1. Protocolo para el uso de varios frascos de cultivo celular <ol><li> Preparación de los buques y la adición de la suspensión de células <ol><li> Preparar el medio de crecimiento de las células según sea necesario. </li><li> Alinee Multi-frasco vertical sobre su lado con la tapa hacia arriba sobre la superficie de trabajo (campana estéril de flujo laminar). <ol><li> Estos frascos están disponibles en tres formatos y de 5 capas y el uso de un similar patrón de flujo de trabajo como un frasco T-175. </li></ol></li><li> Aflojar y remover las tapas. Añadir la cantidad necesaria de pre-calentado medio en un matraz con una pipeta de 50 o 100 ml o por infusión. <ol><li> Pipetas que son ≤ 10 ml puede llegar al fondo de la vasija, cuando varios frascos se colocan verticalmente con la tapa hacia arriba. ≥ 10 ml pipetas pueda llegar hasta el buque inmediatamente pasado el logotipo en el frasco de Multi-, proporcionando así un portal conveniente de añadir un mayor volumen de medios de comunicación para los buques. </li></ol></li><li> Para evitar la propagación del medio, permiten flujo de líquido a flómo lo largo de la pared interna de la tapa del Multi-frasco (logo-lado). </li><li> Añadir suspensión celular de una reserva de células concentradas en un medio de crecimiento a través de la capa superior con una pipeta de 10 ml y frascos de tapa. Consejos: - Transporte Multi-frascos en un carro al sitio de la incubadora y realizar los pasos restantes. - La densidad de siembra de células puede variar dependiendo del tipo de célula, media y duración de la cultura necesita. Comience con la densidad de siembra y el volumen de los medios de comunicación al que se utiliza en el estándar T-175 frascos y se multiplica por 3 o 5 en función del frasco de Multi-formato utilizado. </li></ol></li><li> La mezcla de células <ol><li> Mix posición: Sostenga el frasco en posición vertical Multi-con el logotipo de frente a usted y a su vez hacia la izquierda a un ángulo de 45 ° con el puerto de mezcla frente a usted. </li><li> La celebración en el mismo ángulo, con cuidado de inclinación Multi-frasco de adelante hacia atrás (en el cuello lejos de usted) hasta que el líquido en la capa superior drena totalmente downwards a través del puerto de mezcla. Giran en torno a la mezcla de babor. </li><li> Del mismo modo, agite suavemente Multi-frasco de atrás hacia adelante (el cuello hacia usted) hasta que esté medio drena por completo de la capa de abajo hacia arriba a través del puerto de mezcla. Repita los pasos 1.2.2 y 1.2.3, una vez más para asegurar una mezcla adecuada. </li><li> Lleva Multi-frasco de nuevo a la posición de mezcla (paso 1.2.1) y vaya al paso 1.3 </li><li> Por otra parte, la suspensión de células se pueden preparar externa del frasco Multi-y la suspensión de células se puede agregar a la embarcación con una pipeta o suave vertido. <ol><li> Puerto de combinación permite en recipiente de mezcla de células con los medios de comunicación y elimina la necesidad de hacer grandes cantidades de células en suspensión externa. </li><li> También permite la igualdad de medios a través de las capas de la Multi-frasco </li></ol></li></ol></li><li> El equilibrio de fluidos <ol><li> Luego de mezclar / adición de la suspensión celular, lugar Multi-frasco vertical sobre una superficie plana para igualar el volumen de líquido ecuaciónlly en todas las capas. </li></ol></li><li> Separación líquido en cada capa <ol><li> Sostenga el frasco de Multi-con el logotipo hacia arriba y gire hacia la derecha a un ángulo de 45 ° a la partición del líquido en cada una de las capas. Consejo: - Para obtener mejores resultados, es importante utilizar una superficie plana para los pasos 1.3-1.4 </li></ol></li><li> Transporte <ol><li> Una vez que los medios de comunicación que contiene la suspensión de células se divide, transporte Multi-frasco en el mismo ángulo de 45 ° (a la derecha) como en el paso 1.4.1 con los medios de comunicación del puerto mezcla en incubadora. Esta posición también es adecuado cuando la eliminación de frascos de la incubadora para su visualización en un microscopio. </li></ol></li><li> La colocación de Multi-frasco en incubadora útil <ol><li> La celebración de Multi-frasco en el ángulo de 45 ° (a la derecha, lejos de la mezcla de un puerto), gire suavemente hacia abajo en posición horizontal en la superficie de la incubadora (utilizando la esquina del puerto como una mezclapivote). Coloque Multi-frasco con el logotipo hacia arriba. <ol><li> Falcon Multi-frasco de diseño permite a los barcos que se apilan y se las mantiene en su lugar por una costilla resistente de apilamiento. </li></ol></li></ol></li><li> Distribución de células y los reactivos <ol><li> Después de la colocación de Multi-frasco de planos en la superficie de trabajo, con suavidad hacia adelante y atrás y de lado a lado para