en un modelo de ratón ortotópico de cáncer de ovario

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Metabolic Profile Analysis of Zebrafish Embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.

Análisis del perfil metabólico de embriones de pez cebra

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Metabólicas etiquetado y de membrana de fraccionamiento para Comparativo Proteómica Análisis de<em&gt; Arabidopsis thaliana</em&gt; Culturas suspensión celular

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding primary 3D epithelial structures in vitro. Both the architecture and physiological properties of these &#39;mini-guts&#39;, also called organoids, closely resemble their in vivo counterparts. This makes them an attractive model system for the small intestinal epithelium. Using retroviral transduction, functional genetics can now be performed by conditional gene overexpression or knockdown. This video demonstrates the procedure of organoid culture, the generation of retroviruses, and the retroviral transduction of organoids to assist phenotypic analysis of the small intestinal epithelium in vitro. This novel organotypic model system in combination with retroviral mediated gene expression provides a valuable tool for rapid analysis of gene function in vitro without the need of costly and time-consuming generation for transgenic animals.

This protocol explains primary Lgr5-positve organoid culture and the subsequent performance of retroviral transduction. This enables Cre-inducible overexpression or knockdown of the delivered transgene and allows functional studies to be carried out in the novel in vitro organotypic model system.

Un Protocolo de vídeo de la infección retroviral en Primaria Intestinal organoide Cultura

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

La medición de la rigidez de<em&gt; Ex Vivo</em&gt; Ratón aortas Usando microscopía de fuerza atómica

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells. These primary cultures have been used to identify the signaling pathways underlying the stimulation and inhibition of gut peptide secretion in response to a number of nutrients and neuropeptides as well as pharmacological agents. Furthermore, in combination with the use of transgenic fluorescent reporter mice, we have demonstrated that these primary cultures become a powerful tool for the examination of fluorescently-tagged enteroendocrine cells at the intracellular level, using methods such as patch clamping and single-cell calcium and cAMP-FRET imaging.

This protocol describes the isolation and culture of mixed murine small intestinal cells. These primary intestinal cultures enable the investigation of signaling pathways underlying gut peptide secretion using a number of techniques.

Mezcla de cultivos primarios de murino intestino delgado previstos para el Estudio de Gut Hormone secreción y Imágenes de células vivas de células enteroendocrinas

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal ...

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant defense response to microbial elicitors and to wounding caused by chewing insects, eliciting systemic calcium signals in plants. However, the role of calcium in vivo during biotic stress is still unclear. This protocol describes the use of a genetically-encoded calcium sensor to detect calcium signals in plants during feeding by a hemipteran pest. Hemipterans such as aphids pierce a small number of cells with specialized, elongated sucking mouthparts, making them the ideal tool to study calcium dynamics when a plant is faced with a biotic stress, which is distinct from a wounding response. In addition, fluorescent biosensors are revolutionizing the measurement of signaling molecules in vivo in both animals and plants. Expressing a GFP-based calcium biosensor, GCaMP3, in the model plant Arabidopsis thaliana allows for the real-time imaging of plant calcium dynamics during insect feeding, with a high spatial and temporal resolution. A repeatable and robust assay has been developed using the fluorescence microscopy of detached GCaMP3 leaves, allowing for the continuous measurement of cytosolic calcium dynamics before, during, and after insect feeding. This reveals a highly-localized rapid calcium elevation around the aphid feeding site that occurs within a few minutes. The protocol can be adapted to other biotic stresses, such as additional insect species, while the use of Arabidopsis thaliana allows for the rapid generation of mutants to facilitate the molecular analysis of the phenomenon.

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

This protocol outlines a simple method for analyzing calcium signals in plants generated by feeding hemipteran insects, such as aphids. Arabidopsis thaliana transformed with the GFP calcium biosensor GCaMP3 allow for the real-time in vivo imaging of calcium dynamics with a high temporal and spatial resolution.

En tiempo real en Vivo la grabación de señales de calcio Arabidopsis durante la alimentación de insectos utilizando un Biosensor fluorescente

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant ...

An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell (EC) proliferation assays using human retinal microvascular endothelial cells (HRMECs) or isolated primary retinal ECs are widely used in vitro models to study retinal angiogenesis. However, isolating pure murine retinal endothelial cells is technically challenging and retinal ECs may have different proliferation responses than choroidal endothelial cells and different cell/cell interactions. A highly reproducible ex vivo choroidal sprouting assay as a model of choroidal microvascular proliferation was developed. This model includes the interaction between choroid vasculature (EC, macrophages, pericytes) and retinal pigment epithelium (RPE). Mouse RPE/choroid/scleral explants are isolated and incubated in growth-factor-reduced basal membrane extract (BME) (day 0). Medium is changed every other day and choroid sprouting is quantified at day 6. The images of individual choroid explant are taken with an inverted phase microscope and the sprouting area is quantified using a semi-automated macro plug-in to the ImageJ software developed in this lab. This reproducible ex vivo choroidal sprouting assay can be used to assess compounds for potential treatment and for microvascular disease research to assess pathways involved in choroidal micro vessel proliferation using wild type and genetically modified mouse tissue.

This protocol presents choroid sprouting assay, an ex vivo model of microvascular proliferation. This assay can be used to assess pathways involved in proliferating choroidal micro vessels and assess drug treatments using wild type and genetically modified mouse tissue.

Un ensayo ex vivo de brotes de coroides de angiogénesis microvascular ocular

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell ...

Organoid-Derived Epithelial Monolayer: A Clinically Relevant In Vitro Model for Intestinal Barrier Function

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations as the former do not faithfully represent original tissue physiology, and the availability of the latter is limited. Hence, their application hampers fundamental and drug development research. Adult stem-cell-based organoids (henceforth referred to as organoids) are miniatures of normal or diseased epithelial tissue from which they are derived. They can be established very efficiently from different gastrointestinal (GI) tract regions, have long-term expandability, and simulate tissue- and patient-specific responses to treatments in vitro. Here, the establishment of intestinal organoid-derived epithelial monolayers has been demonstrated along with methods to measure epithelial barrier integrity, permeability and transport,&#160;antimicrobial protein secretion, as well as histology. Moreover, intestinal organoid-derived monolayers can be enriched with proliferating stem and transit-amplifying cells as well as with key differentiated epithelial cells. Therefore, they represent a model system that can be tailored to study the effects of compounds on target cells and their mode of action. Although organoid cultures are technically more demanding than cell lines, once established, they can reduce failures in the later stages of drug development as they truly represent in vivo epithelium complexity and interpatient heterogeneity.

Here, we describe the preparation of human organoid-derived intestinal epithelial monolayers for studying intestinal barrier function, permeability, and transport. As organoids represent original epithelial tissue response to external stimuli, these models combine the advantages of expandability of cell lines and the relevance and complexity of primary tissue.

Monocapa epitelial derivada de organoides: un modelo in vitro clínicamente relevante para la función de barrera intestinal

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations ...