This project was support in part by Johns Hopkins University, Office of the Provost through the Gateway Science Initiative.

1. Login <ol><li> Encienda el biorreactor. Haga clic en cualquier lugar para abrir la página de inicio de sesión. Seleccione el nombre de usuario y escriba la contraseña y haga clic en &quot;Iniciar sesión&quot;. </li></ol> 2. Calibración <ol><li> Calibrar sensor de pH (2 puntos antes de la esterilización en autoclave). </li><li> Inspeccione sensor de pH y confirme la punta del sensor se llena con solución electrolítica. Prepare dos vasos con soluciones de calibración pH (electrolitos) pH 4 y pH 7 y disponer de una botella de lavado con agua destilada. </li><li> Conectar el cable de pH para el sensor de pH. Vaya a la pestaña &quot;Acciones&quot; en la interfaz de Hola y haga clic en &quot;calibrar&quot;. Ingrese temperatura del tampón en el campo de temperatura solución de calibración. </li><li> Coloque el sensor de pH en tampón 1 (pH 4) e introduzca el valor en el campo &quot;cero&quot;. Espere a que el gráfico se estabilice y el clic en el botón &quot;calibrar 1&quot;. Enjuague el sensor de pH con agua destilada. </li><li> Coloque el sensor en tampón 2 (pH 7). Ingrese búfer2 valor en el campo &quot;span&quot;. Espere a que se estabilice el gráfico y haga clic en la calibración de 2 botones. </li><li> Haga clic en &quot;Guardar&quot; y luego haga clic en &quot;Cerrar&quot;. </li><li> Calibrar el sensor de oxígeno disuelto (OD). </li><li> Asegúrese de que el sensor de oxígeno se ha polarizado al ser conectado al sistema durante varias horas. Vaya a la pestaña &quot;Acciones&quot; en la interfaz de Hola y haga clic en Calibrar. Haga clic en el botón &quot;HACER A&quot;. </li><li> Desconecte el sensor de oxígeno y escriba 0 en el campo &quot;cero&quot;. Espere a que el gráfico se estabilice y el clic en el botón &quot;Calibrar 1&quot;. </li><li> Vuelva a conectar el sensor de oxígeno y escriba 100 en el campo &quot;Span&quot;. Espere a que el gráfico se estabilice y el clic en el botón &quot;Calibrar 2&quot;. </li><li> Haga clic en &quot;Guardar&quot; y luego haga clic en &quot;cerrar&quot;. </li></ol> 3. Autoclave e instalación de sensores y recipientes de reactivos <ol><li> Después de la calibración, los sensores de lugar y así térmica en autocLave y bolsas de autoclave durante 30 min a 121 ° C, 15 psi. </li><li> Desinfectar las bolsas de autoclave con 70% alcohol isopropílico (IPA) y bolsas de transferencia a cabina de seguridad biológica (BSC). Retire el embalaje exterior del buque. </li><li> Desinfectar envase interior con un 70% de IPA y transferir buque cabina de seguridad biológica (BSC). Retire el embalaje interior e inspeccionar el buque y la tubería por los daños infligidos durante el envío. </li><li> Instale el pH y DO sensores en los dos puertos frontales. Instale el bienestar térmico en el puerto lateral izquierdo. Abra la tapa del sensor. </li><li> Guía del sensor a través del puerto del sensor. Pase el sensor firmemente en el puerto. </li><li> Transferencia recipiente de BSC. </li><li> Cuelgue la DO y cables de los sensores de pH fuera de la manga del vaso y compruebe que no hay nada en la manga. Deslice el recipiente dentro de la funda, los pies por delante. </li><li> Coloque con cuidado el sensor de temperatura en el pozo termal buque. Asegúrese de que el fondo del recipiente se apoya contra los calentadores. </li><li> Retirela tubería fija de sus bolsas. Partido codificación de color en el tubo a los conectores correspondientes y bombas en la unidad de control biorreactor. </li><li> Instale la línea de gas principal presionando el conector en su salida de gas. Instale la línea de gas micro girando el conector hacia la derecha en la salida de gas. Instale el tubo de filtro de escape: </li><li> Abrir la estufa filtro. Asegure el filtro de escape en el canal en U de modo que su tubo pasa por los dos ganchos al filtro y fuera del horno. </li><li> Instale el tubo de la bolsa de condensador en el soporte de la tubería. Cierre la puerta. </li><li> Ruta líneas adición de A y B, ambas líneas de comunicación, y la línea de