distribuir uniformemente sobre las superficies de las células de la cultura teniendo cuidado de no derramar el líquido de cada capa. Pila de frascos. <ol><li> Este frasco es de un material ópticamente transparente y las células de la última capa se pueden ver fácilmente en un microscopio. </li></ol></li></ol></li><li> Medios de intercambio <ol><li> Aspirar mientras se inclina el frasco Multi-a la izquierda (mezcla de babor) con el logotipo hacia usted. </li><li> A continuación, la inclinación, el frasco de Multi-a la derecha, sigue aspirar todos los medios de comunicación residual. </li><li> Agregue la cantidad adecuada de los medios de comunicación y siga los pasos 1.3-1.7 </li></ol></li></ol> 2.La recolección de células multi-Frascos <ol><li> Para disociar las células, se alinean varios frascos vertical y llevar a cada uno a la mezcla de posición (paso 1.2.1). Invierta suavemente frascos con cuello de frente a usted a fin de que los medios de comunicación para drenar la capa superior de la Multi-Flask. </li><li> Inserte un 5 o 10 ml de la punta de aspiración a través del cuello hasta llegar a nivel de líquido cerca del puerto de mezcla. Aspirar medio agotado. </li><li> Añadir disociación reactivo / tampón de lavado y llevar a la posición de la mezcla como en el paso 1.2.1, a continuación, proceder y seguir los pasos 1.3 a 1.7. Neutralizar con medio de cultivo / agente de neutralización y se vierte la suspensión de células en un tubo receptor o invertir frasco de manera que las células de drenaje para la capa superior de la nave y recoger las células con una pipeta de 10 ml. Consejo: Para mejorar la recuperación de las células o de los reactivos, llevar Multi-frasco para mezclar la posición (paso 1.2.1), invertir con el cuello hacia el operador para permitir el drenaje completo de los medios de comunicación de Al<html> l capas de la capa superior. Luego, incline Multi-frasco de las agujas del reloj a 45 º (distancia desde el puerto de mezcla), mientras que el frasco de Multi-sigue siendo invertida. Utilice una pipeta (1-10 ml) para recoger el resto de los reactivos. </li></ol> 3. Volumen de trabajo recomendado en Falcon Multi-Frascos Medios de cultivo 3-Capa: 75-150 ml por Multi-frasco 5-Capa: 125-250 ml por Multi-frasco La disociación del agente 3-Capa: ≥ 15 mL por Multi-frasco 5-Capa: ≥ 25 ml por frasco de Multi- Sugerencia: Comience con un volumen de medio utilizado en la norma T-175 frascos y se multiplica por 3 o 5 veces dependiendo del frasco de Multi-formato de evaluación para que ml por unidad de superficie sigue siendo el mismo. 4. Los resultados representativos: 1. Diseño de Falcon Multi-frasco files/ftp_upload/3418/3418fig1.jpg &quot;/&gt; . Figura 1: Multi-frasco de cultivo celular barcos están disponibles en 3 - y 5-capa formato apilable para ampliación de las células proporcionando 525 y 875 cm 2 de superficie de crecimiento, respectivamente. Pipeta de acceso facilita la adición y la eliminación de las células y los reactivos en y fuera de la nave. Presencia de la mezcla de puertos permite la rápida en recipiente de mezcla y ecualización de los medios de comunicación en todas las capas de la Multi-frasco. 2. Rendimiento celular utilizando Multi-Frasco: Estas embarcaciones están disponibles en 3 capas y formatos de 5 capas que corresponden a 3 y 5 veces la superficie de la T-175 frascos. En consecuencia, ≥ 3 y 5 veces (130 ± 6,8 x 10 6 y 218 ± 23.6 x 10 6 células, respectivamente), el número de células BHK-21 (riñón de hamster) las células fueron cultivadas y se recuperó de Multi-frasco en comparación con el T-175 frascos (43,2 ± 3,5 x 10 6 células; Fig.2a). Celular rendimiento por uárea de superficie de nit fue equivalente en 3 - y 5-capa y multi-frascos T-175 frascos de BHK-21, LNCaP (adenocarcinoma de próstata humana línea celular), Hep-G2 (línea celular humana hepatocarcinoma), Ecopack 2-293 (humanos línea de células renales) cultivadas por un período de 48-96h (Fig.2b) en medios de cultivo (35 ml por cada capa) según lo recomendado por el fabricante de células (ATCC, Sigma y / o Clontech). Las células fueron enumerados en un sistema automatizado de Vi-contador de células (1). <img alt="Figura 2A" src="/files/ftp_upload/3418/3418fig2A.jpg" /> Figura 2A: de tres y cinco veces el número de células BHK-21 se cultivaron y se recuperó de 3 - y 5-capa Multi-Frascos en comparación con el T-175 frascos. Rendimiento esperado se determinó que el rendimiento en media de células de control T-175 frascos multiplicado por tres veces y cinco para el 3 - y frascos Multi-5-capa, respectivamente (n = 4 frascos / formato). <img alt="Figura 2B" src="/files/ftp_upload/3418/3418fig2B.jpg" /><br/ 2B&gt; Figura: el rendimiento de la célula por cm 2 fue equivalente en 3 - y 5-capa y multi-frascos T-175 frascos de BHK-21, LNCaP, y las células HepG2 EcoPack2-293. Cada barra representa la media de 4 a 6 frascos. Células BHK-21 (11.000 células/cm2) se cultivaron durante 72 horas, las células LNCaP (20.000 células/cm2) y EcoPack2-293 células (~ 35.000 células/cm2) se cultivaron durante 96 horas y las células HepG2 (25.000 células / cm 2 ) se cultivaron durante 48 horas antes de la cosecha. 