Name: culturing human pluripotent and neural stem cells in an enclosed cell culture system for basic and preclinical research
Uploaded: 2016-06-10T13:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research at JoVE.com

Los autores desean dar las gracias al personal de Biospherix por su ayuda en el aprendizaje del uso del sistema de cultivo celular XVIVO cerrado, especialmente Matt Freeman; el personal de Miles &amp; Kelley Construction Company, Inc., por su trabajo en la creación de la infraestructura de laboratorio, especialmente Russ Hughes; el personal del Hospital de Niños del Condado de Orange departamento de instalaciones y servicios de apoyo a su trabajo en la coordinación de la remodelación de laboratorio, especialmente Adam Lukhard y Devin Hugie; el personal del Hospital de Niños del Condado de Orange departamento de Sistemas de Información por su ayuda en la creación de la infraestructura de gestión de datos y el acceso a distancia, especialmente Viet Tran; Hospital de Niños del Condado de Orange Equipo de Gestión Ejecutiva por su constante apoyo del proyecto, especialmente al Dr. Maria Miñon y Brent Dethlefs. Este trabajo fue financiado por el Hospital de Niños del Condado de Orange y el Instituto Regenerativa de California Medicine a través TR3-05476 subvención a PHS. Todos los autores contribuyeron igualmente a este trabajo.

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	<li>Brick, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Autism+Spectrum+Disorders+Stem+Cell+Resource+at+Children's+Hospital+of+Orange+County:+Implications+for+Disease+Modeling+and+Drug+Discovery.">The Autism Spectrum Disorders Stem Cell Resource at Children's Hospital of Orange County: Implications for Disease Modeling and Drug Discovery.</a> Stem Cells Transl.Med. 3 (11), 1275-1286 (2014).</li><li>Nethercott, H. E., Brick, D. J., Schwartz, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+induced+pluripotent+stem+cells+by+lentiviral+transduction.">Derivation of induced pluripotent stem cells by lentiviral transduction.</a> Methods Mol.Biol. 767, 67-85 (2011).</li><li>Pistollato, 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=Hypoxia+and+HIF1alpha+repress+the+differentiative+effects+of+BMPs+in+high-grade+glioma.">Hypoxia and HIF1alpha repress the differentiative effects of BMPs in high-grade glioma.</a> Stem Cells. 27 (1), 7-17 (2009).</li><li>Pistollato, F., Chen, H. L., Schwartz, P. H., Basso, G., Panchision, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+tension+controls+the+expansion+of+human+CNS+precursors+and+the+generation+of+astrocytes+and+oligodendrocytes.">Oxygen tension controls the expansion of human CNS precursors and the generation of astrocytes and oligodendrocytes.</a> Mol.Cell Neurosci. 35, 424-435 (2007).</li><li>Raval, J. S., Koch, E., Donnenberg, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+monitoring+of+non-viable+airborne+particles+correlates+with+airborne+colonies+and+represents+an+acceptable+surrogate+for+daily+assessment+of+cell-processing+cleanroom+performance.">Real-time monitoring of non-viable airborne particles correlates with airborne colonies and represents an acceptable surrogate for daily assessment of cell-processing cleanroom performance.</a> Cytotherapy. 14 (9), 1144-1150 (2012).</li><li>Schwartz, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+of+stem+cell+therapies+for+neurological+diseases.">The potential of stem cell therapies for neurological diseases.</a> Expert.Rev.Neurother. 6, 153-161 (2006).</li><li>Schwartz, P. H., Brick, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+therapies+for+the+lysosomal+storage+diseases+-+the+quintessential+neurodegenerative+diseases.">Stem cell therapies for the lysosomal storage diseases - the quintessential neurodegenerative diseases.</a> Curr.Stem Cell Res.Ther. 3 (2), 88-98 (2008).</li><li>Schwartz, P. H., Brick, D. J., Nethercott, H. E., Stover, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traditional+human+embryonic+stem+cell+culture.">Traditional human embryonic stem cell culture.</a> Methods Mol.Biol. 767, 107-123 (2011).</li><li>Stover, A. E., 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=Process-based+expansion+and+neural+differentiation+of+human+pluripotent+stem+cells+for+transplantation+and+disease+modeling.">Process-based expansion and neural differentiation of human pluripotent stem cells for transplantation and disease modeling.</a> J Neurosci.Res. 91 (10), 1247-1262 (2013).</li><li>Stover, A. E., Schwartz, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+of+human+pluripotent+stem+cells+to+feeder-free+conditions+in+chemically+defined+medium+with+enzymatic+single-cell+passaging.">Adaptation of human pluripotent stem cells to feeder-free conditions in chemically defined medium with enzymatic single-cell passaging.</a> Methods Mol.Biol. 767, 137-146 (2011).</li><li>Yoshida, Y., Takahashi, K., Okita, K., Ichisaka, T., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+enhances+the+generation+of+induced+pluripotent+stem+cells.">Hypoxia enhances the generation of induced pluripotent stem cells.</a> Cell Stem Cell. 5 (3), 237-241 (2009).</li></ol>

