Agradecemos a los miembros de la división de Ciencias de genoma y laboratorio para Biología de sistemas y medicina, RCAST, la Universidad de Tokio. Agradecemos especialmente al Dr. H. Aburatani, Dr. T. Kodama, (LSBM, RCAST, la Universidad de Tokio), Dr. K. Tomizuka, Dr. T. Yoshida y Dr. A. Kunisato (Kyowa Hakko Kirin Co., Ltd.) para discusiones útiles y apoyo. Este trabajo fue financiado en parte por subvenciones para joven científico (A) (26710005, T.O.), subvenciones para la investigación científica en áreas innovadoras (26116711 y 16 H 01567, T.O.) y subvenciones para difíciles Investigación exploratoria (16K 14605, T.O.) desde el Ministerio de educación, cultura, deportes, ciencia y tecnología de Japón, la Fundación de ciencia de Takeda (T.O.), la Fundación de Kobayashi de investigación del cáncer (T.O.) y el proyecto de investigación del cáncer y Evolución terapéutica (P-crear) y la investigación práctica para el Control del cáncer innovadoras de Japón Agencia de investigación médica y desarrollo, AMED (T.O.).

1. preparación de medio de cultivo ácidos
<ol><li>
Preparación de control (pH 7.4) y de medio de cultivo de pH bajo (pH 6.8)
	<ol>
<li>Disolver 4,75 g de polvo DMEM sin L-glutamina en 500 mL de agua.</li>
		<li>Preparar 0,33 M NaHCO3 solución en una botella de presión-apretado. PRECAUCIÓN: Es importante utilizar una botella de presión apretado ya que NaHCO3 emite CO2 gas cuando se calienta y se descompone térmicamente.</li>
		<li>Autoclave el medio y la solución. Permitir estos búferes enfriar a 25 ° C.</li>
		<li>Añadir 5 mL de 200 mM L-glutamina, 5 mL de penicilina-estreptomicina y 50 mL de suero fetal bovino a 500 mL de DMEM sin bicarbonato.</li>
		<li>Por medio de control (pH 7.4), agregar 2,4 mL de solución de 0,33 M NaHCO3 por cada 100 mL de DMEM sin bicarbonato. Por medio de pH bajo (pH 6.8), Añadir 0.6 mL de solución de 0,33 M NaHCO3 por cada 100 mL de DMEM sin bicarbonato.</li>
		<li>Comprobar el pH del medio después de 24 h de incubación a 37 ° C debajo del 5% CO2. Nota: La cantidad de solución de NaHCO3 debe ajustarse según el pH del medio, que puede variar dependiendo del contenido de CO2 de la incubadora y la presión de aire. Idealmente, por primera vez, es mejor medir el pH del medio con diferentes concentraciones de bicarbonato para determinar la cantidad apropiada de solución de NaHCO3 añadir a DMEM.<ol>
<li>Recoge el medio de cultivo inmediatamente después de retirar la placa de cultivo de la incubadora de CO2 y medir el pH usando un pH-metro. Mantenga la temperatura a 37 ° C utilizando un baño de agua si es necesario. Medir el pH del medio tres veces independientemente. Nota: Solución de NaHCO3 debe almacenarse a 4 ° C y cada medio debe ser recién preparado. Productos disponibles comercialmente que pueden ser utilizados en este estudio se enumeran en la Tabla de materiales.</li>
		</ol>
</li>
	</ol>
</li>
	<li>
Preparación de medio de cultivo ácido inducidas por lactato o inducida por el ácido clorhídrico
	<ol>
<li>Añadir 74 μl de solución de lactato o 125 μl de 1 M de HCl a 100 mL de medio control (preparado en el paso 1.1.5).</li>
		<li>Comprobar el pH del medio después de 24 h de incubación a 37 ° C debajo del 5% CO2. Nota: La cantidad de lactato o solución de ácido clorhídrico también tiene que ser ajustado según el pH del medio, que puede variar dependiendo del contenido de CO2 en la incubadora y la presión de aire. Idealmente, es mejor medir el pH del medio con lactato serial o concentraciones de HCl para determinar la cantidad apropiada para ajustar al pH deseado del medio.</li>
	</ol>
</li>
</ol>2. recolección y tratamiento de las células
<ol><li>Empezar a cultivar las células a 37 ° C debajo del 5% de CO2 por lo menos 1 semana antes de realizar experimentos. Nota: Las líneas celulares utilizadas en este estudio se enumeran en la Tabla de materiales.</li>
	<li>Células de paso al llegar a 70-90% de confluencia. No permita que las células a convertirse en confluentes. Cambiar el medio cada 2-3 días durante el cultivo diario.</li>
	<li>Lavar las células añadiendo 5 mL de PBS. Aspire el PBS y añadir enzima desprendimiento como 1 mL de tripsina.</li>
	<li>Incubar a 37 ° C hasta que las células se redondean y empiezan a separar.</li>