3. Medios de distribución entre las capas de Multi-frasco Medio de cultivo celular (DMEM, Invitrogen) a cinco capas múltiples frascos (250 buques mL/5-layer) y se separó en capas de acuerdo con el protocolo descrito anteriormente. Distribución de los medios de comunicación se midió mediante la perforación de agujeros en cada capa y los medios de comunicación bombea desde las capas individuales. Peso del fluido recuperado de cada capa se encontró que era relativamente uniforme de capa a capa, como se muestra en la Fig. 3. Son los siguientes: 510,8 ± 0,73, 50,3 ± 0,58, 50,21 ± 0,13, 49,88 ± 0,35, 49,45 ± 0,37 (g). <img alt="Figura 3" src="/files/ftp_upload/3418/3418fig3.jpg" /> Figura 3. Distribución de los medios de comunicación uniforme en cada una de las cinco capas de una capa de 5-Multi-Flask. Medio de cultivo celular fue introducido en varios matraces (250 buques ml/5-layer), equilibrado y dividido en capas individuales. Los medios de comunicación se bombea a través de agujeros perforados en las capas individuales y el peso de fluido se registró a partir de cada capa (n = 6 frascos). 4. Celular distribución entre las capas de Multi-frasco Las células pueden ser añadidos y mezclados dentro del frasco de Multi-. Nos distribución simulada de las células entre las capas de Multi-frasco con gotas (10μm; Polysciences Inc.) de tamaño similar a las células. Suspensión de bolas se añadió en el recipiente Frasco de Multi-utilizando una pipeta de 10 ml a través de la capa superior y se mezcla con los medios de comunicación en el barco, como described en el protocolo anterior. Distribución de bolas se midió por los agujeros de perforación en cada capa y el líquido que contiene suspensión de bolas se bombea desde las capas individuales. Concentración de bolas recuperadas de cada capa se leyó en un contador Coulter y grabado. Se muestra a continuación son equivalentes entre capas distribuciones de grano de 3 capas múltiples frascos (Fig.4a). La mezcla de puertos permite la distribución homogénea de las células y los reactivos entre las capas Multi-Flask. <img alt="Figura 4A" src="/files/ftp_upload/3418/3418fig4A.jpg" /> Figura 4: Distribución de bolas en cada una de las tres capas de una capa de 3 Multi-Flask. Una suspensión de cuentas (3,6 x10 6 / ml) se añadió al medio se distribuye en varios matraces (suspensión de perlas: el volumen de los medios de comunicación es de 1:10, vol: vol) y se mezcla, seguido por el equilibrio y los pasos partición utilizando el protocolo descrito. Concentración de bolas recuperadas de cada capa (medio bombeado a través de agujeros perforados en cada capa) fue leído en un recuento de Coulterer y grabado. Se muestra a continuación son equivalentes entre capas distribuciones de cuentas en 3 - Multi-capa de Frascos (n = 5 frascos) A continuación se muestran imágenes representativas de los patrones de coloración Ecopack 2-293 de las células cultivadas a la confluencia&gt; 80% en tres capas múltiples frascos de medios de cultivo suplementados. Monocapas de células fueron fijadas y teñidas con cristal violeta y Multi-frasco capas fueron cortadas y las imágenes escaneadas (2). Tenga en cuenta, los patrones de células fue homogénea en todas las capas de Multi-Frasco (Fig.4B). Se obtuvieron resultados similares con múltiples tipos de células evaluados (datos no mostrados). <img alt="Figura 4B" src="/files/ftp_upload/3418/3418fig4B.jpg" /> Figura 4B: Esta figura ilustra el crecimiento de células homogéneas entre las capas de varios frascos. Ecopack-2-293 células cultivadas a la confluencia&gt; 80% en 3 capas múltiples frascos y T-175 fueron fijadas y teñidas con cristal violeta. Frasco de múltiples vasos fueron cortadas y cada capa manchada escaneado fue. 5 El suministro de aire en el Multi-Flask.: El análisis de los medios de comunicación dedicado al uso de Bioprofile FLEX analizador (3,4) (Nova Biomedical) de EcoPack2-293 células cultivadas durante 96 horas no mostraron diferencias en la saturación del aire de las células cultivadas en varios frascos de 5 capas vs T-175 frascos (81.03 ± 1,9 vs 83,4 ± 5,8% del ambiente O 2). <img alt="Figura 5" src="/files/ftp_upload/3418/3418fig5.jpg" /> Figura 5: saturación del aire (ambiente% O 2) de los medios de comunicación pasaron fueron similares en los medios de comunicación pre-mezclado de varios frascos de 5 capas vs T-175 frascos. EcoPack2-293 células se sembraron a una densidad de 35.000 células / cm 2 y se cultivan durante 96 horas antes a los medios de análisis (n = 3 frascos).

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Todos los autores son empleados de la empresa Becton Dickinson, segmento Biosciences, Discovery diagnóstico, de laboratorios de la Unidad.

El presente estudio demuestra el aumento de la productividad que el diseño del frasco de Multi-ofrece a los investigadores. Si bien es importante que siga los pasos descritos anteriormente para un rendimiento óptimo cuando se utilizan varios matraces, son pocos los pasos críticos en este protocolo que se consideran más esenciales. Estos incluyen: (i) la mezcla de las células y los reactivos a través del puerto de mezcla en el recipiente (ii) el transporte de Multi-frasco en un ángulo de 45 ° hacia la derecha después de la partición de líquido en cada una de las capas (iii) por el Multi-frasco después de la partición en el plano incubadora. El uso adecuado de Multi-frasco lleva a la producción de una población celular homogénea dentro de cada buque que se cultiva en un ambiente que es congruente con el T-175 frascos (5). Estas embarcaciones ofrecen las células 3 y 5 veces más en un tamaño similar a la del frasco T-175. El recipiente 3 y 5 capas proporciona 525 y 875cm 2 de área de crecimiento, respectivamente, y ofrecen el espacio y mano de obra savings a los usuarios. Cultivo de Tejidos Tratamiento de la superficie es comparable a frascos estándar lo que permite ampliación sin necesidad de volver a la optimización de las condiciones actuales la cultura o comprometer la calidad, la homogeneidad o la realización de las células (6, 7). Esto también proporciona la comparabilidad con los datos recogidos anteriormente. Estos vasos pueden ser también revestidos con reactivos tales como colágeno, fibronectina, poli-D-lisina para proporcionar un sustrato especializado para la fijación, crecimiento y diferenciación de ciertos tipos celulares como los hepatocitos 8, 9 kertainocytes, las células madre cultivadas en medio libre de suero formulaciones. Soluciones de revestimiento se puede quitar con una retención mínima de líquido residual con el fin de reducir el desperdicio de reactivos de gran valor, así como la eliminación eficaz de solución de revestimiento que antes el cultivo de células. El volumen recomendado del papel óptimo de las células de cultivo en varios frascos de rango 0,142 a 0,287 ml / cm 2, que se traducen en 25 a 50 ml por cada capa. A diferencia de otro frasco de varias capas, tsu producto ofrece una combinación de puerto en el vaso que le permite rápida en recipiente de mezcla, así como una distribución uniforme de las células y los reactivos dentro y entre las capas de cada buque. Diversas líneas celulares, cultivos primarios y las células madre se han ampliado de manera eficiente el uso de varios frascos. Estos vasos sanguíneos son particularmente ventajoso en aplicaciones que requieren un gran número de células, como en el cribado de alto-, la producción de vacunas, transfecciones vector viral y terapia celular. Ahorro de tiempo, espacio, trabajo y generación de residuos se reducen las ganancias fundamentales de la Petaca Multi-contra las culturas tradicionales en una sola capa de vasos sanguíneos. Podemos cultura aproximadamente tres veces el número de células recolectadas a partir de 5, T-175 frascos en el mismo espacio con 3, 5 capas múltiples frascos. Esta ventaja en ahorro de espacio no se limita sólo a la T-175, pero también se puede extender a otros buques: un aparato giratorio botella estándar que encaja en común incubadoras de laboratorio casas ~ 4 roller botellas (2200 ml) cada una de ellas ofrece 850cm 2 de superficie. En la misma zona, aproximadamente 20, de 5 capas de varios frascos pueden ser mantenidos por lo tanto proporciona cinco veces la superficie de crecimiento de las células de cultivo. Por otra parte, con un aumento en el &quot;Go-Green&quot; la conciencia, las opciones para reducir la generación de residuos es altamente deseable. A este respecto, hay un 38% (5, T-175 frascos de peso ~ 640g, mientras que uno, de 5 capas Multi-frasco pesa ~ 400 g) disminución en la generación de residuos a través de múltiples cofres en comparación con el T-175 frascos y estas ventajas conducen a la disminución de almacenamiento de residuos y los costes de eliminación y el resultado en un ahorro económico para el usuario.