The authors declare that they have no competing financial interests.

Las células cultivadas dentro de los CPF ver ningún cambio en las concentraciones de oxígeno o dióxido de carbono a medida que se mueven de incubadora a la cámara de procesamiento al microscopio cámara y la espalda. Es fundamental que las condiciones en cada cámara se adaptan a la incubadora particular en la que se mantienen las células antes de que las células se retiran de la incubadora. La atmósfera dentro del aparato es continuamente filtra-HEPA y es adaptable con respecto a las concentraciones de oxígeno y dióxido de carbono. Las células pueden ser cultivadas en concentraciones estándar para PSCs o NSCs, 5% y 9%, respectivamente; o concentraciones alternativas pueden ser elegidos para los diferentes tipos de células o para experimentos específicos. Así, el aparato se suministra con fuentes constantes de grado médico de oxígeno, dióxido de carbono, y nitrógeno (Figura 4). Los tres de estos gases son suministrados por los sistemas colectores de gases específicos que aseguran un abastecimiento constante. El aparato también se suministra con una mezcla de gas de calibración que consiste en10% (± 0,01%) de dióxido de carbono en oxígeno. Los sistemas múltiples están alojados fuera de la planta de producción de células y los gases se canalizan en la instalación a través del techo. El gas de calibración se encuentra dentro de la instalación. El aparato se suministra adicionalmente con vacío de la casa, también a través del techo. El uso de un sistema de control electrónico e inalámbrico unidades de transmisión, las presiones de salida de los colectores son monitoreados constantemente. En el caso de que cualquier presión cae fuera del rango, los operadores de las instalaciones de producción de células se llamaron por teléfono de forma automática y notificados de tal manera que la acción apropiada puede ser tomada. Los requisitos de alimentación del aparato se cumplen por seis dedicados 120 V circuitos que descienden desde el techo y conectados a generadores de emergencia del hospital para garantizar un suministro constante. El funcionamiento del aparato se controla mediante software en un equipo basado en PC alimentado a través de un sistema de alimentación ininterrumpida. Estos arreglos de poder y de la computadoragarantizar que el sistema funciona de forma continua, incluso en caso de un fallo del sistema de alimentación pública. El software que controla el aparato cuenta con una interfaz gráfica fácil de usar (Figura 1), que permite el control de las concentraciones de oxígeno y dióxido de carbono, así como las presiones de la temperatura, la humedad, y de cámara. Los valores de todos estos parámetros se registran continuamente para proporcionar un registro actualizado de todos los parámetros del aparato. Estos datos son una copia de seguridad en un servidor remoto todas las noches para proteger su integridad. El ordenador y el software se puede acceder de forma remota por los usuarios administrativos para evaluar y / o cambiar cualquier parámetro. Además, el equipo y el software se puede acceder de forma remota, lo que permite la evaluación interactiva de parámetros del aparato y solución de problemas con los usuarios locales. Una unidad de envío adicional de alarma está conectado al aparato de tal manera que los operadores de instalaciones de producción de células son notificados de cualquier condición de fuera de alcance del aparato. El acceso remoto capabilities permiten conectarse y evaluación de las características específicas de la condición de fuera de rango. El aparato está diseñado como un sistema modular tanto en una macro y micro un sentido. módulos de cultivo de células individuales, tales como incubadoras y cámaras de procesamiento, se pueden personalizar en cuanto a sus dimensiones y requisitos, así como en su diseño con respecto a la otra. Además, la mayoría de las funciones de control de los módulos individuales son en sí mismos modular de tal manera que los controladores individuales de gases atmosféricos, por ejemplo, puede reemplazarse fácilmente sin interrupción significativa al sistema. cámaras de tratamiento especializados, tales como uno para visualización y manipulación de los cultivos celulares al microscopio, se adaptan fácilmente al sistema. Tanto de contraste de fase y microscopio de fluorescencia están en el interior del sistema (Figura 6), de modo que las células se pueden teñir en vivo, y las colonias pueden ser diseccionados en las mismas condiciones atmosféricas como dentro de tél incubadoras. Enrutamiento de cables a través de los ojales selladas en las paredes laterales de la cámara de procesamiento permite a equipos tales como fuentes de alimentación y equipos para mantenerse fuera del aparato, por lo general en un carro (Figura 6). Las cámaras de tratamiento en las instalaciones de producción de células tienen un patrón de flujo de aire diferente que BSC convencionales. En BSC convencionales, el flujo de aire fluye hacia abajo desde una salida de escape central y se divide en dos corrientes separadas, que se recoge a continuación por dos entradas de aire diferentes en la parte de proa y popa del piso del gabinete. Por el contrario, la CPF tiene una sola abertura en la parte delantera del techo. El aire fluye hacia abajo y hacia la parte posterior de la cámara, donde luego se arrastra hacia arriba en una ventilación de entrada. Aunque la CPF es inherentemente muy limpio, este patrón de flujo de aire único significa que los técnicos tienen que ajustar ligeramente su técnica para reducir el riesgo de contaminación. Al igual que con un BSC convencional, un laboratorio trabajador should evitar colocar sus manos aguas arriba de las placas de cultivo de células abiertas y botellas de medios de comunicación. Sin embargo, la dirección que está aguas arriba ha sido alterado de la ACB El laboratorio planta de producción de células en sí es bastante estándar y viene equipado con un congelador -20 °, un congelador a -80ºC, a 4 ° C refrigerador, una centrífuga y un baño de agua. El laboratorio también tiene un fregadero con pedales de control para la operación de manos libres integrada. A fin de que este laboratorio para convertirse en una planta de producción de células clínico funcional, sin embargo, deben aún ser hechas varias modificaciones adicionales. En primer lugar, el aparato en sí mismo debe ser actualizado a tener la capacidad de supervisar los compuestos orgánicos volátiles, partículas, y las concentraciones de dióxido de cloro que se utiliza para la descontaminación. En segundo lugar, una cámara de procesamiento que contiene una máquina de FACS puede estar alojado y conectado al resto del aparato a través de un módulo de memoria intermedia. Esto permitirá la clasificación de células y purificación de trpoblaciones de células ansplantable en las condiciones ambientales adecuadas. Por último, todo el aparato debe ser alojado dentro de una pared blanda de sala limpia. Esto proporciona una Organización Internacional de Normalización clase (ISO) 8 entorno de los 5 aparatos. La alta esterilidad y la naturaleza controlada por ordenador de la ACB lo convierte en un sistema ideal para aplicaciones futuras con terapia basada en células y buenos procesos de fabricación. El riesgo de contaminación es mitigado en gran medida, pero lo más importante, las condiciones de la expansión celular se registran y archivada por el sistema de ordenador de forma automática. Las desviaciones en las concentraciones de gases, la temperatura, la humedad y todos los eventos de acceso en el sistema son rigurosamente documentados. Esto puede ser de gran ayuda en la investigación de problemas de calidad del producto. Sin embargo, todavía hay limitaciones. El uso de cualquier y todos los reactivos y materiales de construcción (por ejemplo, componentes de medios, pipetas, placas) se debe documentar por separado. Añadiritionally, hay una multitud de problemas potenciales (incluyendo muchas formas de error humano) que se vayan surgiendo y que no tienen ninguna relación con las variables documentadas por sistema de monitoreo de la ACB. Por lo tanto, la necesidad de personal altamente capacitado y documentación manual detallado de tareas permanece en su lugar.