	<li>Añadir 5 mL de medio de cultivo para las células separadas y desactivar la enzima. Transferir la suspensión de células a un tubo de 15 mL y centrifugar durante 3 min a 300 x g. aspirar el sobrenadante y resuspender las células con medio de cultivo.</li>
	<li>Contar las células viables usando un hemocitómetro y azul de tripano.</li>
	<li>Semilla 5.0 x 105 células/pocillo de un plato de 10 cm en 10 mL de medio. Incube las células durante 24 h a 37 ° C debajo del 5% CO2.</li>
	<li>Aspirar el medio de cultivo, lavar las células con PBS de 5 mL y añadir 10 mL del ácido de medio de cultivo preparado en la sección 1. Incube las células durante 24 h a 37 ° C debajo del 5% CO2.</li>
</ol>3. aislamiento de RNA
<ol><li>Medio de cultivo de aspirar y lavar las células con 5 mL de PBS. Romper las células mediante la adición de reactivo de extracción de RNA (véase Tablas de materiales).</li>
	<li>Raspar el plato brevemente, luego retire el reactivo de extracción de RNA con una pipeta y depositar la RNA extracción reactivo/célula lisada en un tubo de 1,5 mL. Dejar a temperatura ambiente durante 5 minutos.</li>
	<li>Añadir cloroformo μl 250 y agitar el tubo vigorosamente para unos 15 s. deje a temperatura ambiente durante 5 minutos y centrifugar a ≥8, 000 x g durante 5 minutos.</li>
	<li>Cuidadosamente Remueva la fase acuosa mediante una pipeta. Dejar detrás de algunas de la fase acuosa. Colocar en otro tubo de 1,5 mL.</li>
	<li>Añadir 550 de μl isopropanol para la fase acuosa y mezclar suavemente. Dejar a temperatura ambiente durante 5 minutos centrifugar a máxima velocidad (≥ 20, 000 x g) durante 20 min a 4 ° C.</li>
	<li>Quite el isopropanol y añadir 1 mL 75% de etanol en DEPC agua tratada. Mezclar suavemente. Centrifugue a 8.000 x g durante 5 minutos.</li>
	<li>Pipeta el etanol y dejar que secar al aire las pelotillas.</li>
	<li>Agregar que aproximadamente 15-25 μl (dependiendo de la producción) de DEPC agua tratada para el pellet de RNA. Mezclar bien.</li>
	<li>Medir la concentración de RNA aislado mediante la medición de OD en el 260 nm usando un espectrofotómetro.</li>
</ol>4. síntesis de cDNA
<ol><li>Combinar los siguientes componentes en un tubo de reacción de PCR: hasta 5 μg de ARN total, 1 μl de Oligo dT (50 μm) o mezcla al azar de cartilla (60 μm), 1 μl 10 mM dNTP y agua libre de nucleasas hasta un volumen total de 14 μl.</li>
	<li>Mezclar y centrifugar brevemente los componentes utilizando una centrífuga de sobremesa pequeño (~ 5 s). Desnaturalice la muestra cartilla de RNA durante 5 min a 65 ° C. Girar brevemente (~ 5 s) utilizando una mesa pequeña centrífuga y poner la muestra inmediatamente en hielo durante al menos 1 minuto.</li>
	<li>Añadir los siguientes componentes: 4 μL 5 x buffer de transcripción inversa, 1 μl de transcriptasa reversa (200 U/μL), 1 μl de inhibidor de la Rnasa (40 U/μL). Nota: Ejemplos de kits disponibles en el mercado se muestran en la Tabla de materiales
</li>
	<li>Cerrar el tubo, mezclar y luego centrifugar brevemente el contenido utilizando una centrífuga de sobremesa pequeño (~ 5 s). Incubar la mezcla de reacción combinado a 50-55 ° C durante 10 minutos.</li>
	<li>Inactivar la reacción por la incubación a 80 ° C durante 10 minutos.</li>
</ol>5. medición de ácido-responsivo genes mediante PCR en tiempo real
<ol><li>Combinar los siguientes componentes en una placa de 384 pocillos de reacción: 1 μl plantilla cDNA, 0,75 μl de 10 X N',N'-dimethyl-N-[4-[(E)-(3-methyl-1,3-benzothiazol-2-ylidene)methyl]-1-phenylquinolin-1-ium-2-yl]-N-propylpropane-1,3-diamine, 0,3 μl 50 μm hacia adelante cartilla, 0,3 μl 50 μm inversa primer, 1 μl Taq polimerasa, 2,5 μl 10 x buffer de Taq polimerasa, 2,5 μl 20 mM dNTP, 16.65 μl de agua. Nota: Ejemplo de kits disponibles en el mercado se muestran en la Tabla de materiales. En la tabla 1se muestra una lista de los primers. Una placa de 96 pocillos de reacción puede ser también utilizada en esta etapa. También pueden utilizarse otros sondeos (por ejemplo, sonda Taqman).</li>
	<li>Configurar el experimento y el siguiente programa PCR en un sistema de PCR en tiempo real: 50 ° C por 2 min, 1 ciclo; 95 ° C durante 10 min, 1 ciclo; 95 ° C por 15 s y 60 ° C por 1 min, 40 ciclos; 95 ° C por 15 s, 1 ciclo; 60 ° C durante 15 s, 1 ciclo; 95 ° C por 15 s, 1 ciclo.</li>
	<li>Colocar la placa en el instrumento de la qPCR. Inicie el programa. Nota: IDI1 , Upregulation de los genes sensibles pH MSMO1y INSIG1pueden medirse por los niveles de proteína.</li>
</ol>

<ol>
	<li>Cairns, R. A., Harris, I. S., Mak, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cancer+cell+metabolism.">Regulation of cancer cell metabolism.</a> Nature reviews. Cancer. 11 (2), 85-95 (2011).</li><li>Chang, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+Competition+in+the+Tumor+Microenvironment+Is+a+Driver+of+Cancer+Progression.">Metabolic Competition in the Tumor Microenvironment Is a Driver of Cancer Progression.</a> Cell. 162 (6), 1229-1241 (2015).</li><li>Hsu, P. P., Sabatini, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+cell+metabolism:+Warburg+and+beyond.">Cancer cell metabolism: Warburg and beyond.</a> Cell. 134 (5), 703-707 (2008).</li><li>Gerweck, L. E., Seetharaman, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+pH+gradient+in+tumor+versus+normal+tissue:+potential+exploitation+for+the+treatment+of+cancer.">Cellular pH gradient in tumor versus normal tissue: potential exploitation for the treatment of cancer.</a> Cancer research. 56 (6), 1194-1198 (1996).</li><li>Webb, B. A., Chimenti, M., Jacobson, M. P., Barber, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysregulated+pH:+a+perfect+storm+for+cancer+progression.">Dysregulated pH: a perfect storm for cancer progression.</a> Nature Rev. Cancer. 11 (9), 671-677 (2011).</li><li>Gatenby, R. A., Gillies, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+do+cancers+have+high+aerobic+glycolysis?.">Why do cancers have high aerobic glycolysis?.</a> Nature reviews. Cancer. 4 (11), 891-899 (2004).</li><li>McBrian, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+acetylation+regulates+intracellular+pH.">Histone acetylation regulates intracellular pH.</a> Mol Cell. 49 (2), 310-321 (2013).</li><li>Damaghi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+acidosis+in+the+tumour+microenvironment+selects+for+overexpression+of+LAMP2+in+the+plasma+membrane.">Chronic acidosis in the tumour microenvironment selects for overexpression of LAMP2 in the plasma membrane.</a> Nat Commun. 6, 8752 (2015).</li><li>Corbet, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acidosis+Drives+the+Reprogramming+of+Fatty+Acid+Metabolism+in+Cancer+Cells+through+Changes+in+Mitochondrial+and+Histone+Acetylation.">Acidosis Drives the Reprogramming of Fatty Acid Metabolism in Cancer Cells through Changes in Mitochondrial and Histone Acetylation.</a> Cell Metab. 24 (2), 311-323 (2016).</li><li>Chen, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genomic+analysis+of+lactic+acidosis+and+acidosis+response+in+human+cancers.">The genomic analysis of lactic acidosis and acidosis response in human cancers.</a> PLoS genetics. 4 (12), e1000293 (2008).</li><li>Kondo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+Acidic+pH+Activates+the+Sterol+Regulatory+Element-Binding+Protein+2+to+Promote+Tumor+Progression.">Extracellular Acidic pH Activates the Sterol Regulatory Element-Binding Protein 2 to Promote Tumor Progression.</a> Cell Rep. 18 (9), 2228-2242 (2017).</li></ol>

