Este trabajo fue apoyado por un investigador de Nueva Concesión [2014R1A1A1005553] de la Fundación Nacional de Investigación de Corea (NRF) para JIL; y la Universidad de Yonsei líder futuro desafío de Grant [2015-22-0133] para JIL

1. Preparar soluciones para NGT-3D y 3D-NGB <ol><li> Preparar las siguientes soluciones estériles: 1 L de solución 0,1454 M de NaCl, 1 L de 1 M CaCl 2, 1 L de 1 M MgSO 4, Lisogenia Broth (LB), 1 L de 1 M KPO 4 tampón (108,3 g de KH 2 PO 4 y 35,6 g de K 2 HPO 4, y llenar H2O hasta 1 l). La producción de NGM el uso de estas soluciones se pueden encontrar en un protocolo anterior 10. </li><li> soluciones autoclave a 121 ° C, 15 min. </li><li> Preparar 50 ml de una solución estéril 5 mg / ml de colesterol. En un tubo cónico de 50 ml, mezclar 0,25 g de colesterol y 50 ml de 99,99% de etanol y se mezcla bien. No esterilizar en autoclave. Esterilizar la solución colesterol usando una jeringa de 50 ml con un filtro de jeringa de 0,45 micras. También se eliminará cualquier colesterol no disuelto. </li><li> (Opcional) Preparar 10 ml de una 150 mM de 2&#39;-desoxi-5-fluorouridina (FUdR) stock. En un tubo cónico de 15 ml, mezclar 0,3693 gde FUdR y 10 ml de agua destilada. Agitar bien. </li></ol> 2. Preparar Cultura de las bacterias para NGT-3D y 3D-NGB <ol><li> Inocular 10 ml de caldo LB con bacterias. Las bacterias habituales utilizados para la alimentación de C. elegans es E. coli cepa OP50. </li><li> Cultura de la inoculación de bacterias a 37 ° C en una incubadora con agitación durante la noche. </li><li> En la preparación para una dilución en serie, alícuotas 9 ml de la solución de NaCl en 15 ml tubos cónicos estériles. Por ejemplo, parte alícuota de 9 ml en un total de 7 tubos para hacer una dilución 10 -7 de la cepa OP50 E. coli. </li><li> Usando una pipeta estéril 1,000 l, pipetear 1 ml de cultivo bacteriano o un cultivo bacteriano diluido en el nuevo tubo estéril con 9 ml de solución de NaCl y agitar bien la mezcla de 10 ml. Repita hasta que se alcanza la dilución bacteriana deseada. NOTA: La dilución bacteriana deseada depende del tipo y condición de bacterias, así como las condiciones experimentales exactas.Por ejemplo, un 10 -6 - dilución de la cepa de E. coli OP50 10 -8 era suficiente para producir a partir de 1 - 200 colonias por 6,5 ml tubo NGT-3D. Worms fiable desarrollan y se reproducen normalmente cuando el número de colonias era más de 60, que se produjo a una dilución de 10 -6-10 -7. Para NGB-3D, utilizar una dilución de 10 -8. </li></ol> 3. Hacer NGT-3D y 3D-NGB (200 ml) <ol><li> Después de la preparación del cultivo bacteriano, la mezcla 0,6 g de NaCl, 1 g de agar granulado, y 0,5 g de peptona en un matraz de 500 ml. Insertar una barra de agitación magnética en el matraz. </li><li> Añadir 195 ml de agua destilada y cubrir la boca del matraz con papel de aluminio. </li><li> Autoclave 121 ° C durante 15 min. </li><li> Colocar el matraz en autoclave caliente sobre una placa de agitación, y se agita a una velocidad moderada durante al menos 2 horas. Se enfría el matraz a 40 ° C. Asegúrese de que se enfríe lo suficiente como para continuar de aquí a altas temperaturas puede dar lugar a pérdida de transparencia del agar acabado.Sin embargo, temperaturas más bajas pueden resultar en el endurecimiento prematuro de la agar. <ol><li> (Opcional) Para acelerar el proceso de enfriamiento, colocar el matraz en un baño de agua a 40 ° C 15 min antes de colocar sobre una placa de agitación. </li></ol></li><li> Cuando la temperatura de los medios de agar alcanza 40 ° C, añadir 200 l 1 M CaCl 2, 200 l de solución de colesterol 5 mg / ml, 200 l 1 M MgSO 4 y 5 ml 1 M KPO 4 tampón como la solución sigue agitar a concentraciones finales de 1 mM CaCl colesterol 2, 5 mg / ml, 1 mM MgSO 4, 1 mM KPO 4. <ol><li> (Opcional) Para el ensayo de vida útil NGT-3D añaden 80 l de FUdR 150 mM a una concentración final de 120 mM 12. </li></ol></li><li> Añadir 6 ml de los 10 ml de cultivo bacteriano diluido a partir del paso 2.4 en el matraz directamente. <ol><li> (Opcional) Para NGT-3D, retire 6 ml de medio de agar antes del paso 3.6 y mantenerlo caliente por separado. Este medio se utiliza para los libre de bacteriascapa superior. </li></ol></li><li> El uso de procedimientos estériles, prescindir de los medios de comunicación en una cámara de cultivo estéril. Asegúrese de que la cubierta de la cámara se cierra herméticamente. <ol><li> (Opcional) Para prevenir la formación de colonias de bacterias de la superficie superior de la agar, hacer una capa de libre de bacterias medios de agar de la etapa 3.6.1 en la parte superior antes de los medios de comunicación desde el paso 3.7 se endurece por completo. Esto creará una zona libre de bacterias en la parte superior de la NGT-3D. </li><li> Para NGT-3D, verter 6,5 ml de medio a los 8 ml tubos de ensayo de plástico transparente para hacer una capa de agar bacterias, y dispensar cuidadosamente 200 l de los medios de comunicación libre de bacterias en la parte superior de los medios de comunicación semi-endurecido 3D para hacer una fina bacterias- capa libre en la parte superior. </li><li> Para NGB-3D, verter 65 ml de medio a los 25 cm de botella de cultivo de células de plástico transparente 2. Esta cantidad debe llenar el cuerpo de la 25 cm botella de cultivo 2 celular. </li></ol></li><li> Deja cámaras verticalmente a temperatura ambiente durante una semana para permitir que las colonias bacterianas que crecena un tamaño considerable de al menos 1 mm de diámetro. <ol><li> (Opcional) Para el ensayo de vida útil en NGT-3D, cámaras de cubierta con papel de aluminio para evitar la degradación de la luz de FUdR. </li></ol></li></ol> 4. Medir la aptitud de la población de gusanos en NGT-3D (Cría relativa Ensayo de tamaño) <ol><li> Escoja un gusano de L4 etapa usando un alambre de platino recogida y traslado en una placa de NGM sin bacterias. Permitir que el gusano se mueva libremente alrededor de unos pocos minutos para eliminar bacterias adheridas a su cuerpo. </li><li> Repita el paso 4.1 y asegúrese de que el gusano está libre de bacterias. En general, dos repeticiones de 4.1 son suficientes. </li><li> Con cuidado, coloque el gusano limpia en la superficie de los medios de comunicación en 3D con un pico alambre de platino. El gusano, finalmente, debe entrar en el agar agar en la matriz 3D. </li><li> Cierre la tapa sin apretar que permite un poco de aire para entrar en el tubo, pero evitando el secado de los medios de agar. </li><li> Incubar durante 96 horas a 20 ° C. </li><li> Después de 4 días, cerrar las tapas firmemente y coloque lacámara de cultivo NGT-3D en un baño de agua a 88 ° para fundir el agar. El calor mata los gusanos pero sus cuerpos permanecen intactos. NOTA: El uso de tubos de 8 ml de NGT-3D, 20 - 30 min de incubación suele ser suficiente. </li><li> Usando una pipeta de vidrio, transferir los medios fundidos en un 9 cm placa petri de plástico. No se recomiendan pipetas de plástico, como los gusanos pueden a menudo se adhieren a ellos. </li><li> El uso de un microscopio de disección estéreo de transmisión, contar el número de gusanos en la L3, L4 y etapas adultas. No contar los gusanos que son L2 escenario y más joven, ya que las generaciones F1 y F2 se pueden confundir aquí. Por lo tanto, este ensayo es un ensayo de tamaño de la camada relativa en lugar de un total de ensayo tamaño de la camada. </li></ol> 5. Imagen y comportamiento de estos animales en el Registro NGB-3D <ol><li> Escoja un gusano de L4 etapa usando un alambre de platino recoger y eliminar las bacterias adheridas a la superficie mediante la transferencia a una placa de NGM sin bacterias y permitiendo que se mueva libremente durante unos minutos. </li><li> Repita el paso 5.1 para asegurarse de que el worm está libre de bacterias. </li><li> Colocar con cuidado el gusano limpio en el centro de la superficie de agar de la NGB-3D cerca del cuello de la botella con un pico alambre de platino. El gusano, finalmente, debe entrar en el agar agar en la matriz 3D. </li><li> Cierre la tapa sin apretar que permite un poco de aire para entrar en el tubo, al tiempo que evita el secado de los medios de agar. </li><li> Imagen o grabar el gusano bajo un microscopio de disección estéreo de transmisión. Ajuste el enfoque como el gusano se mueve a través de la matriz 3D. </li></ol>

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The authors have nothing to disclose.

