Esta investigación es apoyada por el Programa de CounterACT, Institutos Nacionales de Salud (NIH), la Oficina del Director, y el Instituto Nacional de Ciencias de Salud Ambiental (NIEHS) Número de Grant U54 ES015678 (CWW). SA también es apoyado por el Hospital de Niños Escuela de Minas de Colaboración Premio Pilot # G0100394 y el Hospital de Niños # Premio Piloto de Investigación de Colorado Institue G0100471 Colorado / Colorado.

1. Culturas Rata cardiomiocitos <ol><li> Todos los experimentos se realizaron bajo protocolos aprobados por el cuidado de los animales y el uso comité institucional, IACUC. </li><li> Obtener cardiomiocitos de rata de los corazones (ventrículos) de ratas macho (240-260 g) empleando los métodos descritos anteriormente 4. En pocas palabras, anestesiar a los animales con una inyección intraperitoneal de pentobarbital (100 mg / kg; confirmar la anestesia por el método pizca dedo del pie) y luego retire corazones en 10,0 ml, 1 mM Ca 2 + que contiene tampón Krebs Ringer, pH 7,4. </li><li> Enjuague los corazones 5-6x para quitar la sangre y luego cambiar a un 2 tampón Krebs Ringer Ca + gratuito que contiene 0,02% de proteasa y 0,06% de colagenasa A (5,0 ml / corazón). Incubar los corazones en esta solución durante 10-15 min a 37 ° C con agitación ocasional. </li><li> Después de 10-15 minutos, lavar la solución enzimática con tampón Ca 2 + libre de Krebs Ringer durante 5 min. Liberar las células del tejido fláccido nosotrosing una pipeta de 25 ml con la pipeta la suspensión arriba y abajo varias veces. </li><li> Separar las células de los tejidos mediante el filtrado a través de una malla de nylon de 70 micras y permitir que se asienten en tampón Krebs Ringer que contenía 0,1 mM Ca 2 +. Suspender el sedimento de células en 1,0 ml de tampón Krebs Ringer que contenía 0,2 mM Ca 2 +. </li><li> Capa cuidadosamente más de 5,0 ml 60 / ml de albúmina de suero bovino mg, BSA, en 15 ml tubos de centrífuga, para separar los cardiomiocitos de nonmyocytes y permitir que se asienten durante 30 min. Los cardiomiocitos son más pesados ​​y se mueven a la parte inferior del tubo. Retire con cuidado las células sobrenadante y medios de comunicación. Repita este paso una vez para purificar aún más las células. </li><li> Transición gradual por lavado (en pasos utilizando 0,25 mM, 0,5 mM, 0,75 mM, y 1,0 mM de Ca 2 tampón + que contiene) los miocitos ventriculares / cardiomiocitos a 1,0 mM de Ca tampón 2 + que contiene y se resuspendieron en medio ACCT que consiste en Medio de Eagle Modificado de Dulbecco , DMEM containing 2 mg / ml de BSA, 2 mM de L-carnitina, creatina 5 mM, 5 mM de taurina, 100 UI / ml de penicilina, y 100 mg / ml de estreptomicina. </li><li> Placa de los cardiomiocitos en medio ACCT a una densidad de 100 a 150 células / mm 2 en 100 mm o 35 mm de laminina recubierto placas de cultivo de plástico o 40 x 22 mm cubreobjetos de vidrio recubiertos previamente con laminina (1 g / cm 2). Después de 1 hora, lavar los platos con ACCT 2,0 ml para eliminar las células que no están conectados. Añadir 10,0 ml de medio fresco y se incuban las células. </li></ol> 2. Interfaz de aire-líquido diferenciada (ALI) Cultivo de células de las vías respiratorias humanas epiteliales basales <ol><li> Adquirir tejidos traqueobronquiales humanos de la enfermedad de Investigaciones Científicas de intercambio bajo la Junta de Revisión Institucional de los protocolos aprobados. Cosecha celular y realizar cultivo utilizando un 5,6 procedimiento publicado. Este procedimiento utiliza cultivos de células, ya que son un modelo preciso de la exposición por inhalación humana a TICs, sin embargo, si es necesario se puede aislar y crecer a ALI moutilizar y / o células epiteliales de las vías respiratorias de rata 7,8. </li><li> Brevemente, hacer piezas pequeñas (~ ¼ de pulgada) de tejido después de la eliminación de todos los nodos de tejido conjuntivo y los ganglios. Lave el tejido varias veces en solución de Ringer con lactato. Añadir los tejidos a un tubo cónico de 50 ml y añadir solución de proteasa (1% Protease/0.01% de DNasa en medio mínimo esencial, MEM, tejido a la proporción de fluido (01:10)). </li><li> Rock the tejido a 4 ° C durante la noche. Fin de la disociación del tejido mediante la adición de suero bovino fetal al 10%. Raspar la superficie epitelial con un bisturí quirúrgico y recoger las células por centrifugación (2000 rpm durante 10 min). </li><li> Las células de la placa en el paso uno en snapwells recubiertas de colágeno en medio especial ALI preparados como se describe en detalle por Fulcher et al 9. Cultura durante 5 días antes de cambiar a la interfase aire-líquido (ALI) mediante la eliminación de los medios apicales. </li><li> Alimentar a las células por medio de los medios de comunicación ALI cada dos días y permitir que las células se diferencien por 2-3 semanas adicionales (observar numerosos cilios paliza y la secreción de moco) antes de realizar las exposiciones. Lave la superficie apical con PBS caliente junto con los cambios de los medios. </li></ol> 3. La exposición al cloro <ol><li> Contener el sistema de exposición al cloro (CES) dentro de una campana química calificada con una velocidad de extracción de funcionamiento de 100 pies por minuto que proporciona la contención secundaria necesaria para evitar la exposición del personal en caso de fugas accidentales de cloro. </li><li> Haga funcionar el sistema bajo presión ligeramente positiva (0,5 pulgadas de agua). El aire seco se alimenta a 15 L / min y un nivel adecuado de cloro se alimenta para alcanzar la concentración de cloro final deseado. Las cámaras tienen una tapa de cierre con 4 mini cerraduras BCU y una junta de silicona de durómetro bajo para proporcionar el sello de presión. </li><li> Entregar la mezcla de Cl 2 a través de un controlador de flujo másico. El CES utiliza un cilindro de gas comprimido que contiene 1,0% de Cl 2 en nitrógeno seco. </li><li> Regular el flujo de aire de dilución utilizando eldiseñado a medida del panel de control y del mismo modo regular la concentración de Cl2 entregado a las cámaras de exposición. Una bomba de muestreo de bajo volumen saca los gases de escape de las cámaras en un analizador de cloro para controlar las concentraciones que luego se graban en un registrador de datos conectado al analizador. </li><li> Medir las tasas de flujo dentro de las cámaras antes de la exposición usando un medidor de flujo para asegurar velocidades de suministro y de escape iguales. </li><li> Preparar los cultivos celulares para la exposición mediante la eliminación de los medios de comunicación de sobrenadante y añadir medio fresco (medio basolateral en culturas ALI). Cualquier pretratamientos con agentes podrían ser realizados en este momento. </li><li> Exponga las culturas ALI de las células epiteliales de la vía aérea a Cl 2 gas (50, 100, o 300 ppm durante 30 minutos) en las dos cámaras bioconfinamiento polisulfona selladas. Los cardiomiocitos (sumergidas o culturas en las membranas confluentes) están expuestos a 50 o 100 ppm de Cl 2 durante 15 min. </li><li> Después de la exposición limpiar las cámaras de aire hasta que la Cl 2 nivel cae por debajo de 1 ppm y se puede abrir de forma segura para eliminar las células (dentro de 5 min). </li></ol> 4. Resistencia eléctrica transepitelial (TER) Medición <ol><li> Mida la TER de aire-líquido-interfaz, ALI, culturas utilizando un voltohmmeter epitelial con un par de electrodos de cloruro de &quot;palillos&quot; de plata. </li><li> Equilibrar el electrodo palillo en el ALI medios 15 min antes de su uso. </li><li> Añadir media caliente al apical (1,0 ml) y basolateral (2,0 ml) de la superficie y medir la TER usando los electrodos palillos del voltohmmeter. </li><li> Sumergir el brazo más corto del electrodo en medios apical y el brazo más largo en los medios de comunicación basolateral. Haga clic en el botón de &quot;medida&quot; en el voltohmmeter para evaluar la resistencia eléctrica. </li><li> El voltohmmeter tiene la opción para medir ohmios o K ohmios. Restar la resistencia a través de un soporte de cultivo libre de células a partir de la resistencia medida a través de cada capa de células para producir el transresistencia epitelial (TER). </li></ol> 5. Medición de Caspasa <ol><li> Añadir medios ALI fresco (2,0 ml) a la exposición basolateral mensaje superficie e incubar la célula a temperatura ambiente. Recoger medio sobrenadante a las 4 y 24 horas. </li><li> Medir la caspasa 3/7 actividad en los sobrenadantes de los medios de comunicación mediante el uso de una caspasa comercial 3/7 kit de ensayo. </li></ol> 6. Western Blot e inmunocitoquímica <ol><li> Realizar transferencias de Western usando lisados ​​de células como se ha descrito previamente 10. Suspender el lisado de proteínas (20 g) en tampón de muestra 5x reducida y hervir durante 5 min. Se somete el lisado de proteínas a SDS-PAGE (4-15%) y transferir las proteínas separadas a una membrana de nitrocelulosa mediante transferencia electroforética. </li><li> Bloquear la unión no específica mediante la incubación de la membrana con leche 5% en tampón de lavado (PBS + 0,1% de detergente) y la sonda las membranas con anticuerpos primarios contra SERCA2 o actina sarcomérica en dilución 1:1000, durante la noche a 4 ° C. A continuación, lavar e incubar las membranas con los respectivos anticuerpos secundarios conjugados con peroxidasa y desarrollar para la detección como se ha descrito antes 10 usando el kit de detección de peroxidasa comercial. </li><li> Por inmunocitoquímica tratan células vivas cultivadas en insertos o cubreobjetos de vidrio en placas de 6 pocillos con 0,4% Triton-X-100 en mM tampón de citrato de sodio 10 durante 20 minutos después de enjuagar con PBS. </li><li> Bloquear la unión no específica tratando las células con 5% de suero de burro durante 20 min, y luego incubar las células con IgG no específica o anticuerpos primarios individuales específica a la actina sarcomérica o Ki-67. </li><li> Lavar las células con PBS y se incuban con anticuerpos secundarios fluorescentes conjugado para detectar el anticuerpo primario y un tinte nuclear (1 g / ml DAPI). </li><li> Lavar con PBS y montar cubreobjetos utilizando medio de montaje comercial. </li><li> Visualizar la tinción utilizando un microscopio de fluorescencia. </li></ol>

