Damos las gracias a U. Ganapati para el establecimiento y pruebas de la línea celular mStrawberry OP9. Además, agradecemos a H. Shafifor su ayuda con el co-cultivo. HP es apoyado por una beca del Instituto Nacional del Corazón, los Pulmones y la Sangre (F31HL087714) (HP). El contenido de esta obra es de la exclusiva responsabilidad del autor y no necesariamente representa la posición oficial del Instituto Nacional del Corazón, los Pulmones y la Sangre o de los Institutos Nacionales de Salud (NIH). El flujo de UCLA Citometría Core Facility es apoyado por el NIH (CA-16042 y AI-28697), el Centro del Cáncer Jonsson, el Instituto de SIDA de UCLA, y la Escuela de Medicina de UCLA.

1. Preparación de células madre embrionarias <ol><li> Cultivo de células ES de acuerdo con el protocolo estándar para la línea celular particular. Asegúrese de que la preparación suficiente de células ES de placa en el mStrawberry OP9 células para el día 0 (células madre embrionarias se sembraron a 1x10 ^ 3 células por cm 2 en el día 0) </li></ol> 2. Preparación de las células OP9 <ol><li> Antes de crecer mStrawberry OP9 células, preparar el OP9 medios de cultivo OP-9 medio de cultivo celular <table border="1"><tbody><tr><td></td><td> Valores </td><td> Conc final. </td><td> Volumen (ml) </td></tr><tr><td> AMEM </td><td></td><td></td><td> 39.5 </td></tr><tr><td> FBS (OP-9 pruebas) </td><td> 100% </td><td> 20% </td><td> 10 </td></tr><tr><td> L-glutamina </td><td> 100X (200 mm) </td><td> 2 mM </td><td> 0.5 </td></tr><tr><td></td><td></td><td></td><td> 50 ml </td></tr></tbody></table> Almacenar a 4 ° C y utilizar dentro de una semana de preparación </li><li> A fin de mantener mStrawberry OP9 las células, los medios de comunicación se debe cambiar cada 2 días y las células se subcultivaron antes de llegar a las células la confluencia (subcultura en 70-90% de confluencia). mStrawberry OP9 cultivos de células no se cultivan más de 90% de confluencia, ya que dará lugar a su diferenciación en adipocitos. Véase la figura 3. <ol class="lalpha"><li> Las células de la subcultura de las células de lavado 2 veces con PBS (1x o con PBS 1x + 0,1% con tripsina-EDTA) </li><li> Añadir pre-calentado 0,1% de tripsina-EDTA a 37 ° C durante 5 minutos (o hasta que las células separar) </li><li> Cuando las células se han desprendido, añada OP-9 el medio de cultivo. La transferencia de células a un tubo de 50 ml cónicos. </li><li> Precipitar las células a 300 xg durante 5 minutos a temperatura ambiente. Desechar el sobrenadante. </li><li> Resuspender las células en los medios de comunicación fresca mStrawberry crecimiento OP9 </li><li> Células de la placa a una densidad mínima de 10 4 células / cm 2 </li><li> Crecer mStrawberry OP9 células hasta la confluencia a partir de 3 días antes del día necesarias para el cultivo de células madre embrionarias co-(día 0, día 5, o Day8 / 9 del experimento) </li></ol></li></ol> 3. ES / mStrawberry OP9 Co-cultura <ol><li> Prepare la cultura co-medio de diferenciación <table border="1"><tbody><tr><td></td><td> Valores </td><td> Final </td><td> Volumen (ml) </td></tr><tr><td> AMEM </td><td></td><td></td><td> 39.5 </td></tr><tr><td> FBS </td><td> 100% </td><td> 20% </td><td> 10 </td></tr><tr><td> L-glutamina </td><td> 200 mM </td><td> 2 mM </td><td> 0.5 </td></tr><tr><td></td><td></td><td></td><td> 50 ml </td></tr></tbody></table></li><li> mStrawberry OP9 células (Día -3) <ol class="lalpha"><li> SIEMPRE Subcultura OP9 células el día -3, de modo que usted tiene capas confluentes de células mStrawberry OP9 para el día 0 de co-cultivo. Vea la Figura 4b. Placa mStrawberry OP9 células a 1x10 4 / cm 2 para la confluencia en el día 0. Asegúrese de subcultura de células adicionales para los días 5 y 8.9 para la continuación de co-cultivo. </li></ol></li><li class="jove_content"> ES / mStrawberry OP9 co-cultivo