cosecha detrás del sensor de oxígeno y en el banco junto a la unidad de control de bioreactor. </li><li> Conecte los cables a los sensores de oxígeno y pH. </li></ol> 4. Añadir medianas y micro-portadores <ol><li> Vaya a la pestaña &quot;acciones&quot; y haga clic en &quot;Control de Bombas&quot; en la interfaz de la computadora. </li><li> Formar un esterilizadorLe conexión entre una línea sin usar medio de adición (1 banda naranja) y la fuente de la botella / bolsa de medio por soldadura el tubo o el uso de accesorios Luer. </li><li> Haga clic en el control deslizante para encender la bomba de los medios de comunicación en. Haga clic en el control deslizante para activar los medios de comunicación de la bomba después de adición deseada cantidad de medio. </li><li> Coloque microportadoras cuentas en Ca 2 +, Mg 2 + PBS libre durante 3 horas a temperatura ambiente. </li><li> Lavar perlas varias veces con Ca 2 +, Mg 2 + PBS libre. </li><li> Autoclave durante 15 min a 115 ° C, 15 psi. NOTA: Añadir 3 g / L (peso en seco) en este experimento. </li><li> Bomba en micro-soportes que han sido hidratadas, lavados y esterilizados en autoclave en el reactor de la misma manera se añadió el medio en el paso 4.1. </li></ol> 5. de equilibrado y de un punto de calibración HACER <ol><li> Establecer los controladores a Automático e introducir los puntos de ajuste deseados. Aquí, utilice Agitación Set Point (SP) = 15 rpm, temperatura SP = 37,0 ° C, pH = 7,2, DO = 100%. Espere a que el parámetros se equilibren. </li><li> Confirmar sensor es completamente polarizada. Confirmar DO valor actual se ha estabilizado. </li><li> Vaya a la pestaña &quot;Acciones&quot; y haga clic en &quot;calibrar&quot;. Haga clic en &quot;DO A&quot;, haga clic en &quot;One-punto&quot;. </li><li> Introduzca &quot;100&quot; en el campo &quot;Span&quot;. Haga clic en el botón &quot;Calibrar 1&quot;, haga clic en &quot;Guardar&quot; y haga clic en &quot;cerrar&quot;: </li></ol> 6. de iniciar una ejecución <ol><li> Vaya a la pestaña Acciones. Haga clic en &quot;lotes&quot;. Utilice el teclado en pantalla o un teclado externo para ingresar un nombre de lote de 16 caracteres o menos. </li><li> Haga clic en el botón &quot;Ocultar&quot; del teclado en pantalla. Haga clic en &quot;Iniciar lote&quot; confirme haciendo clic en &quot;Inicio&quot; en la superposición. </li></ol> 7. inoculan con células <ol><li> Formar una conexión estéril entre un (banda naranja 1) línea de adición de medio sin usar y la botella de células / fuente bolsa por soldadurael tubo o el uso de los accesorios Luer. </li><li> Instale la sección de silicona de la tubería en la bomba de los medios de comunicación por lo que la flecha apunte hacia la tubería entre la bomba y el recipiente. </li><li> Compruebe pinza de la línea está abierta y su pinza de la línea ramificada está cerrado. Haga clic en el control deslizante para encender la bomba de los medios de comunicación en y haga clic en &quot;No&quot;, después de la adición de las células. </li><li> Vaya a la pestaña &quot;Acciones&quot; y haga clic en &quot;Bombas de control&quot;. </li></ol> 8. Muestreo <ol><li> Después de inocular y tan frecuentemente como se desee para controlar la cultura, extraer una muestra de la cultura de la siguiente manera: </li><li> Vaya a la pestaña &quot;acciones&quot; y haga clic en &quot;tomar la muestra&quot;. Coloque el tubo de muestreo en la bomba de muestreo y manipular la llave de paso de muestreo de acuerdo con las instrucciones de la pantalla. </li><li> Realizar al menos una observación microscópica diario y recuento de células en estas muestras. </li><li> Cuando las células han alcanzado la densidad deseada, en este cASE 1,2 x 10 6 células / ml, infectar las células con la adición de un inóculo de virus. </li><li> Agregar asépticamente el inóculo a una jeringa de 20 ml, y conectar la jeringa a uno de los puertos de adición de repuesto en el reactor. Introducir el inóculo en el reactor presionando en el émbolo de la jeringa. </li><li> Continuar el muestreo y el análisis de la cultura de la concentración de partículas intracelulares adenovirus. </li></ol>

<ol>
	<li>Freshney, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culture of Animal Cells A Manual of Basic Techniques and Specialized Applications. , (2010).</li><li>Healthcare, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcarrier+Cell+Culture+Principles+and+Methods.">Microcarrier Cell Culture Principles and Methods.</a> GE Healthcare Data File 18-1140-62. , (2005).</li><li>Simaria, A. S., 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=Allogeneic+cell+therapy+bioprocess+economics+and+optimization+Single-Use+cell+expansion+Technologies.">Allogeneic cell therapy bioprocess economics and optimization Single-Use cell expansion Technologies.</a> Biotechnol and Bioeng. 111 (1), 69-83 (2014).</li><li>Lee, B., Fang, D., Croughan, M., Carrondo, M., Paik, S. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+novel+pneumatic+mixing+for+single-use+bioreactor+application.">Characterization of novel pneumatic mixing for single-use bioreactor application.</a> BMC Proc. 5 (S8), O12 (2011).</li><li>Eibl, R., Eibl, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disposable+bioreactors+in+cell+culture-based+upstream+processing.">Disposable bioreactors in cell culture-based upstream processing.</a> BioProcess Int. 7 (S1), 18-23 (2009).</li><li>Chaubard, J. F., 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=Disposable+bioreactors+for+viral+vaccine+production+challenges+and+opportunities.">Disposable bioreactors for viral vaccine production challenges and opportunities.</a> Biopharm Int Supp. , (2010).</li><li>Croughan, M. S., Hamel, J. F., Wang, D. I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+Effects+on+Animal+Cells+Grown+in+Microcarrier+Cultures.">Hydrodynamic Effects on Animal Cells Grown in Microcarrier Cultures.</a> Biotechnol and Bioeng. 95 (2), 295-305 (2006).</li><li>DePalma, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-use+Equipment+on+Cusp+of+Industrialization.">Single-use Equipment on Cusp of Industrialization.</a> Genet Eng Biotechnol News. 32 (1), (2012).</li><li>Baltz, R. H., Demain, A. L., Davies, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manual of Industrial Microbiology and Biotechnology. , (2010).</li><li>Sousa, M. F., Giroux, D., Clemente, J., Lee, B., Carrondo, M. J., Alves, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Impact of Bioreactor Design on the Performance of Microcarrier Cultures. , (2012).</li></ol>

Autores D. Giroux y Y. Hashimura son empleados de PBS Biotech que produce reactivos e instrumentos utilizados en este artículo.

Este sistema de biorreactor de un solo uso es relativamente sencillo de usar y proporciona análisis en tiempo real para la monitorización y el análisis reactor. Está muy bien adecuado para el cultivo de células de mamíferos y de insectos con densidades celulares que llegan a más de 30 millones de células / ml. Además de las células A549 describen en este informe 11, hemos crecido SF-9 células de insectos en el biorreactor también. La mezcla suave proporcionado por la rueda de aire neumático reduce el daño celular. Varios pasos son críticos al establecer este reactor. Primera calibración, apropiado del pH y DO sensores es importante para la vigilancia óptima de la cultura y para la adición de reactivos para ajustar el pH o el oxígeno en el sistema. En segundo lugar, los frascos de reactivos y de semillas deben ser llenados y los accesorios Luer realizan en un ambiente estéril, como un BSC. Una vez que los frascos de reactivos se mueven fuera del ambiente estéril, las conexiones a las líneas de alimentación de biorreactores deben hacerse con cuidado para evitar la contaminación microbiana. <pclass = &quot;jove_content&quot;&gt; Si bien este sistema de biorreactor funciona bien para las líneas de células de mamíferos e insectos que no está diseñado para los cultivos bacterianos. El sistema no puede proporcionar la mezcla rápida y la oxigenación que se requiere para células bacterianas. El crecimiento bacteriano se realiza mejor en un biorreactor de tanque agitado. En comparación con otros biorreactores de un solo uso para el cultivo de células de mamífero o insecto, este sistema es fácil de usar, proporciona datos suficientes para el análisis de pistas, y tiene el crecimiento celular similar o mejor que el otro sistema de un solo uso que hemos evaluado. El uso único sistema biorreactor neumático tiene el potencial para satisfacer muchas de las investigaciones y aplicaciones clínicas en los campos de bioterapéuticos, vacunas, las células madre y la medicina personalizada 4. Además, la flexibilidad de este sistema permite por lotes, Fed-batch, la perfusión, y las aplicaciones de biorreactores de transfección basado 5. Por último, de un solo uso sistemas de biorreactores desechables tienen el potenciales para satisfacer las necesidades de la producción industrial a gran escala y para cumplir con las directrices y recomendaciones de los organismos reguladores nacionales e internacionales 10.6.

Líneas de células de mamíferos se pueden clasificar en una de tres categorías basadas en sus características de crecimiento: células que crecen en suspensión, las células que crecen en forma de agregados, y las células que crecen anclado a un sustrato. Aunque el biorreactor de aire de la rueda demostrado en este video es capaz de crecer los tres tipos de células, este video demostrará el uso del biorreactor para cultivar células dependientes de anclaje en micro-soportes. Células de mamíferos dependientes de anclaje pueden ser cultivadas con el propósito de producir más células - donde las propias células son el producto. Por ejemplo, las células madre mesenquimales derivadas de médula ósea humana están siendo cultivadas con el propósito de recoger las células e inyectarlas en el tejido enfermo. El biorreactor neumático demostrado en este vídeo ha demostrado ser adecuado para la producción de dichas células madre mesenquimales para esta aplicación (Serra et al., Comunicación personal, 2013). Anchorage depcélulas de mamífero se cultivan típicamente endent pequeña escala en recipientes de cultivo 2D tales como placas de cultivo celular, frascos de cultivo de células, o botellas de rodillos, donde se adhieren a una superficie de crecimiento especialmente tratado 1. Cuando se desean más células, las placas o frascos se pueden ampliar mediante el uso de más o grandes vasos. Sin embargo, para un cultivo más rentable de grandes cantidades de células dependientes de anclaje, aumentando el área superficial para la fijación celular que se puede lograr mediante el uso de pequeñas perlas sólidas llamados micro-soportes. Dependiendo de las características de fijación de la célula, diferentes tipos de micro-soportes están disponibles comercialmente, tal como dextrano, un péptido, o colágeno recubierto. Micro-soportes tienen una gran área superficial a volumen que proporciona una mayor superficie para el crecimiento celular; y los micro-soportes se pueden mantener en suspensión con agitación, lo que permite que las células se cultivan a altas densidades en sistemas de biorreactores 2. Actualmente, los tipos de bioreactors donde las células adherentes se cultivan en micro-vehículos incluyen matraces rotativos y sistemas de tanque agitado, que utilizan hélices axiales para mantener la suspensión de las células recubierto micro-soportes. Varios factores son importantes para el cultivo con éxito de células, incluyendo la tensión de oxígeno, la tensión de cizallamiento, la matriz de superficie, y de nutrientes y las concentraciones de metabolitos. El uso de biorreactores permite la monitorización en tiempo real de las condiciones de crecimiento y el potencial de significativamente más bajos costos de producción 1. Hay varios diseños de biorreactores común para el cultivo in vitro de células incluyendo, suspensión agitada, recipiente de pared giratorio, de fibra hueca, la bolsa de biorreactor en una plataforma de balancín, y sistemas de lecho fluidizado 3. Muchos de estos sistemas presentan problemas únicos para el cultivo de células y la escala -up, tales como el alto costo, los gradientes de concentración de nutrientes, cizallamiento hidrodinámico, la agregación de células, y la dificultad en el muestreo, monitoreo y control de cell de escala. Varias líneas de células adherentes se utilizan en la producción de virus, ya sea en la producción de vacunas virales o para la producción de vectores virales para aplicaciones de terapia génica. En este video, usando el sencillo uso neumática (aire-ruedas) sistema de biorreactor, se demuestra la cultura de células de carcinoma de pulmón humano (A549) las células en micro-soportes para la producción de un adenovirus oncolítico. El diseño del biorreactor neumática utiliza una rueda de agitación vertical que es alimentado por la flotabilidad del gas burbujeado en la parte inferior del biorreactor. Este método de agitación suave limita las fuerzas de cizallamiento hidrodinámico, pero aún asegura medio y óptima de la célula de mezcla 4. En comparación con el reactor de tanque agitado, el reactor neumático tiene una baja tensión de cizallamiento, incluso con sistemas de biorreactor de aire de la rueda de alto volumen (Figura 1). En contraste con los biorreactores de tanque agitado, el impulsor vertical de este reactor único uso está activado por una corriente de burbujas de gas dentro deel recipiente, lo que permite suave y uniforme de mezcla medio (Figura 2).

<table border="0" cellpadding="0" cellspacing="0" width="626">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:124px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS 3&#160;</td>
			<td style="width:124px;">PBS</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Single Use Assembly</td>
			<td style="width:124px;">PBS</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;">Human Lung Carcinoma Cells (A549)</td>
			<td style="width:124px;">ATCC</td>
			<td style="width:119px;">&#160;CCL-185</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM High Glucose Medium</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin EDTA, 0.25%</td>
			<td style="width:124px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytodex 1 Microcarriers</td>
			<td style="width:124px;">GE</td>
			<td align="right">3781</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antifoam C</td>
			<td style="width:124px;">Sigma</td>
			<td style="width:119px;">A8011</td>
			<td></td>
		</tr>
	</tbody>
</table>

cultivation of mammalian cells using a single-use pneumatic bioreactor system

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized medicine innovations. However, translating these advances into therapeutic applications will require in vitro systems that allow for robust, flexible, and cost effective bioreactor systems. There are several bioreactor systems currently utilized in research and commercial settings; however, many of these systems are not optimal for establishing, expanding, and monitoring the growth of different cell types. The culture parameters most challenging to control in these systems include, minimizing hydrodynamic shear, preventing nutrient gradient formation, establishing uniform culture medium aeration, preventing microbial contamination, and monitoring and adjusting culture conditions in real-time. Using a pneumatic single-use bioreactor system, we demonstrate the assembly and operation of this novel bioreactor for mammalian cells grown on micro-carriers. This bioreactor system eliminates many of the challenges associated with currently available systems by minimizing hydrodynamic shear and nutrient gradient formation, and allowing for uniform culture medium aeration. Moreover, the bioreactor&#8217;s software allows for remote real-time monitoring and adjusting of the bioreactor run parameters. This bioreactor system also has tremendous potential for scale-up of adherent and suspension mammalian cells for production of a variety therapeutic proteins, monoclonal antibodies, stem cells, biosimilars, and vaccines.

En la Figura 3, se muestran los parámetros para el inicio de la carrera biorreactor. Esta figura muestra la pantalla antes de establecer los parámetros de temperatura, oxígeno disuelto, pH y agitación. Una vez que los parámetros se establecen, los parámetros de ejecución son monitoreados continuamente y se pueden hacer ajustes para mantener las condiciones requeridas. El software produce una lectura continua que permite una fácil identificación de problemas. En este experimento utilizando 2,5 L de DMEM que contenía 10% de FBS, 250 ppm de SAFC anti-espuma C, 2 mM L-glutamina y 3 g / L de micro-soportes, el sistema se estabiliza por lo que el porcentaje de oxígeno es 50%, el temperatura es 37 ° C, y el pH es 7,2. El reactor es inoculado con células A549 a una concentración de 7 x 10 4 células / ml (o 10 células / micro-portadoras). Los parámetros elegidos para esta ejecución resultaron en la mayoría de las células que se adhieren a los micro-soportes dentro de 2 horas (Figura 4). Después de 12 h, las células son demoniossignos trating de aplanamiento y la difusión en la superficie micro-portadora (Figura 5). Por 24 horas, micro-soportes tienen una distribución relativamente uniforme de las células, sin micro-soportes sin células y no hay grandes grupos de células en los micro-soportes (Figura 6). El porcentaje de colonización micro-portador por las células A549 es de 75% en 24 horas y el 90% a partir de entonces (Figura 7). Las células continuaron creciendo exponencialmente a ~ 1 millón de células / ml después de 48 horas (Figura 8). Después de la infección con una dosis de adenovirus oncolítico (2 x 10 8 viriones / ml) a 50 h, la densidad aumentó a 1.2 millones de células / ml y luego comenzó a disminuir a medida que la infección lítica progresó. Había ~ 10.000 amplificación pliegue del inóculo viral. En experimentos anteriores, se ha encontrado que el control y regulación del caudal de gas es crítica para maximizar el crecimiento celular (datos no publicados). El rápido crecimiento de las células pueden agotar el sistema de oxígeno con<html> el nivel de OD de caer a cero. Mientras que las células continuaron creciendo, fue a un ritmo mucho más lento. Es crítico para controlar y ajustar el flujo de oxígeno para satisfacer las demandas celular durante la fase de crecimiento logarítmico. Cada serie se puede analizar para un óptimo crecimiento comparando el pH, DO, y la temperatura con recuentos de células diarias. <img alt="Figura 1" src="/files/ftp_upload/52008/52008fig1highres.jpg" /> Figura 1. Dinámica de Fluidos del sistema biorreactor neumático (PBS) en comparación con el tanque biorreactor Agitador medición de fuerzas de cizallamiento de pared en varios volúmenes de reactor. <img alt="Figura 2" src="/files/ftp_upload/52008/52008fig2highres.jpg" /> Figura 2. neumática Aire rueda impulsor reactor. <html> 52008 / 52008fig3highres.jpg &quot;/&gt; Figura 3. software de ruedas Aire neumático captura de pantalla de los parámetros para iniciar la carrera biorreactor. <img alt="Figura 4" src="/files/ftp_upload/52008/52008fig4highres.jpg" /> Figura 4. colonización Micro-portador con células A549 2 horas después de la inoculación. (Diámetro medio de micro-portador ~ 180 m.) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52008/52008fig4highres.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> <img alt="Figura 5" src="/files/ftp_upload/52008/52008fig5highres.jpg" /> Figura 5. 12 horas después del inicio del cultivo, las células A549 están otorgando, aplanar, y la difusión en los micro-soportes.5highres.jpg &quot;target =&quot; _blank &quot;&gt; Haga clic aquí para ver una versión más grande de esta figura. <img alt="Figura 6" src="/files/ftp_upload/52008/52008fig6highres.jpg" /> Figura 6: Micro-portadoras recubiertas con células después de 24 horas en la cultura. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52008/52008fig6highres.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> <img alt="Figura 7" src="/files/ftp_upload/52008/52008fig7highres.jpg" /> Figura 7. Porcentaje de colonización micro-portadora por las células A549 después de la inoculación. <img alt="Figura 8" src="/files/ftp_upload/52008/52008fig8highres.jpg" /> Figura 8. El número de células A549 viables en cultivo antes y después de la infección con adenovirnosotros. Tenga en cuenta que el paso de la infección viral se produjo a las 50 h.

Watch this Scientific Journal Video about Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System at JoVE.com

Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System

Using a pneumatic bioreactor, we demonstrate the assembly, operation, and performance of this single-use bioreactor system for the growth of mammalian cells.

El cultivo de células de mamífero utilizando un solo uso Sistema Neumático biorreactor

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized ...

cultivation-mammalian-cells-using-single-use-pneumatic-bioreactor

Nanyang Technological University

Research

JoVE Journal

Bioengineering

24.3K vistas.  Johns Hopkins University. Using a pneumatic bioreactor, we demonstrate the assembly, operation, and performance of this single-use bioreactor system for the growth of mammalian cells. 

Video: Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological environment, both mechanical and chemical stimulation are required to maintain the proper development and functionality of arterial vessels1,2.
Bioreactor culture systems developed by our group are designed to support vessel regeneration within a precisely controlled chemo-mechanical environment mimicking that of native vessels. Our bioreactor assembly and maintenance procedures are fairly simple and highly repeatable3,4. Smooth muscle cells (SMCs) are seeded onto a tubular polyglycolic acid (PGA) mesh that is threaded over compliant silicone tubing and cultured in the bioreactor with or without pulsatile stimulation for up to 12 weeks. There are four main attributes that distinguish our bioreactor from some predecessors. 1) Unlike other culture systems that simulate only the biochemical surrounding of native blood vessels, our bioreactor also creates a physiological pulsatile environment by applying cyclic radial strain to the vessels in culture. 2) Multiple engineered vessels can be cultured simultaneously under different mechanical conditions within a controlled chemical environment. 3) The bioreactor allows a mono layer of endothelial cells (EC) to be easily coated onto the luminal side of engineered vessels for animal implantation models. 4) Our bioreactor can also culture engineered vessels with different diameter size ranged from 1 mm to 3 mm, saving the effort to tailor each individual bioreactor to fit a specific diameter size. 
The engineered vessels cultured in our bioreactor resemble native blood vessels histologically to some degree. Cells in the vessel walls express mature SMC contractile markers such as smooth muscle myosin heavy chain (SMMHC)3. A substantial amount of collagen is deposited within the extracellular matrix, which is responsible for ultimate mechanical strength of the engineered vessels5. Biochemical analysis also indicates that collagen content of engineered vessels is comparable to that of native arteries6. Importantly, the pulsatile bioreactor has consistently regenerated vessels that exhibit mechanical properties that permit successful implantation experiments in animal models3,7. Additionally, this bioreactor can be further modified to allow real-time assessment and tracking of collagen remodeling over time, non-invasively, using a non-linear optical microscopy (NLOM)8. To conclude, this bioreactor should serve as an excellent platform to study the fundamental mechanisms that regulate the regeneration of functional small-diameter vascular grafts.

Our group has developed a bioreactor culture system that mimics the physiological pulsatile stresses of the cardiovascular system to regenerate implantable small-diameter vascular grafts.

La ingeniería biológica basada en los injertos vasculares El uso de un biorreactor pulsátil

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological ...

Investigación

Bioingeniería

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

Cultivo de Células progenitoras neurales en un hidrogel de péptidos 3-dimensional de auto-montaje

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

Bacterial Detection & Identification Using Electrochemical Sensors

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

Bacterial Detección e Identificación Usando sensores electroquímicos

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

Diseño de un Carga mecánica Biaxial biorreactor para Ingeniería de Tejidos

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

Operation of a Benchtop Bioreactor

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to carefully control temperature, pH, and dissolved oxygen concentrations in particular makes them essential to efficient large scale growth and expression of fermentation products. This video will briefly describe the advantages of the fermentor over the shake flask. It will also identify key components of a typical benchtop fermentation system and give basic instruction on setup of the vessel and calibration of its probes. The viewer will be familiarized with the sterilization process and shown how to inoculate the growth medium in the vessel with culture. Basic concepts of operation, sampling, and harvesting will also be demonstrated. Simple data analysis and system cleanup will also be discussed.

Fermentors are used to increase culture yield and productivity of bioengineered cells. After screening multiple microbial or animal cell culture candidates in shake flasks, the next logical step is to increase the selected culture&#x2019;s biomass with the fermentor. This video demonstrates the setup and operation of a typical benchtop bioreactor system.

La operación de un biorreactor de sobremesa

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to ...

Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an attractive alternative to classic organ transplantation. This technology utilizes an organ&#39;s natural biological extracellular matrix (ECM) as a scaffold onto which autologous or stem/progenitor cells may be seeded and cultured in such a way that facilitates regeneration of the original tissue. The natural ECM is isolated by a process called decellularization. Decellularization is accomplished by treating tissues with a series of detergents, salts, and enzymes to achieve effective removal of cellular material while leaving the ECM intact. Studies conducted utilizing decellularization and subsequent recellularization of rodent lungs demonstrated marginal success in generating pulmonary-like tissue which is capable of gas exchange in vivo. While offering essential proof-of-concept, rodent models are not directly translatable to human use. Nonhuman primates (NHP) offer a more suitable model in which to investigate the use of bioartificial organ production for eventual clinical use.
The protocols for achieving complete decellularization of lungs acquired from the NHP rhesus macaque are presented. The resulting acellular lungs can be seeded with a variety of cells including mesenchymal stem cells and endothelial cells. The manuscript also describes the development of a bioreactor system in which cell-seeded macaque lungs can be cultured under conditions of mechanical stretch and strain provided by negative pressure ventilation as well as pulsatile perfusion through the vasculature; these forces are known to direct differentiation along pulmonary and endothelial lineages, respectively. Representative results of decellularization and cell seeding are provided.

Whole-organ decellularization produces natural biological scaffolds that may be used for regenerative medicine. The description of a nonhuman primate model of lung regeneration in which whole lungs are decellularized and then seeded with adult stem cells and endothelial cells in a bioreactor that facilitates vascular circulation and liquid media ventilation is presented.

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an ...

A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past studies have evaluated the performance of the reactor for the removal of COD1 and nitrogen species2-4 by heterotrophic and chemoautotrophic bacteria, respectively. The HDBR design eliminates the requirement for external flocculation/sedimentation processes while still yielding effluent containing low suspended solids. In this study, the HDBR is applied as a photobioreactor (PBR) in order to characterize the nitrogen removal characteristics of an algae-based photosynthetic microbial community. As previously reported for this HDBR design, a stable biomass zone was established with a clear delineation between the biologically active portion of the reactor and the recycling reactor fluid, which resulted in a low suspended solid effluent. The algal community in the HDBR was observed to remove 18.4% of total nitrogen species in the influent. Varying NH4+ and NO3- concentrations in the feed did not have an effect on NH4+ removal (n=44, p=0.993 and n=44, p=0.610 respectively) while NH4+ feed concentration was found to be negatively related with NO3- removal (n=44, p=0.000) and NO3- feed concentration was found to be positively correlated with NO3- removal (n=44, p=0.000). Consistent removal of NH4+, combined with the accumulation of oxidized nitrogen species at high NH4+ fluxes indicates the presence of ammonia- and nitrite-oxidizing bacteria within the microbial community.

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Here, the HDBR is successfully applied in a photobioreactor (PBR) configuration for the study of nitrogen metabolism by a mixed high density algal community.

Un biorreactor Novela de alta densidad de cultivo de diversas comunidades microbianas

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past ...

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based method is one of the simplest techniques for the mass production of cellular aggregates, but it does not protect the cultures against over-aggregation, which occurs when they gather at the bottom center of the culture vessel. To solve this problem, we developed an O-shaped dish and an O-shaped bag, neither of which contains a central region. Aggregates grown in either O-shaped culture vessel were noticeably more uniform in size than aggregates grown in conventional vessels. Histological analyses showed that aggregates in conventional culture dishes contained necrotic cores most likely caused by a poor oxygen supply. In contrast, aggregates that were grown in the O-shaped bag, even those with similar diameters to aggregates in conventional culture dishes, did not show necrotic cores. These results suggest that the O-shaped bag provides sufficient oxygen to the aggregates due to the oxygen permeability of the bag material. We, therefore, propose that this novel gas-permeable O-shaped culture bag is suitable for the mass production of uniform aggregates that are necessary in various biotechnological fields.

Here we present a protocol for using O-shaped vessels, specialized for suspension cultures of cellular aggregates, with orbital shaking. The HEK293 cells grown in this bag form more homogeneous aggregates than those grown in conventional culture vessels.

Un Orbital agitación cultivo de células mamíferas en vasos en forma de O para producir agregados uniforme

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based ...

Construction of CRISPR Plasmids and Detection of Knockout Efficiency in Mammalian Cells through a Dual Luciferase Reporter System

Although highly efficient, modification of a genomic site by the CRISPR enzyme requires the generation of a sgRNA unique to the target site(s) beforehand. This work describes the key steps leading to the construction of efficient sgRNA vectors using a strategy that allows the efficient detection of the positive colonies by PCR prior to DNA sequencing. Since efficient genome editing using the CRISPR system requires a highly efficient sgRNA, a preselection of candidate sgRNA targets is necessary to save time and effort. A dual luciferase reporter system has been developed to evaluate knockout efficiency by examining double-strand break repair via single strand annealing. Here, we use this reporter system to pick up the preferred xCas9/sgRNA target from candidate sgRNA vectors for specific gene editing. The protocol outlined will provide a preferred sgRNA/CRISPR enzyme vector in 10 days (starting with appropriately designed oligonucleotides).

Here, we present a protocol describing a streamlined method for the efficient generation of plasmids expressing both the CRISPR enzyme and associated single guide RNA (sgRNAs). Co-transfection of mammalian cells with this sgRNA/CRISPR vector and a dual luciferase reporter vector that examines double-strand break repair allows evaluation of knockout efficiency.

Construcción de Plásmidos CRISPR y Detección de Eficiencia Knockout en Células de Mamíferos a través de un Sistema Dual de Reporteros Luciferase

Although highly efficient, modification of a genomic site by the CRISPR enzyme requires the generation of a sgRNA unique to the target site(s) beforehand. ...