El cultivo de laboratorio de C. elegans usando las placas de medio de crecimiento de nematodos clásicos fue crucial para los cientos de descubrimientos importantes que la investigación en C. elegans ha proporcionado. A continuación, presentamos nuevos métodos para cultivar C. elegans en un entorno que refleja con mayor precisión sus hábitats naturales tridimensionales. Aunque otros métodos se han utilizado para observar C. elegans en 3D 13, este es el primer protocolo que permite el cultivo de gusanos en una matriz 3D sólido. Los dos métodos que se muestran aquí, NGT-3D y NGB-3D, permiten a los científicos hacen preguntas de la aptitud, el desarrollo y el crecimiento en cultivo 3D, y también de imagen y grabar los gusanos en 3D. Aunque NGT-3D y 3D-NGB tienen algunas diferencias con respecto a las placas estándar 2D NGM, que son similares a este método original y única ajustada para añadir el cultivo del gusano en el eje z. Por lo tanto, nos encontramos con que el desarrollo, la fertilidad y la vida útil proceder normalmente en nuestras condiciones. losobjetivo era crear un protocolo que cualquier laboratorio puede emplear para su investigación. Estos métodos son muy simples, fácilmente reproducible a un coste muy bajo, y permiten el ajuste de protocolos para adaptarse a experimentos específicos de los usuarios. A pesar de todos los pasos en el protocolo son importantes, el paso crítico en la toma de NGT-3D y NGB-3D es la temperatura de enfriamiento del agar tras autoclave en los pasos 3.4 a 3.6 del protocolo. Si la temperatura se enfría demasiado baja (algunos grados por debajo de 40 ° C), el agar comenzará a endurecerse y las soluciones añadidas no se mezcla correctamente en el agar. Sin embargo, si la refrigeración es insuficiente (varios grados por encima de 40 ° C), las bacterias añadidas pueden morir y la solución colesterol añadido pueden enturbiar el agar agar haciendo que el inútil para los experimentos. Además, una zona de precaución es en el paso 4.6. Cuando fundir el agar NGT-3D en el baño de agua, asegúrese de cerrar las tapas firmemente para ayudar en la fusión. Sin embargo, el calor puede hacer que eltapas para peligrosamente &quot;pop&quot; y convertirse en un proyectil. Por razones de seguridad es mejor para cubrir el baño de agua. Muchas partes del protocolo son muy adaptables al usuario. Por ejemplo, hemos utilizado un tubo de ensayo de plástico transparente para NGT-3D y una botella de cultivo de células claras 25 cm2 (sostiene un total de 65 ml) para la NGB-3D. Otros tipos de tubos transparentes o botellas disponibles para el usuario también debe trabajar. Además, el crecimiento bacteriano es ajustable. La cepa de E. coli OP50 generalmente toma alrededor de una semana a crecer a por lo menos 1 mm, que se prefiere para nuestros experimentos. En nuestra experiencia, no parece que los tiempos más largos de crecimiento ligeramente más cortos e incluso gran parte de las bacterias que afectan el crecimiento de gusano o la fertilidad. Ajuste de la concentración de agar, sin embargo, afectar el movimiento gusano. Concentraciones más bajas de agar resultan en gusanos que muestran un movimiento de natación similar, y las concentraciones más altas pueden inhibir el movimiento en general. Un área de preocupación en NGB-3D fue el tema de la condensacióny la acumulación de agua. A medida que el agar líquido caliente se endurece, inevitablemente algo de condensación se desarrolla con el tiempo, particularmente en la interfase de la botella y el agar. Esto puede plantear problemas para el gusano, que muestra los comportamientos de natación en líquidos 14 y el crecimiento de las bacterias, que pueden extenderse a lo largo de la botella con el líquido. Prevención de la condensación es difícil. Existen dos soluciones posibles para esto. En primer lugar, nos aseguramos de que el gusano puede entrar fácilmente en el agar, colocándolo en el centro de la superficie del agar en el cuello de la botella. En segundo lugar, mantenemos intencionalmente la concentración de bacterias en bajas NGB-3D, disminuyendo así la probabilidad de que una colonia que crece en la interfaz de líquido y agar. Otra preocupación es la obtención de imágenes de gusanos en NGB-3D. Para aumentar la claridad y la calidad de las imágenes de gusanos en 3D, las colonias deben estar cerca de la superficie, pero permanecen completamente incrustado en el agar. Se intentó hacer crecer colonias sólo incrustados cerca de la surface mediante la colocación de una capa delgada de agar que contienen bacterias en la superficie, y el resto del soporte era libre de bacterias. Sin embargo, el problema no se resolvió totalmente por este método. En su lugar, decidimos producir muchas botellas NGB-3D de una sola vez, y utilizar sólo los que contienen colonias cercanas a la superficie incrustados, lo que aumenta la probabilidad de que un gusano se moverá a una colonia cerca de la superficie. Una de las opciones que no hemos explorado está cambiando los medios sólidos a una matriz que no sea el agar granulado empleamos aquí. La formación de imágenes gusanos en la dirección del eje Z también puede ser un reto. Hacemos esto por subir y bajar el grueso del enfoque de forma manual, pero si existe un programa de seguimiento automatizado de esto, puede ser muy útil. La esperanza es que las técnicas que aquí se presentan para el cultivo de C. elegans en 3D serán ampliamente utilizados por los biólogos de nematodos. Quedan muchas preguntas acerca de la biología del gusano, incluyendo el envejecimiento y las conductas en 3D, y si lo que los científicos observan en2D es relevante para el gusano en 3D. De hecho, un estudio previo utilizando NGT-3D mostró el impacto sobre la aptitud reproductiva que el cultivo 3D tenía en un mutante sensorial que reproduce normalmente en condiciones 2D 9. Además, un estudio mostró comportamientos de movimiento alterados en 3D en comparación con 2D 15. Por último, este sistema se puede utilizar para comenzar a responder a las preguntas de si ciertas condiciones, mutantes o manipulaciones confieren ventajas de la aptitud para el animal en su ambiente nativo 3D.

The study of the nematode Caenorhabditis elegans in the laboratory has led to seminal discoveries in the field of biology over the last five decades1. C. elegans was the first multicellular organism to have its genome sequenced in 19982, and it has been invaluable in understanding the contributions of individual genes to the development, physiology, and behavior of a whole organism. Scientists now are looking to further understand how these genes may contribute to the survival and reproductive fitness of organisms in their natural environments, asking questions about ecology and evolution at the genetic level3-5. 
C. elegans once again can provide an excellent system to answer these questions. However, little is known about C. elegans biology in natural nematode habitats, and there are no current methods to simulate controlled natural conditions of C. elegans in the laboratory. In the lab, C. elegans is cultivated on the surface of agar plates seeded with E. coli bacteria6. In nature, however, C. elegans and related nematodes can be found sparsely inhabiting soils throughout the globe, but they are specifically found thriving in rotting fruits and vegetative matter7,8. These three-dimensional (3D) complex environments are quite different from the simple 2D environments to which worms are exposed to in the laboratory. 
To begin to answer questions about the biology of nematodes in a more natural 3D setting, we have designed a 3D habitat for laboratory cultivation of nematodes we called Nematode Growth Tube 3D or NGT-3D for short9. The goal was to design a 3D growth system that allows for comparable growth, development, and fertility to the standard 2D Nematode Growth Media (NGM) plates10. This system supports the growth of bacteria and nematodes over their entire life cycles in 3D, allows worms to move and behave freely in three dimensions, and is easy and inexpensive to manufacture and employ. 
In the current study, we provide a step-by-step method to manufacture NGT-3D and evaluate worm development and fertility. In addition to assessing worm fitness in 3D, we sought to image, video, and assess worm behavior and physiology in 3D cultivation. Thus, in addition to NGT-3D, we present here an alternate method called Nematode Growth Bottle 3D or NGB-3D, for the microscopic imaging of C. elegans during 3D cultivation. This will be especially important for the study of known behaviors identified in 2D, and the identification of novel behaviors unique to 3D cultivation.

<table><tbody><tr><td>LB broth, Miller (Luria-Bertani)</td><td>Difco</td><td>224620</td><td></td></tr><tr><td>Sodium chloride</td><td>DAEJUNG</td><td>7548-4400</td><td>58.44 MW</td></tr><tr><td>Agar, Granulated</td><td>Difco</td><td>214530</td><td></td></tr><tr><td>Peptone</td><td>Bacto</td><td>211677</td><td></td></tr><tr><td>Calcium chloride, dihydrate</td><td>Bio Basic</td><td>CD0050</td><td>2*H&lt;sub&gt;2&lt;/sub&gt;O; 147.02 MW</td></tr><tr><td>Cholesterol</td><td>Bio Basic</td><td>CD0122</td><td>386.67 MW</td></tr><tr><td>Ethyl alcohol</td><td>B&amp;amp;J</td><td>RP090-1</td><td>99.99%; 46.07 MW</td></tr><tr><td>Magnesium sulfate, anhydrous</td><td>Bio Basic</td><td>MN1988</td><td>120.37 MW</td></tr><tr><td>Potassium phosphate, monobasic, anhydrous</td><td>Bio Basic</td><td>PB0445</td><td>136.09 MW</td></tr><tr><td>2&#039;-Deoxy-5-fluorouridine</td><td>Tokyo Chemical Industry</td><td>D2235</td><td>246.19 MW</td></tr><tr><td>Potassium phosphate, dibasic, anhydrous</td><td>Bio Basic</td><td>PB0447</td><td>174.18 MW</td></tr><tr><td>Multi-Purpose Test Tubes</td><td>Stockwell Scientific</td><td>ST.8570</td><td>8 ml</td></tr><tr><td>Test Tube Closures</td><td>Stockwell Scientific</td><td>ST.8575</td><td></td></tr><tr><td>Cell Culture Flask</td><td>SPL Lifescience</td><td>70125</td><td>25 cm&lt;sup&gt;2&lt;/sup&gt;</td></tr><tr><td>Research Stereo Microscope</td><td>Nikon</td><td>SMZ18</td><td></td></tr><tr><td>High-Definition Color Camera Head</td><td>Nikon</td><td>DS-Fi2</td><td></td></tr><tr><td>PC-Based Control Unit</td><td>Nikon</td><td>DS-U3</td><td></td></tr><tr><td>NIS-Elements Basic Research, Microscope Imaging Software</td><td>Nikon</td><td>MQS32000</td><td></td></tr></tbody></table>

cultivation of caenorhabditis elegans in three dimensions in the laboratory

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

La construcción de NGT-3D es un protocolo simple y directo que resulta en un tubo de ensayo de agar-llena de pequeñas colonias bacterianas espaciados en todo el agar (Figura 1A). Los gusanos pueden moverse libremente a través de la matriz de agar, la búsqueda y el consumo de las colonias bacterianas. Para confirmar si C. elegans pueden reproducirse y crecer normalmente en NGT-3D, se comparó la fertilidad y el desarrollo de las larvas en 3D con placas estándar 2D NGM. En el ensayo de cría de tamaño relativo, adultos de C. elegans hermafroditas en NGT-3D se reproducen tan bien como hermafroditas en las placas estándar 2D NGM cuando las colonias bacterianas son abundantes con al menos 60 colonias (Figura 1B). Además, el desarrollo larval en NGT-3D también procede normalmente cuando hay un montón de colonias de bacterias con la mayoría de los gusanos en el estado adulto después de 96 horas (Figura 1C). Sin embargo, si las colonias bacterianas son escasosen la matriz 3D, tanto en la cría de tamaño relativo (Figura 1B, barra de la izquierda) y el desarrollo de las larvas (Figura 1C, barra de la izquierda) se ven afectados negativamente. Para probar si la vivienda en 3D tiene ningún efecto sobre la fisiología a largo plazo del gusano, se realizó un ensayo de vida útil comparar placas NGT-3D y NGM. El promedio de vida de los gusanos en NGT-3D fue de 15,6 ± 3,6 días en comparación a 14,8 ± 3,1 días para NGM placas estándar. Las curvas de vida útil de los gusanos que viven en NGT-3D y NGM fueron casi idénticos. Por lo tanto, parece que los gusanos sobreviven igualmente bien en condiciones de 3D y 2D. La fabricación del NGB-3D también no es difícil y debe resultar en una botella de cultivo clara, agar-llenado como la que se observa en la Figura 2A. Para ver fácilmente las colonias bacterianas, que hemos expresado deoxyviolacein, un metabolito bacteriano de color púrpura oscuro 11, en el OP50cepa de E. coli (Figuras 2A, 2B; cepa disponible bajo petición). Gusanos vivos se pueden obtener imágenes bajo una transmisión estéreo-microscopio de disección (Figura 2B, Suplementario Película 1). El almacenamiento de NGT-3D y NGB-3D a temperatura ambiente es suficiente para mantener estas dos cámaras de cultivo durante un máximo de 1 mes. La desecación del agar no es una preocupación para cualquiera NGT-3D o 3D-NGB si se aplica una de enchufe o de tipo tornillo de cabeza que se ajusta el tubo o botella. <img alt="Figura 1" src="/files/ftp_upload/55048/55048fig1.jpg" /> Figura 1: Desarrollo, la fertilidad y la vida útil de C. elegans cultivadas en NGT-3D es comparable con la del estándar NGM 2D. (A) Imágenes de NGT-3D en varias diluciones de bacterias. a la izquierda yla derecha contiene una dilución de 5 x 10 -7, y el medio es una dilución 1 x 10 -7. (B) El tamaño relativo de cría de gusanos de tipo salvaje en NGT-3D a menos de 60 colonias OP50 (n = 24), mayor o igual a 60 colonias (n = 14), o en placas 2D NGM (n = 29). Las barras de error indican el error estándar. (C) Porcentaje de gusanos generación F1 en el L3, L4 o etapa de desarrollo para adultos en NGT-3D con menos de 60 colonias OP50, mayores o iguales a 60 colonias, o en las placas de NGM 2D. Curva (D) La supervivencia de los de tipo salvaje C. elegans en placas NGT-3D o NGM. SN no indica significativamente diferente calcula mediante la prueba de log-rank. Esta cifra se ha modificado de Lee 9. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55048/55048fig1large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> <img alt="Figura 2" src = &quot;/ files / ftp_upload / 55048 / 55048fig2.jpg&quot; /&gt; Figura 2: Imágenes en 3D C. elegans utilizando NGB-3D. (A, B) Imágenes de NGB-3D; (C) Imágenes de adultos C. elegans cerca de una colonia de la cepa de E. coli que expresan OP50 el deoxyviolacein pigmento púrpura oscuro. La barra de escala es de 500 micras. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55048/55048fig2large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> <img alt="Figura 2" src="/files/ftp_upload/55048/55048supmov.jpg" /> Película complementaria 1: Adultos C. elegans se acercan a una coli OP50-violacé E. en la colonia en NGB-3D. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55048/Jove_Mov_1.mp4">Por favor, haga clic aquí para descargar este archivo.</a>

Watch this Scientific Journal Video about Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory at JoVE.com

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

Se presenta un método sencillo de construir sistemas de cultivo de nematodos 3D llamados NGT-3D y NGB-3D. Éstos se pueden utilizar para estudiar la aptitud de los nematodos y los comportamientos en los hábitats que son más similares a los naturales Caenorhabditis elegans hábitats que las placas de cultivo estándar en 2D elegans C. laboratorio.

El cultivo de<em&gt; Caenorhabditis elegans</em&gt; En tres dimensiones en el Laboratorio

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

cultivation-caenorhabditis-elegans-three-dimensions

Universidad San Sebastian

Research

JoVE Journal

Biology

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

17.8K vistas.  Yonsei University.  Se presenta un método sencillo de construir sistemas de cultivo de nematodos 3D llamados NGT-3D y NGB-3D. Éstos se pueden utilizar para estudiar la aptitud de los nematodos y los comportamientos en los hábitats que son más similares a los naturales Caenorhabditis elegans hábitats que las placas de cultivo estándar en 2D elegans C. laboratorio. 

Video: Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the intestine the primary location for disease. The nematode intestine structurally and functionally mimics mammalian intestines and is transparent making it amenable to microscopic study of colonization. Here we show that pathogens can cause disease and death. We are able to identify microbial mutants that show altered virulence. Its conserved innate response to biotic stresses makes C. elegans an excellent system to probe facets of host innate immune interactions. We show that hosts with mutations in the dual oxidase gene cannot produce reactive oxygen species and are unable to resist microbial insult. We further demonstrate the versatility of the presented survival assay by showing that it can be used to study the effects of inhibitors of microbial growth. This assay may also be used to discover fungal virulence factors as targets for the development of novel antifungal agents, as well as provide an opportunity to further uncover host-microbe interactions. The design of this assay lends itself well to high throughput whole-genome screens, while the ability to cryo-preserve worms for future use makes it a cost-effective and attractive whole animal model to study.

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

El nematodo Caenorhabditis Elegans - un modelo versátil en Vivo para el estudio de las interacciones huésped-microbio

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

Investigación

Genética

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple and inexpensive method to obtain a large number of animals for the isolation of proteins to proceed with a western blot, mass spectrometry, or further proteomics analyses. Furthermore, an increase of nematode numbers for immunostainings and the integration of multiple analyses under the same culturing conditions can easily be achieved. Additionally, a transfer between plates with different experimental conditions is facilitated. Common techniques in plate culture involve the transfer of a single C. elegans using a platinum wire and the transfer of populated agar chunks using a scalpel. However, with increasing nematode numbers, these techniques become overly time-consuming. This protocol describes the large-scale culture of C. elegans including numerous steps to minimize the impact of the sample preparation on the physiology of the worm. Fluid and shear stress can alter the lifespan of and metabolic processes in C. elegans, thus requiring a detailed description of the critical steps in order to retrieve reliable and reproducible results. C. elegans is a model organism, consisting of neuronal cells for up to one-third, but lacking blood vessels, thus providing the possibility to investigate solely neuronal alterations independent of vascular control. Recently, early neurodegeneration in diabetic retinopathy was found prior to vascular alterations. Thus, C. elegans is of special interest for studying general mechanisms of diabetic complications. For example, an increased formation of advanced glycation end products (AGEs) and reactive oxygen species (ROS) is observed, which are reproducibly found in C. elegans. Protocols to handle samples of adequate size for a broader spectrum of investigations are presented here, exemplified by the study of diabetes-induced biochemical alterations. In general, this protocol can be useful for studies requiring large C. elegans numbers and in which liquid culture is not suitable.

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

This protocol describes a method for the large-scale cultivation of Caenorhabditis elegans on solid media. As an alternative to liquid culture, this protocol allows obtaining parameters of different scales under plate-based cultivation. This increases the comparability of results by omitting the morphological and metabolic differences between liquid and solid media culture.

Base de placa de cultivo a gran escala de elegans de Caenorhabditis: preparación de muestras para el estudio de alteraciones metabólicas en la Diabetes

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple ...

Bioquímica

Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations

Caenorhabditis elegans (C. elegans) has been and remains a valuable model organism to study developmental biology, aging, neurobiology, and genetics. The large body of work on C. elegans makes it an ideal candidate to integrate into large-population, whole-animal studies to dissect the complex biological components and their relationships with another organism. In order to use C. elegans in collaborative -omics research, a method is needed to generate large populations of animals where a single sample can be split and assayed across diverse platforms for comparative analyses.
Here, a method to culture and collect an abundant mixed-stage C. elegans population on a large-scale culture plate (LSCP) and subsequent phenotypic data is presented. This pipeline yields sufficient numbers of animals to collect phenotypic and population data, along with any data needed for -omics experiments (i.e., genomics, transcriptomics, proteomics, and metabolomics). In addition, the LSCP method requires minimal manipulation to the animals themselves, less user preparation time, provides tight environmental control, and ensures that handling of each sample is consistent throughout the study for overall reproducibility. Lastly, methods to document population size and population distribution of C. elegans life stages in a given LSCP are presented.

Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations

To use Caenorhabditis elegans (C. elegans) in omics research, a method is needed to generate large populations of worms where a single sample can be measured across platforms for comparative analyses. Here, a method to culture C. elegans populations on large-scale culture plates (LSCPs) and to document population growth is presented.

Cultivo Y Ensayo De Las Poblaciones De Caenorhabditis elegans De Etapa Mixta A Gran Escala

Caenorhabditis elegans (C. elegans) has been and remains a valuable model organism to study developmental biology, aging, neurobiology, and genetics. The ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Básico<em&gt; Caenorhabditis elegans</em&gt; Métodos de sincronización y de observación

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Biología

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding these biological processes often requires long-term and repeated imaging of the same animal. Long recovery times associated with conventional immobilization methods done on agar pads have detrimental effects on animal health making it inappropriate to repeatedly image the same animal over long periods of time. This paper describes a microfluidic chip design, fabrication method, on-chip C. elegans culturing protocol, and three examples of long-term imaging to study developmental processes in individual animals. The chip, fabricated with polydimethylsiloxane and bonded on a cover glass, immobilizes animals on a glass substrate using an elastomeric membrane that is deflected using nitrogen gas. Complete immobilization of C. elegans enables robust time-lapse imaging of cellular and sub-cellular events in an anesthetic-free manner. A channel geometry with a large cross-section allows the animal to move freely within two partially sealed isolation membranes permitting growth in the channel with a continuous food supply. Using this simple chip, imaging of developmental phenomena such as neuronal process growth, vulval development, and dendritic arborization in the PVD sensory neurons, as the animal grows inside the channel, can be performed. The long-term growth and imaging chip operates with a single pressure line, no external valves, inexpensive fluidic consumables, and utilizes standard worm handling protocols that can easily be adapted by other laboratories using C. elegans.

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

The protocol describes a simple microfluidic chip design and microfabrication methodology used to grow C. elegans in presence of a continuous food supply for up to 36 h. The growth and imaging device also enables intermittent long-term high-resolution imaging of cellular and sub-cellular processes during development for several days.

Un chip microfluídico simple para el crecimiento a largo plazo y la obtención de imágenes de Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding ...

Bioingeniería

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

Un método para cultivar embrionarias<em&gt; C. elegans</em&gt; Células

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans&#160;nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

C. elegans is usually grown on solid agar plates or in liquid cultures seeded with E. coli. To prevent bacterial byproducts from confounding toxicological and nutritional studies, we utilized an axenic liquid medium, CeHR, to grow and synchronize a large number of worms for a range of downstream applications.

El cultivo<em&gt; Caenorhabditis elegans</em &gt; En axénico Liquid Media y Creación de transgénicos Worms por bombardeo de micropartículas

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media ...

Compost Microcosms as Microbially Diverse, Natural-like Environments for Microbiome Research in Caenorhabditis elegans

The nematode Caenorhabditis elegans is emerging as a useful model for studying the molecular mechanisms underlying interactions between hosts and their gut microbiomes. While experiments with well-characterized bacteria or defined bacterial communities can facilitate the analysis of molecular mechanisms, studying nematodes in their natural microbial context is essential for exploring the diversity of such mechanisms. At the same time, the isolation of worms from the wild is not always feasible, and, even when possible, sampling from the wild restricts the use of the genetic toolkit otherwise available for C. elegans research. The following protocol describes a method for microbiome studies utilizing compost microcosms for the in-lab&#160;growth in microbially diverse and natural-like environments.
Locally sourced soil can be enriched with produce to diversify the microbial communities in which worms are raised and from which they are harvested, washed, and surface-sterilized for subsequent analyses. Representative experiments demonstrate the ability to modulate the microbial community in a common soil by enriching it with different produce and further demonstrate that worms raised in these distinct environments assemble similar gut microbiomes distinct from their respective environments, supporting the notion of a species-specific core gut microbiome. Overall, compost microcosms provide natural-like in-lab environments for microbiome research as an alternative to synthetic microbial communities or to the isolation of wild nematodes.

Compost Microcosms as Microbially Diverse, Natural-like Environments for Microbiome Research in Caenorhabditis elegans

Compost microcosms bring the microbial diversity found in nature into the laboratory to facilitate microbiome research in Caenorhabditis elegans. Provided here are protocols for setting up microcosm experiments, with the experiments demonstrating the ability to modulate environmental microbial diversity to explore the relationships between environmental microbial diversity and worm gut microbiome composition.

Microcosmos de compost como entornos microbianamente diversos y naturales para la investigación del microbioma en Caenorhabditis elegans

The nematode Caenorhabditis elegans is emerging as a useful model for studying the molecular mechanisms underlying interactions between hosts and their ...

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid nematode growth media (NGM), allowing the efficient collection of population-level data for survival and other physiological phenotypes, and periodic sampling of subpopulations for fluorescent biomarker quantification. Limitations to this approach are the inability to (1) follow individual worms over time to develop age trajectories for phenotypes of interest and (2) monitor fluorescent biomarkers directly in the context of the culture environment. Alternative culture approaches use liquid culture or microfluidics to monitor individual animals over time, in some cases including fluorescence quantification, with the tradeoff that the culture environment is contextually distinct from solid NGM. The WorMotel is a previously described microfabricated multi-well device for culturing isolated worms on solid NGM. Each worm is maintained in a well containing solid NGM surrounded by a moat filled with copper sulfate, a contact repellent for C. elegans, allowing longitudinal monitoring of individual animals. We find copper sulfate insufficient to prevent worms from fleeing when subjected to aversive interventions common in aging research, including dietary restriction, pathogenic bacteria, and chemical agents that induce cellular stress. The multi-well devices are also molded from polydimethylsiloxane, which produces high background artifacts in fluorescence imaging. This protocol describes a new approach for culturing isolated roundworms on solid NGM using commercially available polystyrene microtrays, originally designed for human leukocyte antigen (HLA) typing, allowing the measurement of survival, physiological phenotypes, and fluorescence across the lifespan. A palmitic acid barrier prevents worms from fleeing, even in the presence of aversive conditions. Each plate can culture up to 96 animals and easily adapts to a variety of conditions, including dietary restriction, RNAi, and chemical additives, and is compatible with automated systems for collecting lifespan and activity data.

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Here, we present a protocol to culture isolated individual nematodes on solid media for lifelong physiological parameter tracking and fluorescence quantification. This culture system includes a palmitic acid barrier around single-worm wells to prevent animals from fleeing, allowing the use of aversive interventions, including pathogenic bacteria and chemical stressors.

Cultivo a largo plazo de Caenorhabditis elegans individuales en medios sólidos para la monitorización longitudinal de fluorescencia e intervenciones aversivas

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid ...

Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices

The nematode Caenorhabditis elegans is among the most common model systems used in aging research owing to its simple and inexpensive culture techniques, rapid reproduction cycle (~3 days), short lifespan (~3 weeks), and numerous available tools for genetic manipulation and molecular analysis. The most common approach for conducting aging studies in C. elegans, including survival analysis, involves culturing populations of tens to hundreds of animals together on solid nematode growth media (NGM) in Petri plates. While this approach gathers data on a population of animals, most protocols do not track individual animals over time. Presented here is an optimized protocol for the long-term culturing of individual animals on microfabricated polydimethylsiloxane (PDMS) devices called WorMotels. Each device allows up to 240 animals to be cultured in small wells containing NGM, with each well isolated by a copper sulfate-containing moat that prevents the animals from fleeing. Building on the original WorMotel description, this paper provides a detailed protocol for molding, preparing, and populating each device, with descriptions of common technical complications and advice for troubleshooting. Within this protocol are techniques for the consistent loading of small-volume NGM, the consistent drying of both the NGM and bacterial food, options for delivering pharmacological interventions, instructions for and practical limitations to reusing PDMS devices, and tips for minimizing desiccation, even in low-humidity environments. This technique allows the longitudinal monitoring of various physiological parameters, including stimulated activity, unstimulated activity, body size, movement geometry, healthspan, and survival, in an environment similar to the standard technique for group culture on solid media in Petri plates. This method is compatible with high-throughput data collection when used in conjunction with automated microscopy and analysis software. Finally, the limitations of this technique are discussed, as well as a comparison of this approach to a recently developed method that uses microtrays to culture isolated nematodes on solid media.

Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices

Presented here is an optimized protocol for culturing isolated individual nematodes on solid media in microfabricated multi-well devices. This approach allows individual animals to be monitored throughout their lives for a variety of phenotypes related to aging and health, including activity, body size and shape, movement geometry, and survival.

Cultivo a largo plazo y monitorización de Caenorhabditis elegans aislada en medios sólidos en dispositivos multipocillos

The nematode Caenorhabditis elegans is among the most common model systems used in aging research owing to its simple and inexpensive culture techniques, ...

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