Name: layered alginate constructs: a platform for co-culture of heterogeneous cell populations
Uploaded: 2016-08-07T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations at JoVE.com

This work was funded by the National Science Foundation (CBET 0845754, AHH).

1. Preparación para la Formación de Discos de alginato <ol><li> Preparar un 4% (w / v) en solución de alginato de 1x solución salina tamponada con fosfato de Dulbecco sin cloruro de calcio añadido o cloruro de magnesio y el lugar en un baño de agua C 37 °. Las concentraciones de la solución de alginato pueden variar, pero 1-4% (w / v) se recomiendan las soluciones de alginato. </li><li> Mezclar la solución de alginato en una proporción de 1: 1 con la base de medios de cultivo celular caliente (por ejemplo, de Eagle modificado por Dulbecco.) Para el tipo de célula deseado. La concentración de alginato es ahora la mitad de la concentración original, es decir., 2% (w / v). Esterilizar solución de alginato / media usando filtros de jeringa de nylon 0,2 micras estériles. </li><li> Preparar un baño de estéril cloruro de calcio dihidrato 102 mM en agua estéril con una solución suficiente para sumergir moldes (~ 200 - 300 ml). </li><li> Recoger el &quot;molde de formación de gel&quot; estéril (diámetro x 3 mm de altura pozos cilíndricos de 6 mm en un 3 x 3 en la placa de aluminio, véase <strong&gt; Figura 1) y placas de extremo (inferior: uno de 3 x 3 en la placa de aluminio, superior: uno de 1,5 pulgadas x 3 en la placa de aluminio) y preparar los moldes de la siguiente manera: <ol><li> Cortar papel grueso filtro (filtro de papel secante) y la membrana celular microtamiz (10 micras de tamaño de poro) para los tamaños de la parte superior e inferior placas terminales y colocarlos en el baño de cloruro de calcio hasta saturado, aproximadamente 1 min. Si se desea, esterilizar el papel de filtro grueso y la membrana a través de microtamiz autoclave o la luz ultravioleta durante 30 minutos antes de su uso. </li><li> Puesta en marcha de la mitad (la mitad inferior) de la construcción de moldes. <ol><li> Coloque los siguientes artículos encima de la otra en el orden siguiente y suavizar con una espátula estéril: gran placa de extremo (3 x en 3 in), papel de filtro grueso, y luego la membrana microtamiz. </li><li> Invertir y pulse el molde sobre una pila de toallas de papel para asegurar la pérdida de exceso de solución de cloruro de calcio. </li><li> Coloque el &quot;molde de formación de gel&quot; con los pozos cilíndricos en la parte superior de la cell membrana microtamiz y suavemente sujetar el molde juntos en dos lados con clips de la carpeta en los lados derecho e izquierdo. Asegúrese de dejar suficiente espacio para que la placa terminal superior (1,5 pulgadas x 3 pulgadas) para cubrir los pozos por completo más adelante. </li></ol></li><li> Puesta en marcha de la segunda mitad (la mitad superior) de la construcción de moldes. <ol><li> Coloque los siguientes elementos uno encima de otro en el siguiente orden y suavizar: pequeña placa terminal, papel de filtro grueso, microtamiz membrana. </li><li> Invertir y pulse este medio del molde sobre una pila de toallas de papel para asegurar la pérdida de exceso de solución de cloruro de calcio. No fije esta mitad del molde usando clips de la carpeta en este momento. </li></ol></li></ol></li><li> células de cultivo, tal como recomiendan las instrucciones del fabricante. Recoger las células deseadas para empotrar en los hidrogeles de alginato en capas. La densidad celular recomendada es de 1 - 2 x 10 6 células / ml, pero esto se puede variar dependiendo de los experimentos deseados. Nota: Cuando se utiliza este métodopara la fabricación de capas con diferentes tipos de células, asegúrese de que la cultura dos tipos de células en paralelo para todas las ocasiones. Las células madre mesenquimales (MSCs) se sembraron a una densidad celular de las células 1 x 10 6 / ml se utilizará como un ejemplo para el siguiente protocolo. El número de células y el número de disco que se pueden escalar para los experimentos según sea necesario. <ol><li> De una a dos semanas antes de la incrustación, las células madre mesenquimales de semillas a una densidad celular de 3.000 - 5.000 células / cm 2 en 10 ml de medio de crecimiento basal (de Eagle modificado por Dulbecco, 10% de suero fetal bovino, 2% de L-glutamina, 1 % no esenciales Aminoácidos, 1% penicilina / estreptomicina) en matraces T-75. </li><li> Retire el medio pasó cada 2 - 3 días y reemplazar con 10 ml de medio de cultivo basal hasta que las células son un 80 - 90% de confluencia. </li><li> Para cosechar las células, eliminar el medio gastado, y la pipeta de 3 ml de 1x solución salina tamponada con fosfato de Dulbecco sin cloruro de calcio añadido o cloruro de magnesio para enjuagar las células. Eliminar a esta solución, pipeta de 3 ml de 0,25% Trypsin-EDTA en células que crecen en matraces T-75, y se incuba durante 5 min a 37 ° C. Después las células se han levantado de la superficie adherente, añadir 6 - 9 ml de medio de crecimiento basal. </li><li> MSC centrifugar a 600 xg durante 5 minutos a temperatura ambiente, aspirar el sobrenadante y resuspender en 1 - 5 ml de medio de cultivo basal. Contar las células utilizando un hemocitómetro usando las instrucciones del dispositivo. </li><li> Eliminar 1 x 10 6 células de la resuspensión celular total, de centrifugación utilizando las mismas condiciones, y aspirar el sobrenadante. </li></ol></li></ol> 2. Formación de sembrado de células discos de alginato <ol><li> Vuelva a suspender el sedimento de células con 1 ml de la solución de alginato / media estéril para alcanzar una densidad celular de 1 x 10 6 células / ml. La mezcla de células-alginato será homogénea nublado en apariencia cuando las células se mezclan en forma apropiada. </li><li> Pipetear 130 l de la mezcla de células de alginato en los pocillos de altura seis 6 mm de diámetro x 3 mm de la mitad inferior del moldeconstruir gota a gota, a fin de no crear burbujas. Una ligera menisco convexo debe ser visible por encima del borde de cada pocillo. </li><li> suavizar cuidadosamente la mitad superior de la construcción del molde con una espátula estéril y darle la vuelta, por lo que la membrana celular es microtamiz en la parte superior de los pozos. Coloca el molde construir en la parte superior de los pozos, asegurándose de cubrir los pozos que contienen el celular y la mezcla de alginato por completo. </li><li> Levante la construcción del molde cargado y, mientras presiona firmemente en el centro, Clip de la carpeta de los otros dos lados (superior e inferior) para sujetar las mitades superior e inferior de la construcción del molde. Las soluciones de alginato por células deben ser enclavado de forma segura en los pocillos en este momento. </li><li> Sumergir el constructo molde fijado en el baño de cloruro de calcio 102 mM, asegurándose de que toda la estructura se sumerge. Incubar en la campana de cultivo de células a temperatura ambiente durante 90 min. </li><li> Al final de la 90 min, eliminar el constructo del molde a partir del cloruro de calcio 102 mMbaño y el lugar en una pila de toallas de papel en la campana de cultivo de células. Retire los cuatro clips de la carpeta y separar las dos mitades de la construcción del molde. Los hidrogeles formados en los pozos no deben tener ningún burbujas y deben llenar los pozos por completo. </li><li> Usando una espátula, rastrear cuidadosamente el borde de los pocillos que contienen los hidrogeles para aflojar cuidadosamente y calzar los hidrogeles. Después de la eliminación de los hidrogeles de la construcción del molde, la caída de los hidrogeles directamente en un baño de de 1x solución salina tamponada con fosfato de Dulbecco con cloruro de calcio y cloruro de magnesio. Cubrir por completo los geles para lavar el exceso de solución de cloruro de calcio durante 1 - 5 min. </li><li> La transferencia de los hidrogeles en solución basal medio de crecimiento en el plato deseado (por ejemplo., Placas de 6 pocillos) que cubre completamente los hidrogeles. Incubar durante al menos 1 hr en la incubadora de cultivo celular a 37 ° C y CO 2 antes de continuar a la etapa de capas de 5%. Nota: Los hidrogeles que contienen células se pueden mantener ense desea la incubadora de cultivo celular indefinidamente en este momento hasta la estratificación de los geles con otra población de células, siempre que los cambios de medios de comunicación se han completado cada 2 - 3 días. </li></ol> 3. Estratificación de Discos de alginato <ol><li> Durante la última media hora de la incubación de 1 hora, recogida, preparación de moldes y solución estéril para recocer los geles. <ol><li> Preparar el &quot;molde de corte&quot; por la fijación de un molde que tiene pozos medio de la altura de la &quot;molde de formación de gel&quot; (pozos de altura 6 mm de diámetro x 1,5 mm en un 3 en x 3 en la placa de aluminio) a una placa de extremo (3 en x 3 en la placa de aluminio) utilizando clips de la carpeta en los lados izquierdo y derecho. </li><li> Preparar el &quot;molde de recocido&quot; por la fijación de un molde que es de 3 mm de diámetro mayor que el &quot;molde de formación de gel&quot; (9 mm de diámetro x 3 mm pozos altos en un 3 en x 3 en la placa de aluminio) a una placa de extremo (3 en x 3 en la placa de aluminio) utilizando clips de la carpeta en los lados izquierdo y derecho. </li><li> Preparar una solución de100 mM de citrato de sodio / 30 mM EDTA (dihidrato de citrato de sodio, ácido etilendiaminotetraacético tetrasódico dihidrato de sal) en agua estéril. </li></ol></li><li> Eliminar geles de poblaciones de células deseadas (en este ejemplo, dos discos con hMSCs) de los medios de comunicación en el plato, y el lugar en los pocillos de corte &quot;de molde&quot;. Cada gel deben encajar perfectamente en los pocillos con medio de gel que sobresale por encima del molde. </li><li> El uso de un bisturí, cortar el gel a lo largo de la superficie del molde (esto reducirá el hidrogel en medio). Voltear la mitad superior del gel y colocarlo en un pozo de molde abierto. La media del gel también deben encajar perfectamente en el molde así, pero ahora ambos geles medio debe ser la altura del molde con la superficie interna de corte visible. Repita con el segundo gel. Nota: Advertencia: Solo las superficies de corte interiores formarán discos en capas. El uso de las superficies externas dará lugar a la separación mitades. Se sospecha que la textura de la membrana microtamiz transferido sobre la superficie del gel impidens éxito del proceso de recocido. </li><li> Coloque un trozo de membrana microtamiz celular seco en la parte superior de los pocillos, asegurándose de que la membrana microtamiz está en contacto con todas las mitades de gel para ser recocido y que cubre por completo. Colocar el papel filtro grueso en la parte superior de la membrana microtamiz, asegurándose de cubrir por completo los geles. </li><li> Pipetear una solución de citrato de sodio 100 mM / mM EDTA 30 (dihidrato de citrato de sodio, ácido etilendiaminotetraacético tetrasódico dihidrato de sal) en el papel de filtro grueso hasta que se satura. Aproximadamente 750 l es suficiente para cuatro pozos. </li><li> Incubar los geles durante 1 min a temperatura ambiente. A continuación, retire la membrana celular y microtamiz papel de filtro grueso y desecharlos. Retire los clips de la carpeta y abrir el molde. </li><li> Para cada gel de recocido, transferir un citrato de sodio / la mitad tratada con EDTA del hidrogel preparado a la &quot;molde de recocido&quot; con la superficie cortada hacia arriba. Esta será la mitad de la const recocidoRUCT. </li><li> Usando una espátula, levantar y colocar un citrato segundo sódico / EDTA tratados con medio-gel (puede contener un tipo de célula diferente) en el gel ya en el &quot;molde de recocido&quot;, volteando este segundo medio-gel de manera que la superficie de corte está en contacto con la superficie de corte del gel ya en el &quot;molde de recocido&quot;. </li><li> Presione suavemente hacia abajo los dos geles utilizando una espátula para eliminar las burbujas entre los dos geles. Además, cambiar la posición de los geles según sea necesario para asegurarse de que son directamente una encima de la otra. </li><li> Levante con cuidado el &quot;molde de recocido&quot; y sumergirlo en el baño de cloruro de calcio 102 mM durante 30 min. No cubra los pocillos con una placa terminal. </li><li> Después de la incubación de 30 minutos, retire el &quot;molde de recocido&quot; y colocarlo sobre toallas de papel. Retire los clips de la carpeta y separar el molde de la placa terminal. </li><li> Usando una espátula, recoger los geles recocidos y los situará en una de Dulbecco 1x Tampón fosfato salino con cloruro de calcio y mbaño de cloruro de agnesio para lavar los geles. Posteriormente, transferir hidrogeles recocidos para medios de cultivo celular en un plato deseado (por ejemplo., Placa de 6 pocillos) para la cultura. </li></ol>