El autor Ayano Kondo es un empleado de Kyowa Hakko Kirin Co., Ltd.

Aquí, describimos un sistema de cultivo simple pH ácido y su proceso de evaluación. La combinación de tres métodos de acidificación del medio, es decir, concentración de bicarbonato menor, adición de lactato y adición de HCl, nos permitió investigar el mecanismo de respuesta de pH fondo y comparar la respuesta celular a otro tumor microambiental condiciones como hipoxia o alimento del hambre.
La clave de este método es determinar las concentraciones apropiadas de ácido clo...

El microambiente tumoral juega un papel crítico en la progresión del tumor y cáncer de la célula metabolismo1,2,3. Las células cancerosas a menudo se exponen a condiciones como hipoxia, la privación de nutrientes y pH extracelular ácido (pHe). Sin embargo, el papel de pHe en la progresión tumoral no ha aclarado tan extensivamente como en hambre hipoxia o nutrientes. El pHe en el tejido del tumor puede ser ácida, llegando a aproximadamente pH 6.84,5. El pH ácido se presenta de aeróbica y anaeróbica glicolítica excreción de protones (H+) y lactato por la proliferación del cáncer las células5,6.
Estudios recientes revelaron que histona ácida inducida por pHe desacetilación y oxidación del ácido graso exocitosis de lisosomas ácidas para la adaptación al ambiente ácido fuerte7,8,9,10. Sin embargo, los mecanismos a través de la cual acidificación extracelular afecta comportamiento de cáncer y la identidad de clave de los reguladores en el microambiente tumoral de pH ácido no han sido completamente determinados. Además, varios informes describen varios medios de pH ácido, usando claro concentraciones de bicarbonato, tampón Tris, tubos y HEPES o lactato y ácido clorhídrico, pero hay pocos informes para demostrar la estabilidad de la ajustada media ni integral comparación de varios medios de cultivo ácidos distintos7,8,9,10
Para aclarar los principales reguladores y cambios metabólicos en las células cancerosas en el contexto de acidificación extracelular, estableció un modelo de cultura simple en vitro para mantener un pHe ácido y examinó el papel de pHe en células de cáncer11. Usando este método, mantuvimos un ácido medio de cultivo con un pHe de 6,8 a 37 ° C debajo del 5% CO2, usando las concentraciones de bicarbonato menor en el medio de cultivo. pH 7.4 se utilizó como normal y medio de control. El pH del medio fue sostenido durante al menos 24 h y poco a poco disminuyó en 72 h en cultivo de células de cáncer de páncreas 1 PANC y AsPC-1. Porque glicólisis mejorada acelera la excreción de protones, pero también lactato7,8,9, también estableció un método de cultura mímico acidosis inducida de lactato por la adición de lactato en lugar de reducir la concentración de bicarbonato. Además, inducida por el ácido clorhídrico ácido pHe en el medio nos permite excluir la posibilidad de que las respuestas celulares a pH ácido medio de cultivo no son debido a una disminución de la concentración de bicarbonato. Por otra parte, utilizando distintos medios con un pH de 6.4 a 7.4 con concentración diferente de bicarbonato, podemos evaluar la extensión de efectos de pHe en respuestas celulares.