<table border="1"><tbody><tr><td> Nombre del reactivo / Equipo </td><td> Empresa </td><td> Catálogo # </td><td> Comentarios </td></tr><tr><td> 3-capa de Cultivo de Tejidos tratados con 525cm 2 </td><td> BD Biosciences </td><td> 353143 </td><td rowspan="21"> <a href="http://www.bdbiosciences.com/cellculture/multiflasks/index.jsp">BD Biosciences cultivo celular - BD Falcon Multi-Frascos</a> </td></tr><tr><td> De 5 capas de Cultivo de Tejidos tratados con 875cm 2 </td><td> BD Biosciences </td><td> 353144 </td></tr><tr><td> 100 ml pipeta </td><td> BD Biosciences </td><td> 357600 </td></tr><tr><td> Pipeta de 10 ml </td><td> BD Biosciences </td><td> 357551 </td></tr><tr><td> 5 ml de aspiración pipeta </td><td> BD Biosciences </td><td&gt; 357501 </td></tr><tr><td> 50 ml tubo de polipropileno cónico </td><td> BD Biosciences </td><td> 352070 </td></tr><tr><td> T-175 frasco de cultivo de tejidos tratados con </td><td> BD Biosciences </td><td> 353028 </td></tr><tr><td> Gramo de cristal violeta </td><td> BD </td><td> 212525 </td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 11885 </td></tr><tr><td> GMEM </td><td> Sigma </td><td> G5154 </td></tr><tr><td> RPMI-1640 </td><td> ATCC </td><td> 30-2001 </td></tr><tr><td> MEM </td><td> ATCC </td><td> 30-2003 </td></tr><tr><td> Tripsina-EDTA </td><td> Lonza </td><td> CC-5012 </td></tr><tr><td> El suero fetal bovino </td><td> Invitrogen </td><td> 16000-044 </td></tr><tr><td> Triptosa fosfato caldo </td><td> Sigma </td><td> T8159 </td&gt; </tr><tr><td> Células BHK-21 </td><td> Sigma </td><td> 85011433 </td></tr><tr><td> Hep-G2, LNCaP </td><td> ATCC </td><td></td></tr><tr><td> Ecopack 2-293 </td><td> Clontech </td><td></td></tr><tr><td> Paraformaldehído </td><td> Microscopía electrónica de Ciencias </td><td> 15710 </td></tr><tr><td> Poliestireno bolas </td><td> Polysciences Inc. </td><td> 24628-20 </td></tr><tr><td> Bio-Flex Analizador de Perfil </td><td> Nova Biomedical </td><td></td></tr></tbody></table>