de configuración (día 0) <ol class="lalpha"><li class="jove_content"> Lavar las células ES 2 veces con PBS </li><li> Trypsinize células madre embrionarias con pre-calentado 0,25% de tripsina-EDTA, se incuba durante 5 min a 37 ° C </li><li> Añadir la cultura co-medio de diferenciación de las células </li><li> Pellet ES células en 400xg, 5-7min, RT. Desechar el sobrenadante </li><li> Resuspender las células en medio de diferenciación de co-cultivo </li><li> Quite el papel del mStrawberry OP9 células </li><li> Añadir la cultura co-medio de diferenciación para mStrawberry frascos OP9 </li><li> Placa de 1x10 3 células ES / cm 2 al matraz de mStrawberry OP9 células. Se incuban las células a 37 ° C, 5% de CO 2 con la humedad. </li></ol></li><li> mStrawberry OP9 células (Día 2) <ol class="lalpha"><li> MStrawberry subcultura OP9 para que tenga capas confluentes de células mStrawberry OP9 para el día 5 de co-cultivo. Placa mStrawberry OP9 células a 1x10 4 / cm 2 para la confluencia en el Día 5 (como en el paso 3.2). Asegúrese de subcultura de células adicionales para el día 8 / 9, si es necesario (como en el paso 2.2). </li></ol></li><li> ES / mStrawberry OP9 co-cultivo (Día 3) <ol class="lalpha"><li> Retire con cuidado el medio de co-cultivo y reemplazar con nuevos co-medio de cultivo de diferenciación </li></ol></li><li> ES / mStrawberry OP9 co-cultivo (día 5) <ol class="lalpha"><li> Lave cuidadosamente co-cultivos con PBS 2 veces </li><li> Trypsinize células pre-calentado 0,25% de tripsina-EDTA </li><li> Lavar las células de frasco con medio de diferenciación de co-cultivo </li><li> Precipitar las células en 400xg, 7 minutos, temperatura ambiente. Desechar el sobrenadante </li><li>Añadir la cultura co-medio de diferenciación de las células. Use una aguja de calibre 21 bluntended para volver a suspender las células y para garantizar la suspensión de células individuales </li><li> Recuento de células utilizando un hemocitómetro. En caso de tener 100 a 150 veces más en el número de células ES a partir del día 0. </li><li> Re-semilla 6.48x10 4 / cm 2 - 7.76x10 4 / cm 2 de co-cultivo las células a una nueva capa de confluencia, las células mStrawberry OP9 </li><li> Día 5 ENSAYOS-Otros co-cultivo las células se pueden utilizar para el análisis de citometría de flujo, cytopreps (30K células), ensayos de chip, etc </li></ol></li><li> ES / mStrawberry OP9 co-cultivo (Día 8 / 9 y el día 12) <ol class="lalpha"><li> Retire el medio de diferenciación de la botella y guardar. Lavar 1x monocapa con PBS para eliminar las células hematopoyéticas en suspensión. No tirar las células hematopoyéticas. </li><li> Trypsinize células adherentes con pre-calentado 0,25% de tripsina-EDTA </li><li> Lavar las células de frasco con medio de diferenciación ES y añadir a las células hematopoyéticas se encuentran en suspensión = </li><li> Precipitar las células en 400xg, 7 minutos, temperatura ambiente. Desechar el sobrenadante </li><li> Añadir Co-cultura diferenciación medios de comunicación o PBS a las células. Use una aguja de calibre 21 bluntended para volver a suspender las células y para garantizar a las células individuales del recuento de células suspensión utilizando un hemocitómetro. </li><li> Co-cultivo se puede continuar durante 14 días para evaluar progenitores hematopoyéticos más maduro por co-cultivo de siembra de nuevo las células en una nueva capa de confluencia, mStrawberry OP9 células en el Día 8 o 9 y luego otra vez el día 12. Re-semilla de células optimizado basado en la recuperación de células necesarias para el día de ensayo. </li></ol></li><li> Evaluación de la ES-derivados progenie (Día 1-14). ES-progenie derivada se puede evaluar en cualquier día de co-cultivo de flujo clasificando mStrawberry células negativas (ES derivados de la progenie). <ol class="lalpha"><li> Retire el medio de diferenciación