Standard stem cell culture techniques suffer from several environmental constraints that place undue stresses on the cells and expose the cells to unacceptable risks of contamination. Among the stresses that cells may endure under standard cell culture conditions are precipitous changes in the levels of carbon dioxide and oxygen concentrations3,4. This occurs when the cells are moved from the incubator to the biosafety cabinet and/or microscope which may not be optimal for the cells. Previous studies have confirmed the advantages of culturing both pluripotent and neural stem cells in hypoxic conditions4,11, and for best results, these conditions need to be continuous. Moreover, risks of cellular contamination are higher as the laboratory environment and personnel impinge upon the cells at almost every step of their culture and manipulation. Traditional clean rooms comprise one effective method to greatly decrease contamination risks but they are expensive, have a large footprint and fail to address stressors related to carbon dioxide and oxygen concentrations. 
A cell production facility that can address both contamination risks and gas concentrations and that can be qualified to meet cGMP criteria9 provides high quality cells for basic science research as well as clinical applications1,6,7. Such a cell production facility consists, at a minimum, of the following components: a process chamber, which acts as a heated workspace for the feeding and manipulation of cell cultures; a laminar flow hood, for the initial sterilization of reagents, tubes, and tools; two buffering airlock chambers in between the hood and the process chamber; two cell culture incubators accessible from the process chamber; a microscope chamber adjacent to the process chamber; and finally, computer software to set and monitor the conditions within these modules. Using this basic infrastructure, a wide variety of tasks can be performed, such as standard feeding and passaging of pluripotent stem cells and multipotent neural stem cells, as well as more specialized methods like Sendai virus-based reprogramming, in vitro migration studies, and differentiation of neural stem cells for electrophysiological characterization.

<table><tbody><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;i&gt;Xvivo System&lt;/i&gt;</td><td>Biospherix</td><td>custom made</td><td></td></tr><tr><td>&lt;i&gt;Xvivo&lt;/i&gt; Software</td><td>Biospherix</td><td>version i.o.2.1.2.1</td><td></td></tr><tr><td>O&lt;sub&gt;2&lt;/sub&gt; Manifold</td><td>Amico</td><td>P-M2H-C3-S-U-OXY</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Manifold</td><td>Amico</td><td>M2H-C3-D-U-CO2</td><td></td></tr><tr><td>N&lt;sub&gt;2&lt;/sub&gt; Manifold</td><td>Western Innovator</td><td>CTM75-7-2-2-BM</td><td></td></tr><tr><td>Microscope with DP21 camera and fluorescence</td><td>Olympus Corporation</td><td>CKX41</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DMEM/F12 Glutamax</td><td>Life Technologies</td><td>10565-018</td><td></td></tr><tr><td>StemPro hESC Supplement</td><td>Life Technologies</td><td>A100006-01</td><td></td></tr><tr><td>Accutase</td><td>Millipore</td><td>SCR005</td><td></td></tr><tr><td>Phosphate-Buffered Sodium</td><td>Hyclone</td><td>9236</td><td></td></tr><tr><td>Fibroblast Growth Factor 2</td><td>R&amp;amp;D Systems</td><td>AFL233</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Protide</td><td>PP1130</td><td></td></tr><tr><td>Hank&#039;s-based Cell dissociation Buffer</td><td>Life Technologies</td><td>13150-016</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Life Technologies</td><td>21985-023</td><td></td></tr><tr><td>Epidermal Growth Factor</td><td>R&amp;amp;D Systems</td><td>AFL236</td><td></td></tr><tr><td>Oct-3/4 Antibody</td><td>Millipore</td><td>AB3209</td><td></td></tr><tr><td>TRA-1-60 Antibody</td><td>Millipore</td><td>MAB4260</td><td></td></tr><tr><td>SSEA4 Antibody</td><td>Millipore</td><td>MAB4304</td><td></td></tr><tr><td>BIT-9500 Serum Supplement</td><td>Stemcell Technologies</td><td>9500</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Consumable Supplies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2 ml Serological pipet</td><td>VWR</td><td>89130-894</td><td></td></tr><tr><td>5 ml Serological pipet</td><td>Olympus Plastics</td><td>12-102</td><td></td></tr><tr><td>10 ml Serological pipet</td><td>Olympus Plastics</td><td>12-104</td><td></td></tr><tr><td>25 ml Serological pipet</td><td>Olympus Plastics</td><td>12-106</td><td></td></tr><tr><td>50 ml Serological pipet</td><td>Olympus Plastics</td><td>12-107</td><td></td></tr><tr><td>6-well plate</td><td>Corning</td><td>353046</td><td></td></tr><tr><td>12-well plate</td><td>Corning</td><td>353043</td><td></td></tr><tr><td>T25 flask</td><td>TPP</td><td>90026</td><td></td></tr><tr><td>T-75 flask</td><td>TPP</td><td>90076</td><td></td></tr><tr><td>20 &amp;micro;l pipet tips</td><td>Eppendorf</td><td>22491130</td><td></td></tr><tr><td>200 &amp;micro;l pipet tips</td><td>Eppendorf</td><td>22491148</td><td></td></tr><tr><td>1,000 pipet tips</td><td>Eppendorf</td><td>22491156</td><td></td></tr><tr><td>Cryovials</td><td>Thermo Scientific</td><td>5000.102</td><td></td></tr><tr><td>70% ethanol</td><td>BDH</td><td>BDH1164-4LP</td><td></td></tr><tr><td>Sanimaster 4</td><td>Ecolab</td><td>65332960</td><td></td></tr><tr><td>Bleach</td><td>Clorox</td><td>A714239</td><td></td></tr></tbody></table>