scale-up of mammalian cell culture using a new multilayered flask

Un número creciente de aplicaciones basadas en Cell requieren grandes cantidades de células. El uso de una sola capa frascos T, que son adecuadas en pequeña escala, la expansión, puede llegar a ser complicado, laborioso y requiere mucho tiempo, cuando un gran número de células se necesitan. Para satisfacer esta necesidad, la realización de un nuevo multi-capas recipiente de cultivo de células para facilitar la escala fácil de las células de capa única frascos T serán discutidos. Los frascos de prueba están disponibles en 3 - y el formato de 5 capas y permitir la recuperación de la cultura y completa de tres y cinco veces el número de células, respectivamente, en comparación con el T-175 frascos. Una característica clave de la BD Multi-frasco es un puerto de mezcla / de equilibrio que permite una rápida en recipiente de mezcla, así como una distribución uniforme de las células y los reactivos dentro y entre las capas de cada buque y constantemente producen las células que pueden ser cultivados en un ambiente que es congruente con el T-175 frascos. El diseño de estos frascos multi-también permite cacceso pipeta onvenient para la adición de reactivos y las células directamente en los frascos, así como la recuperación eficiente de valiosas células y los reactivos y reduce el riesgo de contaminación por el vertido. Para aplicaciones donde se prefiere verter más de pipeteado, el diseño permite una retención mínima de líquido residual con el fin de reducir el desperdicio de valiosas células y los reactivos.

Watch this Scientific Journal Video about Scale-Up of Mammalian Cell Culture using a New Multilayered Flask at JoVE.com

Scale-Up of Mammalian Cell Culture using a New Multilayered Flask

Células juegan un papel fundamental y creciente en la investigación y el descubrimiento y desarrollo de nuevos tratamientos. Con esta creciente necesidad de un mayor número de células que necesitamos maneras más eficientes y eficaces para el cultivo y la recolección de células apego dependiente. Un matraz de múltiples capas con las características adecuadas puede servir para este propósito.

La ampliación de los cultivos celulares de mamíferos con un frasco de varias capas de Nueva

A growing number of cell-based applications require large numbers of cells. Usage of single layer T-flasks, that are adequate during small-scale ...

scale-up-of-mammalian-cell-culture-using-a-new-multilayered-flask

Research

JoVE Journal

Biology

37.5K vistas.  BD Biosciences. Células juegan un papel fundamental y creciente en la investigación y el descubrimiento y desarrollo de nuevos tratamientos. Con esta creciente necesidad de un mayor número de células que necesitamos maneras más eficientes y eficaces para el cultivo y la recolección de células apego dependiente. Un matraz de múltiples capas con las características adecuadas puede servir para este propósito.