<table>
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium&#8220;Nissui&#8221; 2</td>
			<td>Nissui</td>
			<td>05919</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher&#160;</td>
			<td>10438</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Wako</td>
			<td>191-01305</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine solution</td>
			<td>Thermo Fisher&#160;</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Mixed Solution(Stabilized)</td>
			<td>Nacalai</td>
			<td>09367-34</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5g/l-Tripsin/0.53mmol/l-EDTA Solution, with Phenol Red</td>
			<td>Nacalai</td>
			<td>32778-05</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5%-Trypan Blue Stain Solution</td>
			<td>Nacalai</td>
			<td>29853-34</td>
			<td></td>
		</tr>
		<tr>
			<td>Lactic acid solution</td>
			<td>Sigma-Aldrich</td>
			<td>252476</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid solution</td>
			<td>Sigma-Aldrich</td>
			<td>H9892</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Plus Mini Kit</td>
			<td>QIAGEN</td>
			<td>74134</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript IV First-Strand Synthesis System</td>
			<td>Thermo Fisher&#160;</td>
			<td>18091</td>
			<td></td>
		</tr>
		<tr>
			<td>Power SYBR Green PCR Master Mix&#160;</td>
			<td>Applied Biosystems&#160;</td>
			<td>4368702&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Cell lines</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PANC-1</td>
			<td>ATCC</td>
			<td>CRL-1469</td>
			<td></td>
		</tr>
		<tr>
			<td>AsPC-1</td>
			<td>ATCC</td>
			<td>CRL-1682</td>
			<td></td>
		</tr>
		<tr>
			<td>KMS-6</td>
			<td>JCRB Cell Bank</td>
			<td>JCRB0432</td>
			<td></td>
		</tr>
		<tr>
			<td>TIG</td>
			<td>JCRB Cell Bank</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MRC9</td>
			<td>ATCC</td>
			<td>CCL-212</td>
			<td></td>
		</tr>
		<tr>
			<td>HUVEC</td>
			<td>ATCC</td>
			<td>CRL-1730</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop One Microvolume UV-Vis Spectrophotometer with Wi-Fi</td>
			<td>Thermo Fisher</td>
			<td>ND-ONE-W</td>
			<td></td>
		</tr>
		<tr>
			<td>QuantStudio 5 Real-Time PCR System</td>
			<td>Thermo Fisher</td>
			<td>CRL-1682</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishment of an extracellular acidic ph culture system

Condiciones del microambiente tumoral, tales como hipoxia o nutrientes hambre, juegan un papel crítico en la progresión del cáncer y la malignidad. Sin embargo, el papel del pH extracelular ácido en la agresividad del tumor y su mecanismo subyacente no ha sido extensivamente estudiado en comparación con las condiciones de hambre hipóxico o nutrientes. Además, un método de cultivo bien definidas para imitar el microambiente tumoral extracelular ácido no se ha completamente divulgado.
Aquí presentamos un método de cultivo simple en vitro para mantener el pH extracelular ácido utilizando reducción bicarbonato y lactato aumento o concentración de HCl en el medio de cultivo. El pH del medio fue sostenido durante al menos 24 h y poco a poco disminuyó en 72 h después de cultivo de células de cáncer de páncreas 1 PANC y AsPC-1. Tres condiciones de distintos medios ácidos en este estudio muy upregulated genes pH-sensibles tales como MSMO1, INSIG1y IDI1 compararon con hipoxia o hambre de nutrientes. La regulación al alza de estos genes puede utilizarse como marcador de pH ácido. Estas técnicas sencillas son beneficiosas para dilucidar los mecanismos subyacentes de la malignidad del tumor en el microambiente tumoral ácidos. Por lo tanto, nuestro sistema de cultura extracelular pH ácido permite descubrimiento de respuestas celulares de pH ácido en las células cancerosas, pero también en células primarias, como las células tubulares renales, en relación con los otros ácidos trastornos incluyendo, cetoacidosis diabética, acidosis láctica, la acidosis tubular renal y acidosis respiratoria.