de la botella y guardar. Lavar 1x monocapa con PBS para eliminar las células hematopoyéticas en suspensión. No tirar las células hematopoyéticas. </li><li> Trypsinize células adherentes con pre-calentado 0,25% de tripsina-EDTA </li><li> Lavar las células de frasco con medio de diferenciación de co-cultivo y añadir a las células hematopoyéticas se encuentran en suspensión </li><li> Pellet células en 400xg, 7 minutos, RT. Desechar el sobrenadante </li><li> Resuspender las células con aguja calibre 21 bluntended y contar </li><li> ES progenie derivada (mStrawberry células negativas) pueden ser ordenados a partir del flujo mStrawberry positivo OP9 células. ES-progenie derivada puede entonces ser caracterizados por citometría de flujo, cytopreps, ensayos de chip, etc </li></ol></li></ol> 4. Los resultados representativos: <img src="/files/ftp_upload/2559/2559fig1.jpg" alt="Figura 1" /> Figura 1. ES / mStrawberry OP9 Co-cultura del sistema. Nuestro laboratorio utiliza un in vitro de células ES de co-cultivo para el desarrollo del modelo del sistema hematopoyético. En este sistema de co-cultivo, las células ES diferenciar, en hemangioblastos en Día 5, cuando se coloca sobre una capa confluente de células mStrawberry OP9. A medida que avanza la cultura co-, los precursores hematopoyéticos presentes en el día 8, mientras terminales diferenciadas linajes hematopoyéticos aparecer el día 14. Las flechas indican el día de la diferenciación, cuando pases de ES-progenie derivada a nuevas capas confluentes de células en mStrawberry OP9 necesario. <img src="/files/ftp_upload/2559/2559fig2.jpg" alt="Figura 2" /> Figura 2. ES correcta diferenciación de las células. Las células ES son sembradas en una capa confluente de mStrawberry OP9 el día 0. ES-progenie derivada comienzan a ser visibles en el día 3 de diferenciación, con el ES-progenie derivada aspecto de un conjunto de células con un borde definido o principios para formar un patrón espiral. Hemangioblastos, que son el precursor común de los linajes mesodérmicos endotelial y hematopoyético, debería aparecer como amontonados espirales de células en el día 5. Para los períodos de co-cultivo de más de cinco días, ES derivados de descendencia debería aparecer como grupos de células hematopoyéticas. De dos a cuatro grupos de unicelulares, en el Día 6 / 7, que aparecen y se desarrollan en grupos de células grandes en el Día 8 / 9 de co-cultivo. ES progenie derivada también se encuentran otros estrechamente vinculada a la capa de células del estroma adherentes. <img src="/files/ftp_upload/2559/2559fig3.jpg" alt="Figura 3" /> Figura 3. La diferenciación de células ES éxito. Los resultados de este co-cultivo reflejan la diferenciación inadecuada de las células ES. Nótese cómo la progenie derivada ES-no aparecen en un patrón espiral en el día 5. La ausencia de células en un patrón espiral indica la formación hemangioblasto inadecuado. Si hemangioblasto no se desarrollan correctamente, y luego continuó co-cultivo se traducirá en la diferenciación de retraso en el crecimiento linaje hematopoyético. Co-cultivos con la formación de hemangioblasto inadecuado debe ser desechada. <img src="/files/ftp_upload/2559/2559fig4.jpg" alt="Figura 4" /> Figura 4. mStrawberry OP9 cultivos celulares. (A) mStrawberry celular OP9 se subcultivan en70-90% de confluencia. (B) Para la diferenciación adecuada, las células ES y ES-progenie derivada se colocan sobre una capa demasiado confluente de mStrawberry OP9 células. <img src="/files/ftp_upload/2559/2559fig5.jpg" alt="Figura 5" /> Figura 5. Representante ES / mStrawberry OP9 Co-cultura perfil de flujo. Toda la progenie derivada de ES son mStrawberry células negativas y pueden ser ordenados flujo de la mStrawberry OP9 células. puerta mStrawberry celular negativa se determinó mediante un tradicional ES/OP9 co-cultivo.