Video: Scale-Up of Mammalian Cell Culture using a New Multilayered Flask

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

A largo plazo de imágenes usando las células de mamíferos Wide-Field Microscopía

Investigación

Biología

Ole Isacson: Development of New Therapies for Parkinson's Disease

Ole Isacson gives a concise overview of Parkinsons's disease, its causes, therapeutic strategies, and advances in Parkinson's research.

Ole Isacson: desarrollo de nuevas terapias para la enfermedad de Parkinson

Laser Capture Microdissection of Mammalian Tissue

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or formalin-fixed, paraffin-embedded tissue sections. LMD techniques rely on a thermo labile membrane placed either on top of, or underneath, the tissue section. In one method, focused laser energy is used to melt the membrane onto the underlying cells, which can then be lifted out of the tissue section. In the other, the laser energy vaporizes the foil along a path "drawn" on the tissue, allowing the selected cells to fall into a collection device. Each technique allows the selection of cells with a minimum resolution of several microns. DNA, RNA, protein, and lipid samples may be isolated and analyzed from micro-dissected samples. In this video, we demonstrate the use of the Leica AS-LMD laser microdissection instrument in seven segments, including an introduction to the principles of LMD, initializing the instrument for use, general considerations for sample preparation, mounting the specimen and setting up capture tubes, aligning the microscope, adjusting the capture controls, and capturing tissue specimens. Laser-capture micro-dissection enables the investigator to isolate samples of pure cell populations as small as a few cell-equivalents. This allows the analysis of cells of interest that are free of neighboring contaminants, which may confound experimental results.

Láser microdisección de captura de tejidos de mamíferos

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or ...

Development of New Therapeutic Applications Using Microfluidics

Desarrollo de nuevas aplicaciones terapéuticas con microfluídica

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

Método para la Cultura de primeros embriones del polluelo ex vivo (Nueva Cultura)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Determining Cell Number During Cell Culture using the Scepter Cell Counter

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and accuracy. Cell counts are important for monitoring cell health and proliferation rate, assessing immortalization or transformation, seeding cells for subsequent experiments, transfection or infection, and preparing for cell-based assays. It is important that cell counts be accurate, consistent, and fast, particularly for quantitative measurements of cellular responses.
Despite this need for speed and accuracy in cell counting, 71% of 400 researchers surveyed1 who count cells using a hemocytometer. While hemocytometry is inexpensive, it is laborious and subject to user bias and misuse, which results in inaccurate counts. Hemocytometers are made of special optical glass on which cell suspensions are loaded in specified volumes and counted under a microscope. Sources of errors in hemocytometry include: uneven cell distribution in the sample, too many or too few cells in the sample, subjective decisions as to whether a given cell falls within the defined counting area, contamination of the hemocytometer, user-to-user variation, and variation of hemocytometer filling rate2.
To alleviate the tedium associated with manual counting, 29% of researchers count cells using automated cell counting devices; these include vision-based counters, systems that detect cells using the Coulter principle, or flow cytometry1. For most researchers, the main barrier to using an automated system is the price associated with these large benchtop instruments1.
The Scepter cell counter is an automated handheld device that offers the automation and accuracy of Coulter counting at a relatively low cost. The system employs the Coulter principle of impedance-based particle detection3 in a miniaturized format using a combination of analog and digital hardware for sensing, signal processing, data storage, and graphical display. The disposable tip is engineered with a microfabricated, cell- sensing zone that enables discrimination by cell size and cell volume at sub-micron and sub-picoliter resolution. Enhanced with precision liquid-handling channels and electronics, the Scepter cell counter reports cell population statistics graphically displayed as a histogram.

The Scepter Cell Counter is a handheld automated device that can be used to count cells, monitor cell diameter and volume, and be used to check the health and quality of cellular populations from one culture to the next.