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The authors have nothing to disclose.

Aquí, se describe un protocolo para la formación de discos de hidrogel de alginato en capas para el estudio de co-cultivos de múltiples poblaciones de células, tales como los de tejidos fisiológicamente en capas, por ejemplo, el cartílago. estructuras en capas, tales como la plataforma de cultivo descrito, se pueden utilizar para examinar la interacción entre las dos poblaciones de células distintas sometidas al mismo entorno de cultivo o bajo carga. El alginato es un polisa...

los tejidos de soporte de carga de compresión tales como el cartílago articular o discos intervertebrales consisten en regiones de tejido heterogéneas que son críticos tanto para la función biomecánica y apropiado mecano-transducción en el tejido. No sólo es la organización celular y función distinta en las diferentes regiones, pero las matrices extracelulares (ECM) son también variados en su composición y organización. Por ejemplo, el cartílago articular se compone de tres zonas principales con diferentes morfología celular, la función mecánica, y ECM. Las diferencias en su ventaja ECM a la diferencia de las responsabilidades de soporte de carga; la capa superficial está implicado principalmente en respuesta a la tracción a la carga, mientras que las zonas medias y profundas son principalmente responsables de la respuesta a la compresión 1. Del mismo modo, en el disco intervertebral, un núcleo pulposo de gel está rodeada por una fibrosis lamelar anillo y las células dentro de estas dos áreas distintas experimentar diferentes tipos de estímulos biofísicos2. En estos tipos de tejidos, células y las matrices extracelulares dentro de las capas de tejido interactúan entre sí ya que el tejido se somete a y responde a fuerzas mecánicas. Recapitulación de tales estructuras de tejidos heterogéneos sigue siendo un desafío en ingeniería de tejidos y medicina regenerativa, y nuestra comprensión de su importancia biológica es limitada. Hay una necesidad para el cultivo de las plataformas para el análisis de tejidos estratificados, así como co-cultivos de diferentes poblaciones de células dentro de una construcción. En la ingeniería de tejido de cartílago articular, las construcciones de andamio-menos capas se han construido mediante el aprovechamiento de la capacidad de los condrocitos de zona para depositar variada ECM para imitar las diferentes capas de este tejido 3,4. Sin embargo, las construcciones de hidrogel en capas proporcionan una oportunidad para la investigación de la interacción de diversos tipos de poblaciones de células que carecen de la capacidad para formar un tejido robusto de forma independiente. Por ejemplo, el pop diferenteulations de células madre mesenquimales se puede co-cultivadas dentro de construcciones en capas. Tales andamios capas se han utilizado con ambos condrocitos y diferenciación de las células madre mesenquimales para la ingeniería de tejidos mejorada 5. No sólo se pueden cultivar conjuntamente diferentes poblaciones de células en capas de hidrogel similares, pero un solo tipo de células también pueden ser cultivadas dentro de las capas que han sido manipulados para tener rigidez variable o contenido bioquímico para provocar diferentes respuestas de las células 6,7. Muchos diferentes hidrogeles de biomateriales se han utilizado para la capa poblaciones de células para la ingeniería de tejido de cartílago, tales como los que utilizan polietilenglicol o bases alcohol polivinílico 7-9. Sin embargo, los hidrogeles de alginato son uno de los biomateriales más simples desde el que crear andamios en capas para el estudio de las poblaciones de células heterogéneas en co-cultivo. Mientras que los hidrogeles de agarosa también se forman fácilmente, los hidrogeles de alginato tienen la ventaja añadida de que permite un fácil esolation de células de la construcción de 3-D para el análisis de células individuales como se ha descrito previamente 10. En estudios anteriores, los hidrogeles de alginato bi-capas se han formado en láminas delgadas y de estas hojas, se cortaron secciones (por ejemplo., Usando un punzón de biopsia) para aplicaciones particulares, tales como para el análisis de contenido bioquímico o propiedades de cizallamiento interfacial 11,12. Otro método para la formación de hojas de alginato delgadas se ha descrito con el potencial para la estratificación en múltiples capas, pero aún así se requiera la modificación al hidrogel para su uso en ensayos mecánicos 13. A continuación, presentamos un método para crear de forma reproducible discos de hidrogel de alginato bi-capas para su uso en el cocultivo de diferentes poblaciones de células. Esta plataforma disco alginato posee varias ventajas. En primer lugar, la forma reproducible y pequeño tamaño es propicio para la estimulación mecánica de las células embebidas sin necesidad de una biopsia de la PUnch u otra alteración física al hidrogel para muchas aplicaciones. Además, la viabilidad celular se mantiene alta durante el proceso de estratificación, y después de la formación de gel de una clara separación de las dos poblaciones de células dentro del gel es visible sin región de superposición inicial.

<table><tbody><tr><td>Alginic Acid sodium salt</td><td>Sigma Aldrich</td><td>A1112</td><td>Solution made in wt% using DPBS (-/-)</td></tr><tr><td>1x Dulbecco&#039;s Phosphate Buffered Saline (-/-)</td><td>Gibco Life Technologies&amp;nbsp;</td><td>14190</td><td></td></tr><tr><td>1x Dulbecco&#039;s Phosphate Buffered Saline (+/+)</td><td>Gibco Life Technologies&amp;nbsp;</td><td>14190</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium</td><td>Gibco Life Technologies&amp;nbsp;</td><td>11965</td><td>Example. Use desired medium type</td></tr><tr><td>Syringe Filters (0.02 &amp;#956;m Nylon)</td><td>FisherBrand</td><td>0979C</td><td></td></tr><tr><td>Calcium Chloride Dihydrate</td><td>Fisher Bioreagents</td><td>BP510</td><td>Prepare solution in sterile water</td></tr><tr><td>Criterion Blotter Filter Paper</td><td>Biorad</td><td>1704085</td><td>Cut to size of endplates for mold formation</td></tr><tr><td>Cell Microsieve Membrane (10 &amp;#956;m pore size)</td><td>Biodesign Inc of New York</td><td>N10R</td><td>Cut to size of endplates for mold formation</td></tr><tr><td>Sodium citrate dihydrate</td><td>FisherScience</td><td>S93364</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid tetrasodium salt dihydrate (EDTA)</td><td>FisherBioreagent</td><td>BP121</td><td></td></tr><tr><td>Fetal Bovine Serum&amp;nbsp;</td><td>Gibco Life Technologies&amp;nbsp;</td><td>26140</td><td>Used in example mesenchymal stem cell basal growth media</td></tr><tr><td>Penicilin/Streptomycin (10,000 U/ml)</td><td>Gibco Life Technologies&amp;nbsp;</td><td>15140</td><td>Used in example mesenchymal stem cell basal growth media</td></tr><tr><td>L-Glutamine (200 mM)</td><td>Gibco Life Technologies&amp;nbsp;</td><td>25030081</td><td>Used in example mesenchymal stem cell basal growth media</td></tr><tr><td>Non-essential Amino Acids (100x)</td><td>Gibco Life Technologies&amp;nbsp;</td><td>11140050</td><td>Used in example mesenchymal stem cell basal growth media</td></tr></tbody></table>

layered alginate constructs: a platform for co-culture of heterogeneous cell populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

La Figura 1 muestra la formación de capas y de los hidrogeles de alginato. Completado geles bi-capas exhiben una separación inicial completa de las poblaciones de células como se muestra en la Figura 2. La viabilidad celular de las células madre mesenquimatosas humanas) embebido dentro de estos hidrogeles y capas sigue siendo alta y comparable a los hidrogeles a granel como se muestra en la Figura 3. La viabilidad se evaluó después...

Watch this Scientific Journal Video about Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations at JoVE.com

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

Las construcciones en capas de alginato: Una plataforma para el co-cultivo de poblaciones celulares heterogéneas

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

layered-alginate-constructs-platform-for-co-culture-heterogeneous

Research

JoVE Journal

Bioengineering

7.8K vistas.  University of Maryland. Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct. 

Video: Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

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

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

Los hidrogeles de alginato para la cultura del órgano en tres dimensiones de los ovarios y oviductos

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

La encapsulación de alginato de células madre pluripotentes utilizando una boquilla-Co axial

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

Biología del Desarrollo

Generation of Alginate Microspheres for Biomedical Applications

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and physical architecture. Applications include cell encapsulation, drug delivery, stem cell culture, and tissue engineering scaffolds. In fact, clinical trials are currently being performed in which islets are encapsulated in PLO coated alginate microbeads as a treatment of type I diabetes. However, large numbers of islets are required for efficacy due to poor survival following transplantation. The ability to locally stimulate microvascular network formation around the encapsulated cells may increase their viability through improved transport of oxygen, glucose and other vital nutrients. Fibroblast growth factor-1 (FGF-1) is a naturally occurring growth factor that is able to stimulate blood vessel formation and improve oxygen levels in ischemic tissues. The efficacy of FGF-1 is enhanced when it is delivered in a sustained fashion rather than a single large-bolus administration. The local long-term release of growth factors from islet encapsulation systems could stimulate the growth of blood vessels directly towards the transplanted cells, potentially improving functional graft outcomes. In this article, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. In addition, we describe a method we developed for generating multilayered alginate microbeads. Cells can be encapsulated in the inner alginate core, and angiogenic proteins in the outer alginate layer. The release of proteins from this outer layer would stimulate the formation of local microvascular networks directly towards the transplanted islets.

In the following sections, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. We specifically illustrate a technique for creating multilayered alginate microspheres for the dual purpose of cell and protein encapsulation as a potential treatment for type 1 diabetes.

Generación de microesferas de alginato para aplicaciones biomédicas

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and ...

Medicina

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, ...

Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue geometry. The resulting 3D hydrogel-based cellular constructs have been introduced as an alternative to animal experiments for advanced biological studies, pharmacological assays and organ transplant applications. Although hydrogel-based particles and fibers can be easily fabricated, it is difficult to manipulate them for tissue reconstruction. In this video, we describe a fabrication method for micropatterned alginate hydrogel sheets, together with their assembly to form a macro-scale 3D cell culture system with a controlled cellular microenvironment. Using a mist form of the calcium gelling agent, thin hydrogel sheets are easily generated with a thickness in the range of 100 - 200 &#181;m, and with precise micropatterns. Cells can then be cultured with the geometric guidance of the hydrogel sheets in freestanding conditions. Furthermore, the hydrogel sheets can be readily manipulated using a micropipette with an end-cut tip, and can be assembled into multi-layered structures by stacking them using a patterned polydimethylsiloxane (PDMS) frame. These modular hydrogel sheets, which can be fabricated using a facile process, have potential applications of in vitro drug assays and biological studies, including functional studies of micro- and macrostructure and tissue reconstruction.

We describe the fabrication of micropatterned hydrogel sheets using a simple process, which can be assembled and manipulated in a freestanding form. Using these modular hydrogel sheets, a simple macro-scaled 3D cell culture system can be generated with a controlled cellular microenvironment.

Construcción de Hojas modular de hidrogel para la arquitectura celular 3D a escala macro micropatterned

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue ...

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 ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

Tridimensional de alginato-grano Cultura de células humanas del adenoma pituitario

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Core/shell Printing Scaffolds For Tissue Engineering Of Tubular Structures

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the interface with a liquid core. The latter is removed post-printing, leaving behind a hollow tube. Integrating an additive manufacturing technique (like the one described here with tailor-made [bio]inks, which structurally and biochemically mimic the native extracellular matrix [ECM]) is an important step towards advanced tissue engineering. However, precise fabrication of well-defined structures requires tailored fabrication strategies optimized for the material in use. Therefore, it is sensible to begin with a set-up that is customizable, simple-to-use, and compatible with a broad spectrum of materials and applications. This work presents an easy-to-manufacture core/shell nozzle with luer-compatibility to explore core/shell printing of woodpile structures, tested with a well-defined, alginate-based scaffold material formulation.

Presented here is a simple-to-use, core/shell, three-dimensional bioprinting set-up for one-step fabrication of hollow scaffolds, suitable for tissue engineering of vascular and other tubular structures.

Andamios de impresión de núcleo/concha para la ingeniería de tejidos de estructuras tubulares

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the ...

Microgel-Extracellular Matrix Composite Support for the Embedded 3D Printing of Human Neural Constructs

The embedded 3D printing of cells inside a granular support medium has emerged in the past decade as a powerful approach for the freeform biofabrication of soft tissue constructs. However, granular gel formulations have been restricted to a limited number of biomaterials that allow for the cost-effective generation of large amounts of hydrogel microparticles. Therefore, granular gel support media have generally lacked the cell-adhesive and cell-instructive functions found in the native extracellular matrix (ECM).
To address this, a methodology has been developed for the generation of self-healing annealable particle-extracellular matrix (SHAPE) composites. SHAPE composites consist of a granular phase (microgels) and a continuous phase (viscous ECM solution) that, together, allow for both programmable high-fidelity printing and an adjustable biofunctional extracellular environment. This work describes how the developed methodology can be utilized for the precise biofabrication of human neural constructs.
First, alginate microparticles, which serve as the granular component in the SHAPE composites, are fabricated and combined with a collagen-based continuous component. Then, human neural stem cells are printed inside the support material, followed by the annealing of the support. The printed constructs can be maintained for weeks to allow the differentiation of the printed cells into neurons. Simultaneously, the collagen continuous phase allows for axonal outgrowth and the interconnection of regions. Finally, this works provides information on how to perform live-cell fluorescence imaging and immunocytochemistry to characterize the 3D-printed human neural constructs.

This work describes a protocol for the freeform embedded 3D printing of neural stem cells inside self-healing annealable particle-extracellular matrix composites. The protocol enables the programmable patterning of interconnected human neural tissue constructs with high fidelity.

Soporte compuesto de matriz extracelular de microgel para la impresión 3D integrada de construcciones neuronales humanas

The embedded 3D printing of cells inside a granular support medium has emerged in the past decade as a powerful approach for the freeform biofabrication ...