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Diferenciación adecuada de células madre embrionarias en la capa confluente de células mStrawberry OP9 resultados en la producción de Flk1 hemangioblastos + en el día 5 de co-cultivo. Hemangioblastos debe aparecer como amontonados espirales de células, como se observa en la Figura 1. Para el resto de los compañeros de la cultura, visibles ES derivados de descendencia debería aparecer como grupos de células hematopoyéticas (Figura 1). En Day6 / 7 desde dos hasta cuatro grupos de células que aparecen y se desarrollan en grupos de células grandes (tanto en adherentes y en suspensión) en el Día 8 / 9 de co-cultivo. ES progenie derivada también se encuentran otros estrechamente ligadas dentro de la capa de células del estroma adherentes. Aunque no figure en el protocolo, es fundamental poner a prueba el FBS utilizada en el medio de cultivo ES, el mStrawberry OP9 medios de cultivo y el ES / mStrawberry OP9 medio de diferenciación. Sometidos a las pruebas de suero debe garantizar un adecuado crecimiento de mStrawberry OP9 células, así como, la diferenciación correcta de las células madre embrionarias en el co-cultivo hasta el día 5 de diferenciación. Como alternativa o como complemento de mStrawberry OP9 células, OP9 células pueden ser irradiados, a 8000 rads, antes de la siembra en la confluencia (7.8x10 4 células / cm 2). La irradiación de células OP9 permite la eliminación de los repetidos pasos replating, así como, permite la eliminación casi completa de los viejos, contaminantes OP9 las células de los ensayos de aguas abajo. OP9 células fueron modificadas para expresar la proteína de transducción OP9 mStrawberry por células con 3ug/mL de un mStrawberry vector lentiviral expresar bajo el control de un promotor CMV (CMV-pRRLsin vector, vector principal de la UCLA). ES/OP9 estándar de co-cultivo protocolos implican la colocación de células madre embrionarias en un confluente capa OP9 células, así como, un día 5 pasos replating con el fin de reducir la edad, la contaminación de OP9 células del paso de la célula. Además, sólo las células hematopoyéticas se encuentran en suspensión se eliminan para la evaluación de la ES-derivados progenie. Sin embargo, tanto con un paso replating y la recolección de células en suspensión sólo una falla potencialmente un número importante de los países en desarrollo ES derivados de las células hematopoyéticas. Este problema se vuelve importante para hacer frente al tratar de caracterizar los defectos asociados con las líneas hematopoyéticas ES octavos de final. Con el fin de eludir este problema nuestro laboratorio utiliza una versión modificada de co-cultivo, mediante el uso de mStrawberry que expresan las células OP9. Esto asegura que toda la progenie ES se transfieren a la continua cooperación de la cultura en el día 5, y para la cosecha de toda la progenie derivada de ES para los ensayos de aguas abajo. Esta modificación ofrece una herramienta útil en la evaluación y caracterización de la progenie hematopoyéticas derivadas de humanos y líneas de ratones ES. Este ES-mStrawberry OP9 cultura co-modelo las diferentes etapas primitivas y la hematopoyesis definitiva recapitula. Al estudiar las primeras etapas de desarrollo hematopoyético, muchas personas utilizan el BL-CFC (Blast-similares a las células formadoras de colonias) de ensayo, que evalúan el desarrollo de la hemangioblasto de la célula ES. Mientras que el ensayo BL-CFC es una herramienta útil en la investigación de desarrollo hematopoyético temprano, el mStrawberry-ES co-cultivo de sistemas de servicios públicos presenta aumento, ya que permite la evaluación de hemangioblasto, así como, los linajes hematopoyéticos más adulto. El trabajo previo con OP9/ES tradicionales y los protocolos de diferenciación de EB ha demostrado que los factores adicionales tal vez sea necesario, como la sobreexpresión de HOXB4, a fin de obtener a largo plazo repoblar las células madre hematopoyéticas in vitro. El trabajo adicional es necesario para evaluar si mStrawberry negativos, ordenados ES derivados de las poblaciones contienen verdaderas células madre hematopoyéticas.

OP-9 celdas componentes del medio de mantenimiento de archivo <table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> AMEM </td><td> Invitrogen </td><td> 12571-063 </td><td></td></tr><tr><td> FBS </td><td> Omega Científico </td><td> FB01 </td><td> Pre-Prueba </td></tr><tr><td> L-glutamina </td><td> Cellgro </td><td> 25 a 005-CI </td><td> 200nm </td></tr><tr><td> Tripsina-EDTA </td><td> Madre Cell Technologies </td><td> 07901 </td><td></td></tr></tbody></table> ES/OP-9 de diferenciación celular componentes del medio de archivo <table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> AMEM </td><td> Invitrogen </td><td> 12571-063 </td><td></td></tr><tr><td> FBS </td><td> Hyclone </td><td> SH30070 </td><td> Pre-Prueba </td></tr><tr><td> L-glutamina </td><td> Cellgro </td><td> 25 a 005-CI </td><td> 200 mM </td></tr><tr><td> PBS (sin Ca 2 + y Mg 2 +) </td><td> Cellgro </td><td> 21 a 031 CV </td><td></td></tr><tr><td> Tripsina-EDTA </td><td> Madre Cell Technologies </td><td> 07901 </td><td></td></tr></tbody></table>

modified es / op9 co-culture protocol provides enhanced characterization of hematopoietic progeny

La diferenciación in vitro de células madre embrionarias hacia un destino de células hematopoyéticas es útil cuando se estudian poblaciones celulares que son de difícil acceso en vivo y para la caracterización de los primeros genes que participan en la hematopoyesis, sin tener que lidiar con lethalities embrionarias. El co-cultivo ES/OP9 sistema fue diseñado originalmente para producir una progenie hematopoyética, sin el exceso de producción de los macrófagos, como la línea de células del estroma OP9 se deriva de la bóveda craneal de ratones mutantes que carecen de osteopetrosis funcional M-CSF. El in vitro ES/OP9 co-cultura del sistema se puede utilizar con el fin de recapitular el desarrollo hematopoyético temprano. Cuando se cultivan en OP9 las células del estroma, células madre embrionarias se diferencian en Flk-1 + hemangioblastos, progenitores hematopoyéticos, y, finalmente, madura, los linajes de diferenciación terminal. La norma ES/OP9 co-cultivo protocolo implica la colocación de células madre embrionarias en una capa confluente de células OP9, así como, las medidas periódicas replating con el fin de eliminar viejos, contaminantes OP9 células. Además, los protocolos actuales incluyen la evaluación sólo las células hematopoyéticas se encuentran en suspensión y no están optimizados para la evaluación de la ES-derivados de la progenie a cada día de la diferenciación. Sin embargo, con los pasos replating y la recolección de células en suspensión solo potencialmente pierde una gran parte de la progenie derivada de ES y el desarrollo de las células hematopoyéticas. Este problema se vuelve importante para hacer frente al tratar de caracterizar los defectos asociados con las líneas hematopoyéticas ES octavos de final. Aquí se describe una modificación ES / mStrawberry OP9 co-cultivo, lo que permite la eliminación de contaminantes OP9 las células de los ensayos de aguas abajo. Este método permite la evaluación completa de toda la progenie derivada de ES en todos los días de co-cultivo, resultando en un patrón de diferenciación hematopoyética, lo que más se corresponde directamente con el patrón de diferenciación hematopoyética observado en el embrión.

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Modified ES / OP9 Co-Culture Protocol Provides Enhanced Characterization of Hematopoietic Progeny

mStrawberry OP9 células permiten una evaluación completa de toda la progenie derivada de ES-co-cultivo.

Modificado ES / OP9 Co-cultivo Protocolo establece Caracterización mejorada de la progenie hematopoyética

The in vitro differentiation of ES cells towards a hematopoietic cell fate is useful when studying cell populations that are difficult to access in vivo ...

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20.6K vistas.  University of California, Los Angeles. mStrawberry OP9 células permiten una evaluación completa de toda la progenie derivada de ES-co-cultivo.

Video: Modified ES / OP9 Co-Culture Protocol Provides Enhanced Characterization of Hematopoietic Progeny

Freezing Human ES Cells

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.  A healthy, exponentially expanding culture is washed with PBS to remove residual media that could otherwise quench the Trypsin reaction.  Warmed 0.05% Trypsin-EDTA is then added to cover the cells, and the plate allowed to incubate for up to 5 mins at room temperature.  During this time cells can be observed rounding, and colonies lifting off the plate surface.  Gentle repeated pipetting will remove cells and colonies from the plate surface.  Trypsinized cells are placed in a standard conical tube containing pre-warmed hES cell media to quench remaining trypsin, and then spun.  Cells are resuspended growth media at a concentration of approximately one million cells in one mL of media, a concentration such that one frozen aliquot is sufficient to resurrect a culture on a 10cm plate.  After cells are adequately resuspended, ice cold freezing media is added at equal volume.  Cell suspensions are mixed thoroughly, aliquoted into freezing vials, and allowed to slowly freeze to -80C over 24 hours.  Frozen cells can then moved to the vapor phase of liquid nitrogen for long term storage, or remain at -80 for approximately six months.

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.

Congelar las células madre embrionarias humanas

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.  A healthy, exponentially expanding culture is washed with PBS to remove ...

Investigación

Biología

Human ES cells: Starting Culture from Frozen Cells

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator.  A frozen vial of cells from long term liquid nitrogen storage or a -80C freezer is sourced and quickly submerged in a 37C water bath for quick thawing.  Cells in freezing media are then removed from the vial and placed in a large volume of HuES media.  The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate.  Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate.  It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate.  The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.

Células madre embrionarias humanas: Cultura a partir de células congeladas

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  ...

ES Cell-derived Neuroepithelial Cell Cultures

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In general, the differentiation of ES cells follows the concept to first generate immature progenitor cells, which then can be propagated and differentiated into mature cellular phenotypes. This also applies for ES cell-derived neurogenesis, in which the development of neural cells follows two major steps: First, the derivation and expansion of immature neuroepithelial precursors and second, their differentiation into mature neural cells. A common method to produce neural progenitors from ES cells is based on embryoid body (EB) formation, which reveals the differentiation of cells from all germ layers including neuroectoderm. An alternative and more efficient method to induce neuroepithelial cell development uses stromal cell-derived inducing activity (SDIA), which can be achieved by co-culturing ES cells with skull bone marrow-derived stromal cells (1). Both, EB formation and SDIA, reveal the development of rosette-like structures, which are thought to resemble neural tube- and/or neural crest-like progenitors. The neural precursors can be isolated, expanded and further differentiated into specific neurons and glia cells using defined culture conditions. Here, we describe the generation and isolation of such rosettes in co-culture experiments with the stromal cell line MS5 (2-5).

Derivation of neuroepithelial precursors from embryonic stem (ES) cells using stromal cell-derived inducing activity (SDIA).

ES células derivadas de células de las Culturas neuroepiteliales

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In ...

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Yeah, so I'm Lauren Deon. I'm in charge of the Human Amgen stem cell co facility at MGH. Yeah, so I'm Lauren, I'm in charge of the Human AM stem cell co facility at MGH.So I've been working with human Amgen stem cells for years now since I was a postdoc in George De Lab before, from four, four years. And so right now we have two cell lines, H one and H nine, which are both from Y cell. And so the way we are expanding or maintaining these cells in culture is by, at least the way we are splitting these cells is by using collagenous.So what I usually do is for the first few passage after throwing the cells, I use a technical, the picking colonies. So in this case, I can check under the microscope which colonies are good and differentiate and nice shape, and I will collect only these colonies and I will cut them in few pieces and put them back in, in a fresh flat. And the, the colonies that look kind of differentiate it are just left them alone.The other way to pass the cells is by using trypsin. So I don't use this technique here. I think collagenase is a little bit safer because human being stem cells don't like to be individualized or they don't like to be alone.So if you do trypsin, if you use trypsin for a little bit too long, if you use it for like three to four minutes, which is the way we use strips in for other type of cells, human stem cells will be individualized and most of them will die or just differentiate or will not attach to the myth. So the idea is you have to keep the cells as clumps. You don't want them to be alone in the in, in the flask.So using collagenase, the advantage is to keep cells as plants. Using chipsy, you just select for cells that will survive this very, very harsh process of g opsonization. So these cells are usually very highly proliferative.So you get, you can adapt your cells to this tripsin process. The problem is that you have cells that they behave differently, they go much faster. And one risk is that you can get some commercial abnormality to, to this, to this very high division process.So you say that ization can cause selection of cells in culture for most proliferative cells.Right. More ative and probably also higher survival rates than other cells. So after proliferation, basically culture after proliferation in tripsin, after several passages become a different culture, these different properties.So it's, it's hard to figure out if they are exactly identical. If you do the test, the usual test, let's say you check markers, regular markers of end differentiated cells like OC four, nano one 60 SC four C, C3, all this molecule might still be expressed. If you do TER formation, this cells might still differentiate and be formed like al like normal cells.If you look at the karyotype, you might have some abnormality, but you might still have a normal karyotype. The, the, the selective advantage might be due to just a mutation that you cannot see by doing a karyotype. So the fact that they grow much faster is by itself I think a little bit worser.And if we want to use these cells for transplantation at some point, I would definitely not use this type of cells.Okay. So would you like to tell me what are the main technical problems with using this technique for, for passages of cells? So using S, yes.It's just a little bit longer and also it's, since the cells are not going as fast, it's just much longer to get a high number of cells to do some experiment. So it's just less convenient I would say. Would you like to tell me about few words about the goals of your experiment?How do you use CS cells? Okay, so my main project here is to get endothelial cells from human AB stem cells. And the idea is to be able to make blood.And we are working a lot with tissue bioengineering labs and one of their goal is to make tissue in vitro. But one of the limitation to this is the lack of blood vessels going through this tissues. So if we can bring endothelial cells or put endothelial cells in this tissue, we would have better growth of this tissue.So the idea few sources of cells are known for endothelial cells like cold blood or pressure blood, but it would be interesting to have another source of cells like human years. And the advantage is since human years can be expanded and most infinitely, you could modify these cells, make some inducible system and be able to use it. Then in your final product, your material cells, in this case.

Propagación de la madre embrionarias humanas (ES) Células

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

A neuronales y astrocitos Co-cultivo de ensayo para el análisis de alto contenido de neurotoxicidad

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

Un compartimiento de Multi-CNS neuronas-glia Co-cultivo de microfluidos Plataforma

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

Microfluidos Co-cultivo de células epiteliales y bacterias para la Investigación de la señal mediada Soluble Interacciones

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

Aislamiento de células epiteliales mamarias de tridimensional mixto de células Esferoide Co-cultura

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a &#8216;targeting&#8217; vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term &#8216;subcloning plus insertion&#8217;, which can be used to generate custom gene targeting vectors rapidly.

Subclonación Plus Inserción (SPI) - Un nuevo método recombinería para la rápida construcción de la orientación de genes Vectores

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic ...

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

Desarrollo de un Sistema de inserción Co-cultivo de dos tipos celulares en ausencia de la célula-célula Contacto

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...