Determinar el número de células durante el cultivo celular utilizando el contador de células Cetro

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

Análisis de la posición del ciclo celular en células de mamífero

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Determination of Mammalian Cell Counts, Cell Size and Cell Health Using the Moxi Z Mini Automated Cell Counter

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls1-5. A critical breakthrough in cell/particle counting technologies was the development of the Coulter technique by Wallace Coulter over 50 years ago. The technique involves the application of an electric field across a micron-sized aperture and hydrodynamically focusing single particles through the aperture. The resulting occlusion of the aperture by the particles yields a measurable change in electric impedance that can be directly and precisely correlated to cell size/volume. The recognition of the approach as the benchmark in cell/particle counting stems from the extraordinary precision and accuracy of its particle sizing and counts, particularly as compared to manual and imaging based technologies (accuracies on the order of 98% for Coulter counters versus 75-80% for manual and vision-based systems). This can be attributed to the fact that, unlike imaging-based approaches to cell counting, the Coulter Technique makes a true three-dimensional (3-D) measurement of cells/particles which dramatically reduces count interference from debris and clustering by calculating precise volumetric information about the cells/particles. Overall this provides a means for enumerating and sizing cells in a more accurate, less tedious, less time-consuming, and less subjective means than other counting techniques6.
Despite the prominence of the Coulter technique in cell counting, its widespread use in routine biological studies has been prohibitive due to the cost and size of traditional instruments. Although a less expensive Coulter-based instrument has been produced, it has limitations as compared to its more expensive counterparts in the correction for "coincidence events" in which two or more cells pass through the aperture and are measured simultaneously. Another limitation with existing Coulter technologies is the lack of metrics on the overall health of cell samples. Consequently, additional techniques must often be used in conjunction with Coulter counting to assess cell viability. This extends experimental setup time and cost since the traditional methods of viability assessment require cell staining and/or use of expensive and cumbersome equipment such as a flow cytometer.
The Moxi Z mini automated cell counter, described here, is an ultra-small benchtop instrument that combines the accuracy of the Coulter Principle with a thin-film sensor technology to enable precise sizing and counting of particles ranging from 3-25 microns, depending on the cell counting cassette used. The M type cassette can be used to count particles from with average diameters of 4 - 25 microns (dynamic range 2 - 34 microns), and the Type S cassette can be used to count particles with and average diameter of 3 - 20 microns (dynamic range 2 - 26 microns). Since the system uses a volumetric measurement method, the 4-25 microns corresponds to a cell volume range of 34 - 8,180 fL and the 3 - 20 microns corresponds to a cell volume range of 14 - 4200 fL, which is relevant when non-spherical particles are being measured. To perform mammalian cell counts using the Moxi Z, the cells to be counted are first diluted with ORFLO or similar diluent. A cell counting cassette is inserted into the instrument, and the sample is loaded into the port of the cassette. Thousands of cells are pulled, single-file through a "Cell Sensing Zone" (CSZ) in the thin-film membrane over 8-15 seconds. Following the run, the instrument uses proprietary curve-fitting in conjunction with a proprietary software algorithm to provide coincidence event correction along with an assessment of overall culture health by determining the ratio of the number of cells in the population of interest to the total number of particles. The total particle counts include shrunken and broken down dead cells, as well as other debris and contaminants. The results are presented in histogram format with an automatic curve fit, with gates that can be adjusted manually as needed.
Ultimately, the Moxi Z enables counting with a precision and accuracy comparable to a Coulter Z2, the current gold standard, while providing additional culture health information. Furthermore it achieves these results in less time, with a smaller footprint, with significantly easier operation and maintenance, and at a fraction of the cost of comparable technologies.

The Moxi Z miniature automated cell counter is a novel instrument that combines the Coulter Principle with patented thin-film sensor technology and a proprietary software algorithm to perform sizing and counting of a broad size range of particles as well as to determine the overall health of monodisperse mammalian cell cultures. This protocol describes the use of this instrument for counting and assessing the health of cell cultures.

Determinación del recuento de células de mamíferos, el tamaño celular y la salud de la célula mediante el Moxi Z Mini contador automatizado de células

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls1-5. A ...

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

Preparación de suero de rata Adecuado para mamíferos Whole cultivo de embriones

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for ...

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Zinc-finger domains are intrinsically cell-permeable and capable of mediating protein delivery into a broad range of mammalian cell types. Here, a detailed step-by-step protocol for implementing zinc-finger technology for intracellular protein delivery is presented.

Proteína entrega directa de células de mamíferos Utilización de Células permeable Cys<sub&gt; 2</sub&gt; -Su<sub&gt; 2</sub&gt; Dominios de zinc-finger

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged ...