Este trabajo fue apoyado por los EE.UU. Institutos Nacionales de Salud de subvención del NIH / NICHD # HD051998 (de CAM).

1. Preparación de bolas de colágeno <ol><li> Antes de EVTS de carga para 3-D de cultivo de células, se necesita para preparar las cuentas microportadores Cytodex-3: <ol><li> Pesar la cantidad apropiada de Cytodex-3 cuentas necesarios para el experimento. Este protocolo está adaptado para el vaso de 10 ml RCC, en la que 0,05 g de granos se necesitan. Para que un buque 50ml RCC, la escala correspondiente. En un tubo cónico de 50 ml autoclavable, mezclar 250 mg Cytodex-3 perlas con 12 ml de fosfato tamponado Dulbecco (DPBS). Esta cantidad es suficiente para 5 buques RCC. </li></ol></li><li> Asegurar un volumen adecuado está presente en el tubo cónico, como el proceso de autoclave dará lugar a la evaporación. Libremente tapar el tubo y autoclave durante 10 minutos a 110 ° C. </li><li> Retire el tubo cónico en la finalización del ciclo de autoclave y dejar que la solución de cuentas Cytodex-3 que se enfríe. </li><li> Después de que el tubo cónico se ha enfriado a temperatura ambiente, una técnica estéril para obtener un volumen total de 12.5ml utilizando 1X DPBS. </li><li> Cap y almacenar el preparado Cytodex-3 cuentas a temperatura ambiente. Permita que los granos se hinchan y preparado para enfriar a temperatura ambiente antes de su uso. La preparación por encima de Cytodex-3 cuentas se establecen cinco vasos de 10 ml RCC. Hemos observado que el almacenamiento prolongado de Cytodex-3 cuentas a temperatura ambiente se traducirá en una disminución del rendimiento, como el colágeno va a desestabilizar después de aproximadamente un mes. Cytodex-3 cuentas varían en tamaño desde 133 hasta 215 micras. </li></ol> 2. Preparación de medios <ol><li> Preparar 1 litro de RCC-2 optimizado GTSF los medios de comunicación (adaptado de 22), que consiste en un 40% MEM alfa, además de los suplementos y el 60% L-15 de Leibovitz medios de comunicación (adaptado de 22). En primer lugar, preparar 400 ml en el volumen total del MEM alfa complementado con: Bicarbonato de sodio 21.2mM 0,06% de peptona La fructosa 0,7 mm La galactosa 1.4mm Glucosa 5,6 mm HEPES 1% 1% de L-glutamina 0,5% SUS 10% FBS </li><li> Para preparar una solución de reserva de los STI, se disuelven en 5 ml estéril acidificada H 2 O preparados por la adición de ácido acético glacial (aproximadamente 0.05 ml). Agitar para disolver, seguir con 45 ml de agua estéril. </li><li> Pesar suficiente L-15 de Leibovitz en polvo de 600 ml de medio de disolución en el grado de cultivo de tejidos H 2 O. </li><li> Obtener un volumen total de medio de cultivo celular de 1 litro con L-15 de Leibovitz medio. Filtro-esterilizar y almacenar a 4 ° C en la oscuridad. Añadir 1% de penicilina-estreptomicina individualmente a cada parte alícuota del medio utilizado. </li><li> Formulaciones de muchos medios que no sean GTSF-2 medios de comunicación han sido probados con éxito en la RCC. Cada laboratorio debe decidir por sí mismos cuál es el medio óptimo para el experimento que se realiza. </li></ol> 3. Las células y la incubación de bolas <ol><li> Propagar la EVTS a ~ 80% de confluencia, y contar con trypsinize las prácticas aceptadas de cultivo celular. </li><li> Suspender 1x10 6 EVTS en 4 ml de agua tibiaed los medios de comunicación. </li><li> Mezclar cuidadosamente preparado Cytodex-3 cuentas. El uso de técnicas estériles, quitar 2,5 ml de cuentas preparados con una punta ancha, 10ml serológicas pipeta y transferir a un tubo cónico de 15 ml sin usar. Tenga en cuenta que puede haber una pequeña pérdida de cuentas que al unirse a la pipeta. </li><li> Deje que el Cytodex-3 cuentas a instalarse en la parte inferior del tubo. Después de la sedimentación, uso una pipeta para retirar la capa superior de DPBS sin molestar a las cuentas Cytodex-3. </li><li> Mezclar el 1x10 6 EVTS preparado en los medios de comunicación con el preparado Cytodex-3 cuentas. </li><li> Incubar la mezcla de células de perlas a temperatura ambiente durante 30 min. Periódicamente, mezclar suavemente. Incubar durante 30 min a 37 ° C y 5% CO 2. Periódicamente, mezclar suavemente. </li></ol> 4. Carga de la RWV <ol><li> En una cabina de flujo laminar, eliminar un RCC de 10 ml de la caja y colocarlo en una placa estéril, la cultura 6-y para la estabilidad. Consulte la Figura 1 para ver un diagrama etiquetado de la RCCS. </li><li> Retire el tapón grande del puerto. </li><li> Obtener un volumen total de la incubación de células cordón mezcla de 10 ml con medio calentado. </li><li> Cargar el celular / cordón mezcla-en la RCC a través del puerto de gran tamaño. La RCC se debe inclinar a un ángulo de 45 ° (hasta el puerto grande) cuando se carga para facilitar la eliminación de burbujas de aire posible. Evitar la acumulación de la presión positiva al llenar el vaso poco a poco y de manera constante. </li><li> Vuelva a colocar el tapón en el gran puerto. </li><li> Quite los pistones de las jeringas de 3 ml y las jeringas vacías en los pequeños puertos. Añadir 1-3 ml de los medios de comunicación a cada jeringa. Lentamente abra las dos válvulas. Vuelva a colocar los pistones de la jeringa en las jeringas con suavidad. Añadir los medios de comunicación de una jeringa hasta que las burbujas se retiran de la cámara interior. </li><li> Recoger los RCC y golpee suavemente el lado haciéndolo girar en frente de ti para comprobar si hay burbujas. Si hay burbujas, hay que sacarlos de la cámara, como burbujas de aire que interfieren con la formación of agregados de células e introducir las fuerzas de cizallamiento. Eliminar las burbujas haciendo girar el RCC hasta que las burbujas se encuentran bajo el pequeño puerto. A continuación, empuje hacia abajo en la jeringa en el lado opuesto a la fuerza de las burbujas en el puerto y fuera de la cámara. </li><li> Cerca de un puerto lateral. Empuje suavemente el émbolo de la jeringa otras para introducir una pequeña cantidad de presión positiva en el vaso, lo que impide la formación de burbujas. Cierre la válvula del segundo. </li><li> Cargue el RCC en el rotor. Iniciar la rotación a 19rpm en un 37 ° C incubadora de CO 2. </li></ol> 5. Cambio de los medios de comunicación <ol><li> Cambiar los medios de comunicación cada dos días durante los tres primeros días, y luego todos los días a partir de entonces. </li><li> Apague el rotor y quite la RCC. Tire de los pistones para crear algunos de succión, retire las jeringas de cada pequeño puerto y lugar de la RCC en un ángulo para que las cuentas de resolver opuesto del gran puerto. </li><li> Después de las cuentas han resuelto, abra una de las válvulas pequeñas ypermitir que los medios de comunicación a salir de la RCC y en un contenedor de residuos. Quite los 2 / 3 de los medios de comunicación de esta manera. Asegúrese de no molestar a las cuentas y para no descartar cualquier agregado. </li><li> Cierre la válvula de pequeña y abierta, el gran puerto. Añadir los medios de comunicación de nuevo en la RCC a través del puerto de gran tamaño. Vuelva a colocar el tapón en el gran puerto. </li><li> Repita los pasos 4,6 a 4,9. </li><li> A medida que el aumento del tamaño de los agregados, es necesario aumentar la velocidad de rotación para mantener en suspensión en todo momento, a ser posible en un patrón circulatorio pequeños a una velocidad óptima. Por lo general, aumentar la velocidad entre 0,3-0,7 rpm después de cada comida una vez que los agregados comienzan a crecer visiblemente. </li></ol> 6. Recolección de células propagadas <ol><li> Retire la RCC del rotor. Eliminar las jeringas de cada pequeño puerto y lugar de la RCC en un ángulo para que las perlas pueden resolver opuesto del gran puerto. </li><li> Después de las cuentas han resuelto, abrir una de las válvulas pequeñas y permitir que los medios de comunicación para que fluyade los RCC y en un contenedor de residuos. Retire 1 / 3 de los medios de comunicación de esta manera. </li><li> Cierre la válvula de pequeña y abierta, el gran puerto. Hacer girar suavemente el recipiente para dispersar a los agregados de nuevo el producto. Mezcla de vacío líquido en un tubo cónico de 50 ml estéril. Permita que los agregados recopilados para instalarse en el tubo cónico, antes de usar el sobrenadante a lavar bien el recipiente de cultivo para maximizar la recuperación de los agregados. Los agregados pueden ser utilizados para los ensayos de aguas abajo de inmediato, como la citometría de flujo, ensayos de invasión, inmunofluorescencia y otros. </li></ol> 7. Resultados representante Un ejemplo de EVT-como las células (SGHPL-4 la línea celular del trofoblasto) obtenidas en los RCC en Cytodex-3 cuentas se muestra en la Figura 2. La línea celular EVT-como muestra proyecciones que se extienden lejos del grupo principal y vinculados a grupos de vecinos. Muchas de las cuentas están totalmente cubiertas con células de propagación. Una vez retirado de la RCC y la plaTed en una matriz extracelular, el EVT-como el 3-D las células cultivadas agresiva invadir y / o migrar (Figura 3). RT-PCR datos confirman el aumento de la expresión de MMP se ve en 3-D en comparación con los agregados tradicionales monocapas de cultivo celular (Figura 4). Curiosamente, los genes no relacionados con la invasión también aumentada en los RCC (Tabla 1) y puede ayudar a delimitar las zonas, como las interacciones inmune con células invasoras trofoblasto. <img alt="Figura 1" src="/files/ftp_upload/3367/3367fig1.jpg" /> Figura 1. Representación de dibujos animados del sistema de cultivo celular rotación (RCC). <img alt="Figura 2" src="/files/ftp_upload/3367/3367fig2.jpg" /> Figura 2. Micrografía de contraste de fase de un total de representante después de 5 días de crecimiento en un RCC. Las flechas indican las células invasoras y * indica que las cuentas microportadores Cytodex-3. <imgalt = &quot;Figura 3&quot; src = &quot;/ files/ftp_upload/3367/3367fig3.jpg&quot; /&gt; Figura 3. Agregados RCC Crecido fueron incorporados en geles de fibrina. Invasión a través de gel de fibrina se observó ya en (A) 24 horas después de la incorporación de la fibrina y (B) continuó durante 48 horas. Las flechas indican las células invasoras y * indica que las cuentas Cytodex-3 microportadores (Adaptado, con permiso, de la referencia. 7). <img alt="Figura 4" src="/files/ftp_upload/3367/3367fig4.jpg" /> Figura 4. Zimograma comparativo de gelatina de monocapa y RCC propagado SGHPL-4 EVT-como las células. Forma pro-activa y de la MMP-2 y MMP-9 secretada por las células del trofoblasto RCC crecido (Adaptado, con permiso, de la referencia. 7). <img alt="Tabla 1" src="/files/ftp_upload/3367/3367table1.jpg" /> Tabla 1. Resumen de resultados de microarrays de SGHPL-4 células cultivadas en un RCC (Adaptado, con permiso, de la referencia. 7). <ol class="lalpha"><liInducción&gt; Doble en los agregados de 3-D se calculó de la siguiente manera: normalizado en 3-D del gen de valor / valor normalizado gen monlayer. </li><li> Indica que los genes que estaban por debajo del límite de detección en monocapas. </li></ol>

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No tenemos nada que revelar.

La técnica de cultivo que aquí se presenta proporciona a los investigadores altamente invasiva EVT-como las células. Ahora se ha reconocido que la pérdida de la diferenciación se produce en monocapas debido a la inhibición de la respuesta celular a agentes químicos y señales moleculares en tres dimensiones (la superficie celular apical, basal y lateral) 10,13. Esta técnica refleja características señaladas en el útero de invadir las células EVT. Como el procedimiento convencional imita e...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Cytodex microportadores cuentas </td><td> Sigma-Aldrich </td><td> C3275 </td><td></td></tr><tr><td> Celular sistema de rotación Cultura (CRC) </td><td> Synthecon </td><td> RCC-D </td><td> Incluye base de rotor de suministro, la energía, 4 unidades RCC desechables </td></tr><tr><td> RCC unidades desechables </td><td> Synthecon </td><td> Contacto Synthecon </td><td></td></tr><tr><td> 3 ml Luer-Lock punta de la jeringa </td><td> BD </td><td> 309585 </td><td></td></tr><tr><td> 10 ml de ancho de punta de la pipeta serológica </td><td> BD </td><td> 357504 </td><td></td></tr><tr><td> MEM Alfa </td><td> Invitrogen </td><td> 12561-072 </td><td></td></tr><tr><td> L-15 de Leibovitz medio, en polvo </td><td> Invitrogen </td><td> 41300-039 </td><td></td></tr><tr><td> H 2 O, endotoxinas </td><td> Pescador </td><td> MT-25-055-CM </td><td></td></tr><tr><td> Bicarbonato de sodio </td><td> Sigma-Aldrich </td><td> S-7795 </td><td></td></tr><tr><td> Peptona </td><td> Fisher Scientific </td><td> BP1420-100 </td><td></td></tr><tr><td> Fructosa </td><td> Sigma-Aldrich </td><td> F3510-100 </td><td></td></tr><tr><td> Galactosa </td><td> Sigma-Aldrich </td><td> G5388-100 </td><td></td></tr><tr><td> Glucosa </td><td> Sigma-Aldrich </td><td> G7528-250 </td><td></td></tr><tr><td> HEPES </td><td> Invitrogen </td><td> 15630-080 </td><td></td></tr><tr><td> L-glutamina </td><td> Invitrogen </td><td> 25030 </td><td></td></tr><tr><td> La insulina-transferrina-selenita de sodio (ITS) </td><td> Sigma-Aldrich </td><td> I1884 </td><td></td></tr><tr><td> FBS </td><td> Invitrogen </td><td> 10437 </td><td></td></tr><tr><td> Penicilina-estreptomicina </td><td> Invitrogen </td><td> 15140 </td><td></td></tr></tbody></table>

rotating cell culture systems for human cell culture: human trophoblast cells as a model

El campo de la investigación trofoblasto humano ayuda a comprender el complejo entorno establecido durante la placentación. Debido a la naturaleza de estos estudios, humanos en la experimentación in vivo es imposible. Una combinación de cultivos primarios, cultivos de explantes y las líneas de células del trofoblasto 1 para comprender mejor la invasión de la pared uterina 2 y remodelación de las arterias espirales uterinas por las células del trofoblasto 3,4 extravelloso (EVTS), que se requiere para el éxito del embarazo. A pesar de la riqueza de los conocimientos obtenidos de estos modelos, se acepta que en los modelos de cultivo celular in vitro utilizando EVT-como líneas celulares de pantalla alteración de las propiedades celulares en comparación con sus contrapartes en vivo 5,6. Las células cultivadas en el sistema de rotación de cultivo celular (RCC) mostrar las propiedades morfológicas, fenotípicas y funcionales de las líneas celulares EVT-como el que más se asemejan mucho en la diferenciación utero EVTS, con el aumento de expresión de los genes de la mediación invasión (por ejemplo, metaloproteinasas de matriz (MMP)) y la diferenciación del trofoblasto 7,8,9. El Hospital de Saint-Georges-de células placentarias Línea-4 (SGHPL-4) (donado amablemente por el Dr. Guy Whitley y la Dra. Judith Cartwright) es una línea de células EVT-como el que se utilizó para las pruebas de la RCC. El diseño del recipiente de cultivo RCC se basa en el principio de que los órganos y tejidos en función de tres dimensiones (3-D) el medio ambiente. Debido a las condiciones de cultivo en la dinámica del buque, incluidas las condiciones de corte fisiológicamente relevantes, las células cultivadas en tres dimensiones forman agregados basados ​​en afinidades naturales celular y diferenciarse en tejidos como organotípicos asambleas 10,11,12. El mantenimiento de una órbita fluido proporciona un bajo cizallamiento, baja turbulencia entorno similar a las encontradas en vivo. Sedimentación de las células cultivadas se ve contrarrestada por el ajuste de la rotaciónvelocidad de la RCC para asegurar una constante caída libre de las células. El intercambio gaseoso ocurre a través de una membrana permeable hidrofóbica en la parte posterior del biorreactor. Al igual que sus padres en el tejido vivo, RCC células cultivadas son capaces de responder a los químicos y los gradientes moleculares en tres dimensiones (es decir, en su superficie apical, basal y lateral), ya que se cultivan en la superficie de cuentas microportadores porosa. Cuando se cultiva en dos dimensiones monocapas sobre superficies impermeables como el plástico, las células se ven privadas de esta importante comunicación en su superficie basal. En consecuencia, las limitaciones espaciales impuestas por el medio ambiente afectan profundamente a cómo el sentido y las señales de las células de decodificar el microambiente que rodea, lo que implica un papel importante para el medio en 3-D 13. Hemos utilizado la RCC para diseñar biológicamente significativos modelos 3-D de diversos tejidos epiteliales humanas 7,14,15,16. De hecho, muchos informes anteriores han demonstrated que las células cultivadas en el RCC puede asumir fenotipos fisiológicamente relevantes que no han sido posible con otros modelos 10,17-21. En resumen, la cultura de la RCC representa una forma fácil, reproducible y de alto rendimiento plataforma que ofrece un gran número de células diferenciadas que son susceptibles a una variedad de manipulaciones experimentales. En el siguiente protocolo, utilizando EVTS como ejemplo, que describa claramente los pasos necesarios para cultivar células en tres dimensiones adherentes en la RCC.

Watch this Scientific Journal Video about Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model at JoVE.com

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

Tradicional de dos dimensiones técnicas de cultivo de células a menudo resultan en características alteradas con respecto a los marcadores de diferenciación, citoquinas y factores de crecimiento. En tres dimensiones de cultivo de células en el sistema de rotación de cultivo celular (RCC) restablece la expresión de muchos de estos factores, como se muestra aquí con una línea de células trofoblásticas extravelloso.

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

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

rotating-cell-culture-systems-for-human-cell-culture-human

Research

JoVE Journal

Bioengineering

16.4K vistas.  Tulane University Medical School. Tradicional de dos dimensiones técnicas de cultivo de células a menudo resultan en características alteradas con respecto a los marcadores de diferenciación, citoquinas y factores de crecimiento. En tres dimensiones de cultivo de células en el sistema de rotación de cultivo celular (RCC) restablece la expresión de muchos de estos factores, como se muestra aquí con una línea de células trofoblásticas extravelloso.

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

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

Plataformas microfabricated para el cultivo celular mecánico dinámico

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

Investigación

Bioingeniería

Shape Memory Polymers for Active Cell Culture

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

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

Los polímeros con memoria de forma para el cultivo celular activo

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

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

Microcápsulas de alginato como una plataforma 3D para la propagación y diferenciación de las células estaminales embrionarias humanas (CMEH) para los diferentes linajes

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 &mu;m s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.

Tridimensional de la célula modelo de cultivo para medir los efectos del flujo del fluido intersticial en la invasión de células tumorales

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

Co-cultivo celular Patterning Uso acuoso de dos fases Sistemas

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to model well differentiated human airway epithelium and heart muscle cells. This could be an invaluable tool to study the deleterious effects of toxic inhaled chemicals, such as chlorine, that can normally interact with the cell surfaces, and form various byproducts upon reacting with water, and limiting their effects in submerged cultures. Our model using well differentiated human airway epithelial cell cultures at air-liqiuid interface circumvents this limitation as well as provides an opportunity to evaluate critical mechanisms of toxicity of potential poisonous inhaled chemicals. We describe enhanced loss of membrane integrity, caspase release and death upon toxic inhaled chemical such as chlorine exposure. In this article, we propose methods to model chlorine exposure in mammalian heart and airway epithelial cells in culture and simple tests to evaluate its effect on these cell types.

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

This protocol is designed to demonstrate exposure method of cell cultures to inhaled toxic chemicals. Exposure of differentiated air-liquid&#160;interface (ALI) cultures of airway epithelial cells provides a unique model of airway exposure to toxic gases such as chlorine. In this manuscript we describe effect of chlorine exposure on air-liquid&#160;interface cultures of epithelial cells and submerged culture of cardiomyocytes. In vitro exposure systems allow important mechanistic studies to evaluate pathways that could then be utilized to develop novel therapeutic agents.

In vitro modelo de cultivo celular para Toxic inhalado Prueba química

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

Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies of endothelial-epithelial bilayers have traditionally relied on permeable support models that enable bilayer culture, but permeable supports are limited in their ability to replicate the diversity of human basement membranes. In contrast, hydrogel models that require chemical synthesis are highly tunable and allow for modifications of both the material stiffness and the biochemical composition via incorporation of biomimetic peptides or proteins. However, traditional hydrogel models are limited in functionality because they lack pores for cell-cell contacts and functional in vitro migration studies. Additionally, due to the thickness of traditional hydrogels, incorporation of pores that span the entire thickness of hydrogels has been challenging. In the present study, we use poly-(ethylene-glycol) (PEG) hydrogels and a novel zinc oxide templating method to address the previous shortcomings of biomimetic hydrogels. As a result, we present an ultrathin, basement membrane-like hydrogel that permits the culture of confluent cellular bilayers on a customizable scaffold with variable pore architectures, mechanical properties, and biochemical composition.

Current bilayer culture models do not allow for functional in vitro studies that mimic in vivo microenvironments. Using polyethylene glycol and a zinc oxide templating method, this protocol describes the development of an ultrathin biomimetic basement membrane with tunable stiffness, porosity, and biochemical composition that closely mimics in vivo extracellular matrices.

Hidrogeles ultrafino Porated elástico como una membrana del sótano biomiméticos para doble cultura de la célula

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies ...

Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by several researchers. However, most of these investigators have used polyacrylamide hydrogels for stem cell culture in their studies. Therefore, their results are controversial because those results might originate from the specific characteristics of the polyacrylamide and not from the physical cue (stiffness) of the biomaterials. Here, we describe a protocol for preparing hydrogels, which are not based on polyacrylamide, where various stem, cells including human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells, can be cultured. Hydrogels with varying stiffness were prepared from bioinert polyvinyl alcohol-co-itaconic acid (P-IA), with stiffness controlled by crosslinking degree by changing crosslinking time. The P-IA hydrogels grafted with and without oligopeptides derived from extracellular matrix were investigated as a future platform for stem cell culture and differentiation. The culture and passage of amniotic fluid stem cells, adipose-derived stem cells, human ES cells, and human iPS cells is described in detail here. The oligopeptide P-IA hydrogels showed superior performances, which were induced by their stiffness properties. This protocol reports the synthesis of the biomaterial, their surface manipulation, along with controlling the stiffness properties and finally, their impact on stem cell fate using xeno-free culture conditions. Based on recent studies, such modified substrates can act as future platforms to support and direct the fate of various stem cells line to different linkages; and further, regenerate and restore the functions of the lost organ or tissue.

A protocol is presented to prepare polyvinyl alcohol-co-itaconic acid hydrogels with varying stiffness, which were grafted with and without oligopeptides, to investigate the effect of the stiffness of biomaterials on the differentiation and proliferation of stem cells. The stiffness of the hydrogels was controlled by the crosslinking time.

Cultura de células madre humanas pluripotentes en polivinilos hidrogeles ácidos itacónico-Co-Alcohol con rigidez variable en condiciones libres de Xeno

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by ...

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

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

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

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

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

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Medición del potencial de acción óptica unicelular en cardiomiocitos derivados de células madre pluripotentes inducidas por humanos

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...