culturing human pluripotent and neural stem cells in an enclosed cell culture system for basic and preclinical research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Este manuscrito describe con cierto detalle una instalación de producción de células, y los aspectos únicos de cultivo de células dentro de un sistema cerrado. La mayor parte de las instalaciones de producción de células comprende un aparato de medida, cerrado, cultivo celular, que tiene un tamaño reducido y puede alojarse fácilmente dentro de un 6 x 7,5 m 2 habitación (Figura 2). En ningún momento son las células dentro del aparato expuesto a personal de laboratorio o ambiente. El aparato consiste en varios módulos: una cámara de proceso, una campana de flujo laminar, 2 cámaras de esclusa de aire en el almacenamiento en búfer entre la campana y la cámara de proceso, dos incubadoras de cultivo de células, y una cámara de microscopio adyacente a la cámara de proceso (Figura 3). El sistema es controlado por un software que permite a las variables ambientales pueden controlar y monitorizar (Figura 1). El equipo que ejecuta este programa se encuentra en un sistema de alimentación ininterrumpida. Los gases se introducen en THsistema de correo por un conjunto de colectores para oxígeno, nitrógeno y dióxido de carbono (Figura 4). Gases para el dispositivo están alimentados por sistemas múltiples que tienen dos juegos de tanques cada uno - un conjunto activo y un conjunto de reserva. Cuando el conjunto activo se dibuja hacia abajo, el colector cambia automáticamente a los tanques de reserva de reemplazo para fijar y personal. El poder está en un sistema generador de respaldo. Pluripotentes y células madre neurales pueden ser cultivadas en condiciones hipóxicas en esta instalación utilizando protocolos desarrollados anteriormente 9, con las complicaciones añadidas inherentes al equipo novela. Las células madre pluripotentes se pueden derivar usando virus Sendai, usando anticuerpos-pluripotencia específico para identificar colonias totalmente transfectadas, que luego se aislaron y expandieron. (Figura 5A y B), A partir de estos iPSCs, las células madre neuronales y neuronas se pueden derivar usando la inhibición dual SMAD, y su fenotipo USI verificados<html> ng anticuerpos NSC-específicos (Figura 5C y D). <img alt="Figura 1" src="/files/ftp_upload/53685/53685fig1.jpg" /> Figura 1. Interfaz gráfica para la instalación de pilas de Producción. (A) La representación gráfica de la ACB es la pantalla predeterminada y coincide con el dibujo CAD se muestra en la Figura 3. Un clic del ratón sobre el Buffer Módulo 1 (2F en la Figura 1) abre su pantalla de control (B), donde se establecen los valores de O2 para que coincida con la cámara de procesamiento (3 en la Figura 3). Del mismo modo, al hacer clic en el Proceso de Sala o Incubadora 1 trae sus respectivas pantallas de control (C y D, respectivamente), donde los valores pueden ser cambiados o monitoreados según corresponda.<html> rget = &quot;_ blank&quot;&gt; Haga clic aquí para ver una versión más grande de esta figura. <img alt="Figura 2" src="/files/ftp_upload/53685/53685fig2.jpg" /> Figura 2. El mecanismo de producción de células. (A) El sistema como se ve desde una vista frontal derecha. Tenga en cuenta las conexiones eléctricas y de gas en el techo y el carro con los accesorios de microscopio y el ordenador a la derecha. (B) La campana de flujo laminar con las puertas de acceso a los módulos de tampón visto a la derecha. (C) La cámara de proceso con los dos incubadoras (puertas negras) visto en la parte trasera. (D) Una vista a través del lado derecho del sistema que muestra el microscopio y monitor con las puertas de las cámaras de amortiguamiento visto en la distancia. <a href="https://www.jove.com/files/ftp_upload/53685/53685fig2large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> t &quot;fo: keep-together.within-page =&quot; 1 &quot;&gt; <img alt="figura 3" src="/files/ftp_upload/53685/53685fig3.jpg" /> Figura 3. Un dibujo de CAD de la Línea de Producción de la célula. Todos los artículos que entran en el dispositivo se limpió primero con alcohol y secado al aire en la campana de flujo laminar (1). Los productos son entonces la transición a la atmósfera apropiada en el módulo de almacenamiento intermedio frontal (2F) antes de ser pasado en el módulo UCPC, o Proceso de cámara (3). Las células se cultivan en las dos incubadoras (4, 5). Tinción de células vivas, la recolección de la colonia, la evaluación de la morfología celular general y análisis de la migración se lleva a cabo en el módulo de la UPC, o cámara del microscopio (6). Por último, los residuos sale del sistema a través del módulo de amortiguación trasera (2R). <a href="https://www.jove.com/files/ftp_upload/53685/53685fig3large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> &lt;<html> / P&gt; <img alt="Figura 4" src="/files/ftp_upload/53685/53685fig4.jpg" /> Figura 4. Fuentes de gases y el poder. (A) El colector de CO 2 es una configuración de 4 x 4, ya que suministra todos los incubadores de laboratorio. (B) El colector de O 2 es una configuración y materiales de construcción solamente la instalación de producción de células 2 x 2. Nitrógeno para el sistema se genera en un vaporizador (C, justo por debajo de la señal de salida) unida a un colector de nitrógeno líquido (a la derecha), que también llena automáticamente cryofreezers (parte inferior izquierda). El colector es una configuración de 2 x 2 que se suministra por dos pares de tanques de nitrógeno líquido 160 L. CO2, O2 y N2 son suministrados desde el techo a través de válvulas de cierre (D). Casa de vacío también se suministra en esta ubicación. Ve justo detrás de las válvulas de cierre de gas de alimentación son un par de cables de alimentación que suministran energía al sistema.f = &quot;https://www.jove.com/files/ftp_upload/53685/53685fig4large.jpg&quot; target = &quot;_ blank&quot;&gt; Haga clic aquí para ver una versión más grande de esta figura. <img alt="Figura 5" src="/files/ftp_upload/53685/53685fig5.jpg" /> Figura 5. pluripotentes células madre derivadas de la hipoxia con el virus Sendai. (A) SC187-SF4-2I0-E3 iPSCs CMPI (nomenclatura que se encuentran en Stover et al. 9), contraste de fase 10x. (B) SC88.1-UH1-2I0 iPSCs vivo-manchada de marcado AF-594 Tra-1-60, dilución 1: 100 en los medios de comunicación. Todas las células iPS fueron transducidas en las instalaciones de producción de células en 5% de O2 con el virus Sendai. (C) El contraste de fases de NSC-UH0-2I0-SC68.1-M0S13 N2G6 derivados de IPSC-, que se cultiva en el 5% de O2 en la cámara de diapositivas. Las células fueron fijadas en paraformaldehído al 4% y se tiñeron con el anticuerpo primario anti-nestina humano, y el anticuerpo secundario Alex-488. Todas las barras de escala son en 1001;. M <a href="https://www.jove.com/files/ftp_upload/53685/53685fig5large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a>

Watch this Scientific Journal Video about Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research at JoVE.com

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

El cultivo humanas pluripotentes y células madre neurales en un sistema de cultivo celular cerrado para la investigación básica y preclínica

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

culturing-human-pluripotent-neural-stem-cells-an-enclosed-cell

Research

JoVE Journal

Developmental Biology

10.2K vistas.  Childrens Hospital of Orange County Research Institute. Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

Video: Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of interest. Nevertheless, an ideal humanized ex vivo feeder-free propagation method for hPSCs has not been developed; currently, additional exogenous substrates including basic fibroblast growth factor (bFGF), a master hPSC-sustaining factor, is added to all of culture media and synthetic substrata such as Matrigel or laminin are used in all feeder-free cultures. Recently, our group developed a simple and efficient protocol for the propagation of hPSCs using only conditioned media derived from the human placenta on a gelatin-coated dish without additional exogenous supplementation or synthetic substrata specific to hPSCs. This protocol has not been reported previously and might enable researchers to propagate hPSCs efficiently in humanized culture conditions. Additionally, this model obviates hPSC contamination risks by animal products such as viruses or unknown proteins. Furthermore, this system facilitates easy mass production of hPSCs using the gelatin coating, which is simple to handle, dramatically decreases the overall costs of ex vivo hPSC maintenance.

This protocol provides a simple and efficient way to propagate human pluripotent stem cells (hPSCs) using only conditioned media derived from the human placenta in a gelatin-coated dish without additional exogenous supplementation or hPSC-specific synthetic substrata.

Una Cultura Modelo Novela de Pluripotentes Stem Cell Propagación de gelatina en Media Placenta acondicionado

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of ...

Investigación

Biología del Desarrollo

Passaging Human Neural Stem Cells

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for therapeutic purposes as well as to explore many fundamental processes of human neural development and pathology.  This protocol presents a simple method of culturing and passaging hNSPCs in hopes of standardizing this technique and increasing reproducibility of human stem cell research.  The hNSPCs we use were isolated from cadaveric postnatal brain cortices by the National Human Neural Stem Cell Resource and grown as adherent cultures on flasks coated with fibronectin (Palmer et al., 2001; Schwartz et al., 2003).  We culture our hNSPCs in a DMEM:F12 serum-free media supplemented with EGF, FGF, and PDGF and passage them 1:2 approximately every seven days.  Using these conditions, the majority of the cells in the culture maintain a bipolar morphology and express markers of undifferentiated neural stem cells (such as nestin and sox2).

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro allows to investigate their utility as cell transplants for therapeutic purposes and to explore human neural development. This protocol presents a method of culturing and passaging hNSPCs in hopes of increasing reproducibility of human stem cell research.

Pases células madre neurales humanas

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for ...

Biología

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

Generación de las células troncales nerviosas de desechados Humanos tejido cortical fetal

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

Neurociencias

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

Derivación eficiente de los Derechos Humanos progenitores neuronales y neuronas de pluripotentes células madre embrionarias humanas mediante la inducción de moléculas pequeñas

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

Diferenciación neuronal eficiente utilizando el cultivo monocelular de células madre embrionarias humanas

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

Diferenciación inducida de células madre pluripotentes mediante un sistema de cultivo automatizado

An Automated Culture System for Maintaining and Differentiating Human-Induced Pluripotent Stem Cells

Human induced pluripotent stem cells (hiPSCs) with infinite self-proliferating ability have been expected to have applications in numerous fields, including the elucidation of rare disease pathologies, the development of new medicines, and regenerative medicine aiming to restore damaged organs. Despite this, the social implementation of hiPSCs is still limited. This is partly because of the difficulty of reproducing differentiation in culture, even with advanced knowledge and sophisticated technical skills, due to the high sensitivity of iPSCs to minute environmental changes. The application of an automated culture system can solve this issue. Experiments with high reproducibility independent of a researcher's skill can be expected according to a shared procedure across various institutes. Although several automated culture systems that can maintain iPSC cultures and induce differentiation have been developed previously, these systems are heavy, large, and costly because they make use of humanized, multi-articulated robotic arms. To improve on the above issues, we developed a new system using a simple x-y-z axis slide rail system, allowing it to be more compact, lighter, and cheaper. Furthermore, the user can easily modify parameters in the new system to develop new handling tasks. Once a task is established, all the user needs to do is prepare the iPSC, supply the reagents and consumables needed for the desired task in advance, select the task number, and specify the time. We confirmed that the system could maintain iPSCs in an undifferentiated state through several passages without feeder cells and differentiate into various cell types, including cardiomyocytes, hepatocytes, neural progenitors, and keratinocytes. The system will enable highly reproducible experiments across institutions without the need for skilled researchers and will facilitate the social implementation of hiPSCs in a wider range of research fields by diminishing the obstacles for new entries.

Autor destacado: Automatización de la cultura iPSC para mejorar la reproducibilidad 

Here, we present a protocol for an automated cell culture system. This automated culture system reduces labor and benefits the users, including researchers unfamiliar with handling induced pluripotent stem (iPS) cells, from the maintenance of iPS cells to differentiation into various types of cells.

Un sistema de cultivo automatizado para mantener y diferenciar células madre pluripotentes inducidas por humanos

Human induced pluripotent stem cells (hiPSCs) with infinite self-proliferating ability have been expected to have applications in numerous fields, ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

Culturas alternativas para Pluripotentes Stem Cell Production, mantenimiento y análisis genético

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Cultivos celulares mixtos de cerebro de ratón embrionario para estudiar la infección y la inmunidad innata

Establishing Mixed Neuronal and Glial Cell Cultures from Embryonic Mouse Brains to Study Infection and Innate Immunity

Models of the central nervous system (CNS) must recapitulate the complex network of interconnected cells found in vivo. The CNS consists primarily of neurons, astrocytes, oligodendrocytes, and microglia. Due to increasing efforts to replace and reduce animal use, a variety of in vitro cell culture systems have been developed to explore innate cell properties, which allow the development of therapeutics for CNS infections and pathologies. Whilst certain research questions can be addressed by human-based cell culture systems, such as (induced) pluripotent stem cells, working with human cells has its own limitations with regard to availability, costs, and ethics. Here, we describe a unique protocol for isolating and culturing cells from embryonic mouse brains. The resulting mixed neural cell cultures mimic several cell populations and interactions found in the brain in vivo. Compared to current equivalent methods, this protocol more closely mimics the characteristics of the brain and also garners more cells, thus allowing for more experimental conditions to be investigated from one pregnant mouse. Further, the protocol is relatively easy and highly reproducible. These cultures have been optimized for use at various scales, including 96-well based high throughput screens, 24-well microscopy analysis, and 6-well cultures for flow cytometry and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. This culture method is a powerful tool to investigate infection and immunity within the context of some of the complexity of the CNS with the convenience of in vitro methods.

Autor destacado: Establecimiento de cultivos mixtos de células neuronales y gliales a partir de cerebros embrionarios de ratón para estudiar la infección y la inmunidad innata  

This protocol presents a unique way of generating central nervous system cell cultures from embryonic day 17 mouse brains for neuro(immuno)logy research. This model can be analyzed using various experimental techniques, including RT-qPCR, microscopy, ELISA, and flow cytometry.

Establecimiento de cultivos mixtos de células neuronales y gliales a partir de cerebros embrionarios de ratones para estudiar la infección y la inmunidad innata

Models of the central nervous system (CNS) must recapitulate the complex network of interconnected cells found in vivo. The CNS consists primarily of ...

El cultivo humanas pluripotentes y células madre neurales en un sistema de cultivo celular cerrado para la investigación básica y preclínica

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