Para determinar la concentración adecuada de bicarbonato, preparamos DMEM en el rango de 0 - 8 mM NaHCO3 (concentración final en el medio de cultivo) y tuvo éxito en la preparación de medios de cultivo con pH entre 6.4-7.4 (figura 1). Preparamos DMEM con 8 mM NaHCO3 (pH 7.4) como medio de control y 2 mM NaHCO3 (pH 6.8) como un medio con pH ácido, según un informe anterior indicando que el pH extracelular llegue a pH 6.8 ...

Watch this Scientific Journal Video about Establishment of an Extracellular Acidic pH Culture System at JoVE.com

Establishment of an Extracellular Acidic pH Culture System

El microambiente tumoral ácida juega un papel crítico en la progresión tumoral. Para evaluar los efectos del pH extracelular ácido en las células de cáncer in vitro, hemos establecido sistemas de cultivo ácidos simples.

Establecimiento de un pH extracelular ácido sistema de cultivo

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...

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14.5K vistas.  Kyowa Hakko Kirin Co., Ltd. El microambiente tumoral ácida juega un papel crítico en la progresión tumoral. Para evaluar los efectos del pH extracelular ácido en las células de cáncer in vitro, hemos establecido sistemas de cultivo ácidos simples.

Video: Establishment of an Extracellular Acidic pH Culture System

Measuring Plant Cell Wall Extension (Creep) Induced by Acidic pH and by Alpha-Expansin

Growing plant cell walls characteristically exhibit a property known as 'acid growth', by which we mean they are more extensible at low pH (&lt; 5) 1. The plant hormone auxin rapidly stimulates cell elongation in young stems and similar tissues at least in part by an acid-growth mechanism 2, 3. Auxin activates a H+ pump in the plasma membrane, causing acidification of the cell wall solution. Wall acidification activates expansins, which are endogenous cell wall-loosening proteins 4, causing the cell wall to yield to the wall tensions created by cell turgor pressure. As a result, the cell begins to enlarge rapidly. This 'acid growth' phenomenon is readily measured in isolated (nonliving) cell wall specimens. The ability of cell walls to undergo acid-induced extension is not simply the result of the structural arrangement of the cell wall polysaccharides (e.g. pectins), but depends on the activity of expansins 5. Expansins do not have any known enzymatic activity and the only way to assay for expansin activity is to measure their induction of cell wall extension. This video report details the sources and preparation techniques for obtaining suitable wall materials for expansin assays and goes on to show acid-induced extension and expansin-induced extension of wall samples prepared from growing cucumber hypocotyls.
 To obtain suitable cell wall samples, cucumber seedlings are grown in the dark, the hypocotyls are cut and frozen at -80 &deg;C. Frozen hypocotyls are abraded, flattened, and then clamped at constant tension in a special cuvette for extensometer measurements. To measure acid-induced extension, the walls are initially buffered at neutral pH, resulting in low activity of expansins that are components of the native cell walls. Upon buffer exchange to acidic pH, expansins are activated and the cell walls extend rapidly. We also demonstrate expansin activity in a reconstitution assay. For this part, we use a brief heat treatment to denature the native expansins in the cell wall samples. These inactivated cell walls do not extend even in acidic buffer, but addition of expansins to the cell walls rapidly restores their ability to extend.

Measuring Plant Cell Wall Extension Creep Induced by Acidic pH and by Alpha-Expansin

We demonstrate the use of a constant-force extensometer to measure long-term extension (creep) of plant cell wall specimens induced by acidic buffers and expansin protein.

Celda de medición Planta de Extensión de la pared (creep) inducida por el pH ácido y por Alfa-expansina

Growing plant cell walls characteristically exhibit a property known as 'acid growth', by which we mean they are more extensible at low pH (&lt; 5) 1. The ...

Investigación

Biología

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

Un Ex-ovo</emPollo> Embrión de cultivo apropiado para aplicaciones de imagen y sistema de Microcirugía

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development and growth of mammalian embryos are critically dependent on the function of the placenta, WEC technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages during embryonic day (E) 6.5-E12.5 in the mouse or E8.5-E14.5 in the rat 2, 3, 4. In WEC, we can directly target desired areas of embryos using fine glass capillaries because embryos can be manipulated under the microscope. Therefore, rodent WEC is very useful technique when we want to study dynamic developmental processes of postimplanted mammalian embryos. Up to date, several types of WEC systems have been developed 1. Among those, the rotator-type bottle culture system is most popular and suitable for long-term culture of embryos at midgestation, i.e., after E9.5 and E11.5 in the mouse and rat, respectively 1. In this video protocol, we demonstrate our standard procedures of rat WEC after E12.5 using a refined model of the original rotator system, which was designed by New and Cockroft 5, 6, and introduce various applications of WEC technique for studies in mammalian developmental biology.

Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.

El método de cultivo de embriones de roedores enteros utilizando el sistema de tipo de botella rotador Cultura

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development ...

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

Un<em&gt; Ex vivo</em&gt; Sistema de Cultura para estudiar el desarrollo de la tiroides

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

Desarrollo de un Sistema de inserción Co-cultivo de dos tipos celulares en ausencia de la célula-célula Contacto

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH (pHi) is influenced by many ion transporters and thus monitoring pHi is a useful tool for assessing transporter activity. Modern Genetically Encoded pH-Indicators (GEpHIs) provide optical quantification of pHi in intact cells on a cellular and subcellular scale. This protocol describes real-time quantification of cellular pHi regulation in Malpighian Tubules (MTs) of Drosophila melanogaster through ex vivo live-imaging of pHerry, a pseudo-ratiometric GEpHI with a pKa well-suited to track pH changes in the cytosol. Extracted adult fly MTs are composed of morphologically and functionally distinct sections of single-cell layer epithelia, and can serve as an accessible and genetically tractable model for investigation of epithelial transport. GEpHIs offer several advantages over conventional pH-sensitive fluorescent dyes and ion-selective electrodes. GEpHIs can label distinct cell populations provided appropriate promoter elements are available. This labeling is particularly useful in ex vivo, in vivo, and in situ preparations, which are inherently heterogeneous. GEpHIs also permit quantification of pHi in intact tissues over time without need for repeated dye treatment or tissue externalization. The primary drawback of current GEpHIs is the tendency to aggregate in cytosolic inclusions in response to tissue damage and construct over-expression. These shortcomings, their solutions, and the inherent advantages of GEpHIs are demonstrated in this protocol through assessment of basolateral proton (H+) transport in functionally distinct principal and stellate cells of extracted fly MTs. The techniques and analysis described are readily adaptable to a wide variety of vertebrate and invertebrate preparations, and the sophistication of the assay can be scaled from teaching labs to intricate determination of ion flux via specific transporters.

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Cellular ion transport can often be assessed by monitoring intracellular pH (pHi). Genetically Encoded pH-Indicators (GEpHIs)&#160;provide optical quantification of intracellular pH in intact cells. This protocol details the quantification of intracellular pH through cellular ex vivo live-imaging of Malpighian tubules of Drosophila melanogaster with pHerry, a pseudo-ratiometric genetically encoded pH-indicator.

Óptica pH cuantificación de intracelular en epitelios de túbulos de Malpighi de Drosophila melanogaster con fluorescentes genéticamente codificado pH indicador

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH ...

Establishment of a Clonal Culture of Unicellular Conjugating Algae

It is essential to establish clonal cultures of microalgae for use in studies of various topics, such as physiology, genetics, taxonomy, and microbiology. Thus, it is extremely important to develop techniques to establish clonal cultures. In this article, we demonstrate the establishment of clonal cultures of a conjugating alga. Water samples are collected from the field. Subsequently, cells are isolated using a glass capillary pipette, placed in media, and grown under conditions suitable for generating a clonal culture.

In this article, we demonstrate the establishment of clonal cultures of unicellular conjugating algal species collected from a natural field site.

Establecimiento de una cultura clónica de algas unicelulares de conjugación

It is essential to establish clonal cultures of microalgae for use in studies of various topics, such as physiology, genetics, taxonomy, and microbiology. ...

Measuring the Confluence of iPSCs Using an Automated Imaging System

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence in real time has been established without the need to count cells in single cell suspension to avoid any growth perturbation. A high-content image analysis system was used to assess iPCS confluence on 4 different ECMs over time in an automated manner. Different analysis settings were used to assess cell confluence of adherent iPSCs and only a slight difference (at 24 and 48 hours with laminin) has been observed whether a 60, 80 or 100% mask was applied. We also show that laminin lead to the best confluence compared to Matrigel, vitronectin and fibronectin.

The goal of the protocol is to compare different extracellular matrix (ECM) coating conditions to assess how differential coating affects the growth rate of induced pluripotent stem cells (iPSCs). In particular, we aim to set up conditions to obtain optimal growth of iPSC cultures.

Medición de la confluencia de iPSCs utilizando un sistema automatizado de imágenes.

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence ...

Culture of Piglet Intestinal 3D Organoids from Cryopreserved Epithelial Crypts and Establishment of Cell Monolayers

Intestinal organoids are increasingly being used to study the gut epithelium for digestive disease modeling, or to investigate interactions with drugs, nutrients, metabolites, pathogens, and the microbiota. Methods to culture intestinal organoids are now available for multiple species, including pigs, which is a species of major interest both as a farm animal and as a translational model for humans, for example, to study zoonotic diseases. Here, we give an in-depth description of a procedure used to culture pig intestinal 3D organoids from frozen epithelial crypts. The protocol describes how to cryopreserve epithelial crypts from the pig intestine and the subsequent procedures to culture 3D intestinal organoids. The main advantages of this method are (i) the temporal dissociation of the isolation of crypts from the culture of 3D organoids, (ii) the preparation of large stocks of cryopreserved crypts derived from multiple intestinal segments and from several animals at once, and thus (iii) the reduction in the need to sample fresh tissues from living animals. We also detail a protocol to establish cell monolayers derived from 3D organoids to allow access to the apical side of epithelial cells, which is the site of interactions with nutrients, microbes, or drugs. Overall, the protocols described here is a useful resource for studying the pig intestinal epithelium in veterinary and biomedical research.

Here, we describe a protocol to culture pig intestinal 3D organoids from cryopreserved epithelial crypts. We also describe a method to establish cell monolayers derived from 3D organoids, allowing access to the apical side of epithelial cells.

Cultivo de organoides 3D intestinales de lechones a partir de criptas epiteliales criopreservadas y establecimiento de monocapas celulares

Intestinal organoids are increasingly being used to study the gut epithelium for digestive disease modeling, or to investigate interactions with drugs, ...

Aislamiento mejorado de vesículas extracelulares bacterianas para microbiota multiómica

Isolation and Purification of Bacterial Extracellular Vesicles from Human Feces Using Density Gradient Centrifugation

Bacterial extracellular vesicles (BEVs) are nanovesicles derived from bacteria that play an active role in bacteria-bacteria and bacteria-host communication, transferring bioactive molecules such as proteins, lipids, and nucleic acids inherited from the parent bacteria. BEVs derived from the gut microbiota have effects within the gastrointestinal tract and can reach distant organs, resulting in significant implications for physiology and pathology. Theoretical investigations that explore the types, quantities, and roles of BEVs derived from human feces are crucial for understanding the secretion and function of BEVs from the gut microbiota. These investigations also necessitate an improvement in the current strategy for isolating and purifying BEVs.
This study optimized the isolation and purification process of BEVs by establishing two density gradient centrifugation (DGC) modes: Top-down and Bottom-up. The enriched distribution of BEVs was determined in fractions 6 to 8 (F6-F8). The effectiveness of the approach was evaluated based on particle morphology, size, concentration, and protein content. The particle and protein recovery rates were calculated, and the presence of specific markers was analyzed to compare the recovery and purity of the two DGC modes. The results indicated that the Top-down centrifugation mode had lower contamination levels and achieved a recovery rate and purity similar to that of the Bottom-up mode. A centrifugation time of 7 h was sufficient to achieve a fecal BEV concentration of 108/mg.
Apart from feces, this method could be applied to other body fluid types with proper modification according to the differences in components and viscosity. In conclusion, this detailed and reliable protocol would facilitate the standardized isolation and purification of BEVs and thus, lay a foundation for subsequent multi-omics analysis and functional experiments.

Autor destacado: Aceleración de la investigación sobre la separación y heterogeneidad de vesículas extracelulares bacterianas 

This study describes a method to isolate and purify bacterial extracellular vesicles (BEVs) enriched from human feces via density gradient centrifugation (DGC), identifies the physical characteristics of BEVs from morphology, particle size, and concentration, and discusses the potential applications of the DGC approach in clinical and scientific research.