<ol>
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The authors declare that they have no competing financial interests.

El tipo más común de exposición a sustancias tóxicas agudas se produce cuando se respira un producto químico venenoso en los pulmones. Estas sustancias químicas también pueden ser tomadas rápidamente en el torrente sanguíneo y pueden afectar otros órganos, como el cerebro y el corazón. Toxicidad por inhalación de diversos agentes que utilizan modelos animales son estudiados e informó ampliamente, sin embargo los mecanismos son menos conocidos. Esto es un obstáculo importante en el desarrollo de terapias ef...

La exposición a productos químicos tóxicos inhalados (TICs) / gases como el cloro (Cl 2) sigue siendo un problema de salud en curso en exposiciones accidentales, así como en su uso potencial como un agente de amenaza química. Aunque los pulmones son el blanco principal, órganos como el corazón y el cerebro también se ven afectados 1-3. Modelos in vivo se utilizan generalmente para las pruebas de toxicidad de las TIC, pero en los ensayos in vitro para la evaluación de la toxicidad son más simples, más rápido y más rentable. En modelos in vitro también permiten la extensa investigación de las interacciones célula-agente que pueden ser difíciles de evaluar in vivo. Tales sistemas de exposición in vitro en son raros y por otra parte, en algunos modelos convencionales donde se añaden agentes tóxicos al medio de cultivo en el que se sumergen las células, las propiedades de los agentes pueden cambiar debido a las interacciones y la unión a componentes en el medio. En tales sistemas de cultivo de células escenarios tales como la interfaz aire-líquido (ALI) cultivos de células primarias vías respiratorias humanas epiteliales, que aquí se proponen, que pueden estar expuestos directamente a los agentes gaseosos podrían ser prometedores. Las células epiteliales que recubren las vías respiratorias son las primeras líneas de defensa contra sustancias químicas tóxicas inhaladas. El epitelio de la vía aérea humana forma una barrera física entre la luz y las células subyacentes en el pulmón y participa en la respuesta del pulmón. Se produce un número de citoquinas y otros agentes pro-y anti-inflamatorias, así como segrega líquido de la superficie de moco / las vías respiratorias (ASL) que cubre el epitelio. Una de las limitaciones en sumergido en sistemas de cultivo in vitro convencionales es también que la ASL y el moco que cubre la superficie epitelial se quita o se diluyen. Esto no refleja el estado fisiológico de las células epiteliales del pulmón que se exponen al aire. Por lo tanto, un ideal sistema in vitro para las pruebas de toxicidad TIC debe replicar esta arquitectura. Hay un gran interés en el desarrollo de la detección rápida métodos que predicen la toxicidad in vivo. Las células epiteliales cultivadas en el ALI diferenciar y tienen estructuras y funciones bien diferenciadas en comparación con las células cultivadas sumergidas y servir a un modelo superior de las vías respiratorias. En este estudio, se describe el uso de la cultura-líquido interfaz aérea de las vías respiratorias humanas (traqueobronquiales) las células epiteliales para las pruebas de toxicidad por inhalación de gas venenoso y lo comparamos con un cultivo de células sumergida de los cardiomiocitos, estudiando, por lo tanto otro objetivo importante de la toxicidad.

<table border="0" cellpadding="0" cellspacing="0" width="1128">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Name</td>
			<td style="width:329px;">Company</td>
			<td style="width:147px;">Catalog Number</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Rats</td>
			<td style="width:329px;">Harlan Laboratories</td>
			<td>Sprague-Dawley&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Pentobarbital</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">P3761</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Chlorine</td>
			<td style="width:329px;">AirGas, Inc</td>
			<td style="width:147px;">X02NI99CP163LS1</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Caspase 3/7 kit&#160;</td>
			<td style="width:329px;">Promega</td>
			<td style="width:147px;">G8091</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Epithelial voltohmmeter and chopstick electrode</td>
			<td style="width:329px;">World Precision Instruments</td>
			<td style="width:147px;">EVOM and STX2</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Snapwell inserts</td>
			<td style="width:329px;">Corning</td>
			<td style="width:147px;">07-200-708</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">70 micron nylon cell strainer</td>
			<td style="width:329px;">Corning</td>
			<td style="width:147px;">#352360</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polysulfone biocontainment chambers&#160;</td>
			<td style="width:329px;">BCU, Allentown Cage Equipment</td>
			<td style="width:147px;">BCU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td style="width:329px;">Life technologies</td>
			<td style="width:147px;">12491-015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Sarcomeric actin antibody</td>
			<td style="width:329px;">Abcam Cambridge, MA</td>
			<td style="width:147px;">ab28052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SERCA2 antibody</td>
			<td style="width:329px;">&#160;Affinity Bioreagents, Golden, CO</td>
			<td style="width:147px;">MA3-9191</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ki-67 antibody</td>
			<td style="width:329px;">&#160;Dako, Carpinteria, CA</td>
			<td style="width:147px;">M7248</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa-488-conjugated secondary antibody</td>
			<td style="width:329px;">&#160;Invitrogen, Grand Island, NY</td>
			<td style="width:147px;">A11029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">BSA</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">A9418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Carnitine</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">C0283</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Taurine</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">T8691</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Creatinine</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">C6257</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Krebs Ringer Buffer</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">K4002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Protease</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">P5147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Collagenase</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">C6885</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; DNAase</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">DN-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Lactated Ringer solution</td>
			<td style="width:329px;">Abott Laboratories</td>
			<td style="width:147px;">7953</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Donkey serum</td>
			<td style="width:329px;">Fisher Scientific</td>
			<td style="width:147px;">017-000-001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">PBS, phosphate buffered saline</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">D1408</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">4-15% SDS-PAGE gels</td>
			<td style="width:329px;">Bio-Rad</td>
			<td style="width:147px;">456-1083</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Nitrocellulose membrane</td>
			<td style="width:329px;">Bio-Rad</td>
			<td style="width:147px;">162-0115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Dergent, Tween&#160;</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">P1379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Peroxidase detection kit</td>
			<td style="width:329px;">Pierce</td>
			<td align="right" style="width:147px;">3402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">DAPI</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">D9542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Mounting media, Fluormount G</td>
			<td style="width:329px;">eBiosciences</td>
			<td style="width:147px;">00-4958-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Sodium citrate</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">71497</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Collagen</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">C7521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">MEM</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">M8028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Laminin</td>
			<td style="width:329px;">BD biosciences</td>
			<td style="width:147px;">354259</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Penicillin/Streptomycin</td>
			<td style="width:329px;">Life Technologies</td>
			<td style="width:147px;">15070063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">FBS</td>
			<td style="width:329px;">Gibco</td>
			<td style="width:147px;">200-6140AJ</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro cell culture model for toxic inhaled chemical testing

Los cultivos celulares son indispensables para desarrollar y estudiar la eficacia de los agentes terapéuticos, antes de su uso en modelos animales. Tenemos la capacidad única de modelar las células del epitelio de la vía aérea humana y del músculo del corazón bien diferenciadas. Esto podría ser una herramienta muy valiosa para estudiar los efectos nocivos de los productos químicos tóxicos inhalados, como el cloro, que normalmente pueden interactuar con las superficies de las células, y forman varios subproductos al reaccionar con el agua, y la limitación de sus efectos en cultivos sumergidos. Nuestro modelo utilizando cultivos de células epiteliales de la vía aérea humana bien diferenciados en la interfaz aire liqiuid elude esta limitación, así como proporciona una oportunidad para evaluar los mecanismos fundamentales de la toxicidad de los potenciales químicos inhalados venenosas. Describimos una mayor pérdida de la integridad de la membrana, la liberación de caspasa y la muerte sobre químicos tóxicos inhalados, como la exposición al cloro. En este artículo, se propone métodos para modelar la exposición al cloro en el corazón de los mamíferos y de las vías respiratorias epiteliales cells en la cultura y sencillas pruebas para evaluar su efecto en estos tipos de células.

Cardiomiocitos en forma de varilla primaria se adhieren sobre matrices de laminina y se propagan y se diferencian en cultivos confluentes (Figura 1A y su inserción). Estas células se caracterizan además sobre la base de actina sarcomérica y la expresión de SERCA2 (Figuras 1B y 1C). Cardiomiocitos de rata son altamente susceptibles a la toxicidad de cloro como 15 minutos de exposición a 100 ppm de cloro causó grandes redondeo celular y muerte en cultivos sumergido...

Watch this Scientific Journal Video about In vitro Cell Culture Model for Toxic Inhaled Chemical Testing at JoVE.com

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Este protocolo está diseñado para demostrar método de exposición de cultivos de células a productos químicos tóxicos inhalados. La exposición de diferenciada interfaz aire-líquido (ALI) cultivos de células epiteliales de las vías respiratorias proporciona un modelo único de la exposición de las vías respiratorias a los gases tóxicos tales como el cloro. En este manuscrito se describe el efecto de la exposición al cloro en las culturas de la interfaz aire-líquido de las células epiteliales y la cultura sumergida de los cardiomiocitos. In vitro sistemas de exposición permiten importantes estudios sobre el mecanismo para evaluar las vías que podrían entonces ser utilizados para desarrollar nuevos agentes terapéuticos.

In vitro modelo de cultivo celular para Toxic inhalado Prueba química

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to ...

in-vitro-cell-culture-model-for-toxic-inhaled-chemical-testing

Research

JoVE Journal

Bioengineering

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

10.6K vistas.  University of Colorado.  Este protocolo está diseñado para demostrar método de exposición de cultivos de células a productos químicos tóxicos inhalados. La exposición de diferenciada interfaz aire-líquido (ALI) cultivos de células epiteliales de las vías respiratorias proporciona un modelo único de la exposición de las vías respiratorias a los gases tóxicos tales como el cloro. En este manuscrito se describe el efecto de la exposición al cloro en las culturas de la interfaz aire-...

Video: In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

Dispositivo de microfluidos para volver a crear un microambiente tumoral<em&gt; In Vitro</em

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

Investigación

Bioingeniería

Shape Memory Polymers for Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape.
Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture.
Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions.

A method for developing cell culture substrates with the ability to change topography during culture is described. The method makes use of smart materials known as shape memory polymers that have the ability to memorize a permanent shape. This concept is adaptable to a wide range of materials and applications.

Los polímeros con memoria de forma para el cultivo celular activo

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk of thromboemboli formation1,2,3. We have developed a method to line the inside surface of Ti tubes with autologous blood-derived human or porcine endothelial progenitor cells (EPCs)4. By implanting Ti tubes containing a confluent layer of porcine EPCs in the inferior vena cava (IVC) of pigs, we tested the improved biocompatibility of the cell-seeded surface in the prothrombotic environment of a large animal model and compared it to unmodified bare metal surfaces5,6,7 (Figure 1). This method can be used to endothelialize devices within minutes of implantation and test their antithrombotic function in vivo. 
Peripheral blood was obtained from 50 kg Yorkshire swine and its mononuclear cell fraction cultured to isolate EPCs4,8. Ti tubes (9.4 mm ID) were pre-cut into three 4.5 cm longitudinal sections and reassembled with heat-shrink tubing. A seeding device was built, which allows for slow rotation of the Ti tubes. 
We performed a laparotomy on the pigs and externalized the intestine and urinary bladder. Sharp and blunt dissection was used to skeletonize the IVC from its bifurcation distal to the right renal artery proximal. The Ti tubes were then filled with fluorescently-labeled autologous EPC suspension and rotated at 10 RPH x 30 min to achieve uniform cell-coating9. After administration of 100 USP/ kg heparin, both ends of the IVC and a lumbar vein were clamped. A 4 cm veinotomy was performed and the device inserted and filled with phosphate-buffered saline. As the veinotomy was closed with a 4-0 Prolene running suture, one clamp was removed to de-air the IVC. At the end of the procedure, the fascia was approximated with 0-PDS (polydioxanone suture), the subcutaneous space closed with 2-0 Vicryl and the skin stapled closed. 
After 3 - 21 days, pigs were euthanized, the device explanted en-block and fixed. The Ti tubes were disassembled and the inner surfaces imaged with a fluorescent microscope.
We found that the bare metal Ti tubes fully occluded whereas the EPC-seeded tubes remained patent. Further, we were able to demonstrate a confluent layer of EPCs on the inside blood-contacting surface.
Concluding, our technology can be used to endothelialize Ti tubes within minutes of implantation with autologous EPCs to prevent thrombosis of the device. Our surgical method allows for testing the improved biocompatibility of such modified devices with minimal blood loss and EPC-seeded surface disruption.

A method for seeding titanium blood-contacting biomaterials with autologous cells and testing biocompatibility is described. This method uses endothelial progenitor cells and titanium tubes, seeded within minutes of surgical implantation into porcine venae cavae. This technique is adaptable to many other implantable biomedical devices.

Autólogo de células progenitoras endoteliales-Siembra Tecnología y las pruebas de biocompatibilidad para dispositivos cardiovasculares en el modelo animal de gran

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk ...

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

Rotación de sistemas de cultivos celulares de cultivo de células humanas: células humanas trofoblásticas como un modelo de

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

Irritación de los ojos de prueba (IET) para la identificación del peligro de los ojos: Irrita los productos químicos que utilizan Reconstruido humana Córnea-como epitelial (RHCE) Modelo de Tejidos

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Formación luz mediada y el patrón de hidrogeles para aplicaciones de cultivo celular

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

Un<em&gt; In Vitro</em&gt; Órgano Cultura Modelo Murino del disco intervertebral

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are an emerging treatment for photoreceptor degenerative diseases that seek to restore vision by artificially stimulating the surviving retinal neurons in the hope of eliciting comprehensible visual perception in patients. Current retinal prostheses have demonstrated success in restoring limited vision to patients using an array of electrodes to electrically stimulate the retina but face substantial physical barriers in restoring high acuity, natural vision to patients. Chemical neurostimulation using native neurotransmitters is a biomimetic alternative to electrical stimulation and could bypass the fundamental limitations associated with retinal prostheses using electrical neurostimulation. Specifically, chemical neurostimulation has the potential to restore more natural vision with comparable or better visual acuities to patients by injecting very small quantities of neurotransmitters, the same natural agents of communication used by retinal chemical synapses, at much finer resolution than current electrical prostheses. However, as a relatively unexplored stimulation paradigm, there is no established protocol for achieving chemical stimulation of the retina in vitro. The purpose of this work is to provide a detailed framework for accomplishing chemical stimulation of the retina for investigators who wish to study the potential of chemical neuromodulation of the retina or similar neural tissues in vitro. In this work, we describe the experimental setup and methodology for eliciting retinal ganglion cell (RGC) spike responses similar to visual light responses in wild-type and photoreceptor-degenerated wholemount rat retinas by injecting controlled volumes of the neurotransmitter glutamate into the subretinal space using glass micropipettes and a custom multiport microfluidic device. This methodology and protocol are general enough to be adapted for neuromodulation using other neurotransmitters or even other neural tissues.

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

This protocol describes a novel method for investigating a form of chemical neurostimulation of wholemount rat retinas in vitro with the neurotransmitter glutamate. Chemical neurostimulation is a promising alternative to the conventional electrical neurostimulation of retinal neurons for treating irreversible blindness caused by photoreceptor degenerative diseases.

Metodología para la neuromodulación química Biomimetic de Retinas de ratas con el neurotransmisor glutamato In Vitro

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

Un modelo de cultivo de matriz-adipocitos extracelulares 3D humano para el estudio de la conversación cruzada metabólica Matrix-Cell

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

In Vitro Model of Human Cutaneous Hypertrophic Scarring using Macromolecular Crowding

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol applies a macromolecular crowding (MMC) technique to mimic this physiological crowding through the addition of neutral polymers (crowders) to cell cultures in vitro. Previous studies using Ficoll or dextran as crowders demonstrate that the expression of collagen I and fibronectin in WI38 and WS-1 cell lines are significantly enhanced using the MMC technique. However, this technique has not been validated in primary hypertrophic scar-derived human skin fibroblasts (hHSFs). As hypertrophic scarring arises from the excessive deposition of collagen, this protocol aims to construct a collagen-rich in vitro hypertrophic scar model by applying the MMC technique with hHSFs. This optimized MMC model has been shown to possess more similarities with in vivo scar tissue compared to traditional 2-dimensional (2-D) cell culture systems. In addition, it is cost-effective, time-efficient, and ethically desirable compared to animal models. Therefore, the optimized model reported here offers an advanced &#8220;in vivo-like&#8221; model for hypertrophic scar-related studies.

This protocol describes the use of macromolecular crowding to create an in vitro human hypertrophic scar tissue model that resembles in vivo conditions. When cultivated in a crowded macromolecular environment, human skin fibroblasts exhibit phenotypes, biochemistry, physiology, and functional characteristics resembling scar tissue.

Modelo In Vitro de Cicatrices Hipertróficas Cutáneas Humanas usando Crowding Macromolecular

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol ...