Este trabajo fue apoyado por el Saban Research Institute Pre-doctoral Premio (PMDM), y por el NIH RO1 HL75773 (DW).

El cultivo de órganos es una técnica muy útil y versátil para el estudio de genes y expresión de la proteína. Estos métodos ex vivo la cultura puede ser utilizado como preliminar o como complemento de los estudios in vivo. 1. Aislamiento de pulmón embrionario <ol><li> Cronometrada embarazadas ratones son sacrificados en el día postcoitum 11,5 o 12,5 (E11.5-12.5) de CO 2 narcosis de acuerdo con las directrices del NIH y OLAW. El animal se coloca en una cámara y un 100% de CO 2 se introduce. Después de que el animal está inconsciente el flujo de CO2 es mayor. La muerte clínica del animal debe estar garantizada. </li><li> Todos los pasos siguientes deben ser cumplidos bajo condiciones estériles en una campana de flujo laminar. Se extirpa el útero de las hembras preñadas. Para extirpar el útero del animal, las mujeres embarazadas se colocan en la espalda y se rocían con etanol al 70%. Se hace una incisión en el abdomen y la piel se quita tirando de la piel hacia arriba, manteniendo las patas traseras del animal. La cavidad peritoneal se abre y se extirpa el útero del animal. </li><li> La sangre se aclara mediante la colocación de la matriz en un tubo cónico de 50 ml llena de solución salina fría Hanks equilibrada (HBSS), y el tubo se mecía suavemente durante 2 minutos. </li><li> El útero se coloca en una placa de Petri bajo el microscopio estereoscópico de disección, y los embriones dado de alta del útero mediante una incisión en la pared del útero con unas tijeras de primavera. Los embriones se recogen con una cuchara perforada y se coloca en hielo en una nueva placa de Petri que contiene HBSS. </li><li> Bajo el microscopio estereoscópico de disección, pulmones embrionarias se disecan uno por uno en una placa de Petri que contiene HBSS frío (Figura 1). </li><li> El pulmón de embriones aislados se coloca en el hielo en un plato de Petri que contiene HBSS con una pipeta de transferencia estéril. </li></ol> 2. Cultura de pulmón embrionario <ol><li> 500 microlitros de 1 mililitro por pocillo de D-MEM/F-12 suplementado con 50 unidades / ml de penicilina-estreptomicina, se añade en una placa de poliestireno Nunclon. </li><li> A Nuclepore policarbonato pista Etch membrana se coloca en la parte superior de los medios de comunicación. Cuando el Nuclepore policarbonato pista Etch membrana se coloca en la parte superior de los medios de comunicación, asegúrese de que el lado brillante contra los medios de comunicación, y cara rugosa está hacia arriba. </li><li> Un pulmón disecados embrionarias se coloca en la parte superior de la pista Nuclepore policarbonato-Etch membrana con una pipeta de transferencia estéril. </li><li> La posición de pulmón embrionario se ajusta con las pinzas. Pulmón embrionario colocado en el filtro debe tener una traquea intactos y se coloca con su tráquea con una posición recta, permitiendo así que todos los pulmones que tiene la misma presión interna. </li><li> El plato de poliestireno Nunclon se transfiere por el momento la cultura deseada en una incubadora de células (Figura 2). </li></ol> 3. Materiales 1. Aislamiento embrionarias todo el pulmón <ol><li> Tiempo de espera del embarazo (C57BL6 tipo salvaje o transgénicos) en ratones que se sacrificaron el día postcoitum 11,5 a 13,5 (E11.5-13.5). </li><li> Solución salina equilibrada de Hanks (HBSS) (1X), líquido, sin cloruro de calcio, cloruro de magnesio o sulfato de magnesio. (Invitrogen, Carlsbad, CA), complementado con 50 unidades / ml de penicilina-estreptomicina (Invitrogen, Carlsbad, CA). Guarde el HBSS a temperatura ambiente ya 4 ° C después de la apertura. Alícuota y almacenar la penicilina-estreptomicina a -20 ° C. </li><li> Microscopio estereoscópico de disección (Leica, Wetzlar, Alemania). </li><li> Herramientas de disección incluyendo Dumont pinzas, tijeras iris Artes (recto), tijeras Noyes primavera y Moria cuchara ® (perforada) (Herramientas de Bellas Ciencia, Foster City, CA). </li></ol> 2. Cultura embrionarias todo el pulmón <ol><li> Dulbecco modificado del medio Eagle: mezcla de nutrientes F-12 (D-MEM/F-12) (1X), líquido, 01:01 Contiene L-glutamina, pero no tampón HEPES. (Invitrogen, Carlsbad, CA), complementado con 50 unidades / ml de penicilina-estreptomicina (Invitrogen, Carlsbad, CA). Mantenga la botella DMEM/F-12 lejos de la luz (por ejemplo, cubiertas con papel de aluminio) y se almacenan a 4 ° C. Alícuota y almacenar la penicilina-estreptomicina a -20 ° C. </li><li> Nuclepore policarbonato pista Etch de membrana, de 8,0 m, 13 mm (Whatman Nuclepore pista Etch membrana (Whatman, Florham Park, NJ). </li><li> Nunclon plato de poliestireno con tapas, 4 pozos (Rochester, NY). </li><li> Pipetas desechables de transferencia, estéril (Fisher Scientific, Pittsburgh, PA). </li></ol><img src="/files/ftp_upload/2035/2035fig1.jpg" alt="Figura 1" /> Figura 1. La disección de los pulmones E12 embrionarias. (A) E12.5 embrión todo visto desde el lado derecho (B). Extremidad anterior derecha ha sido removido del embrión. (C) Pinzas en poder de la mano izquierda sostiene el embrión constante en el plato. Las flechas indican el plan de la disección (D) de pulmón embrionario se encuentra por detrás del corazón (eliminado) Y por delante de la columna vertebral. Esta figura muestra los pulmones después de la piel y eliminación de corazón. (E) eliminación de la faringe permite la separación del pulmón intacto desde el embrión. (F) los tejidos extraños faringe y el esófago se recorta. Disecados E12.5 pulmón embrionario con la tráquea y la laringe intacta se muestra. (Cr) del lóbulo craneal, (Med) lóbulo (Ca) del lóbulo caudal, (Acc) lóbulo accesorio, (Le) del lóbulo izquierdo. <img src="/files/ftp_upload/2035/2035fig2.jpg" alt="Figura 2" /> Figura 2. FGF9 induce la expansión del mesénquima y la dilatación del epitelio en el pulmón cultivadas in vitro. (AC) E12.5 pulmón se cultiva durante 48 horas en ausencia de FGF9. Tenga en cuenta el aumento de la ramificación. (DF) E12.5 pulmón crecido durante 48 horas en presencia de FGF9. Tenga en cuenta la dilatación del epitelio y el mesénquima tan temprano como a las 24 horas de cultivo (E). Después de 48 horas (F), el efecto sobre el epitelio es aún más pronunciada. Barra de escala: AF 400 m 4. El protocolo de texto completo de este enfoque experimental está disponible en <a href="http://springerprotocols.com/Abstract/doi/10.1007/978-1-59745-019-5_5">los Protocolos Springer</a> .

<ol>
	<li>Alescio, T., Cassini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+in+vitro+of+tracheal+buds+by+pulmonary+mesenchyme+grafted+on+tracheal+epithelium.">Induction in vitro of tracheal buds by pulmonary mesenchyme grafted on tracheal epithelium.</a> J Exp Zool. 150, 83-94 (1962).</li><li>Warburton, D., Schwarz, M., Tefft, D., Flores-Delgado, G., Anderson, K. D., Cardoso, W. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+basis+of+lung+morphogenesis.">The molecular basis of lung morphogenesis.</a> Mech Dev. 92, 55-81 (2000).</li><li>Spooner, B. S., Hardman, P., Paulsen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gravity+in+mammalian+organ+development:+differentiation+of+cultured+lung+and+pancreas+rudiments+during+spaceflight.">Gravity in mammalian organ development: differentiation of cultured lung and pancreas rudiments during spaceflight.</a> J Exp Zool. 269, 212-222 (1994).</li><li>Moral, d. e. l., M, P., De Langhe, S. P., Sala, F. G., Veltmaat, J. M., Tefft, D., Wang, K., Warburton, D., Bellusci, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+role+of+FGF9+on+epithelium+and+mesenchyme+in+mouse+embryonic+lung.">Differential role of FGF9 on epithelium and mesenchyme in mouse embryonic lung.</a> Dev Biol. 293, 77-89 (2006).</li><li>Moral, D. e. l., M, P., Sala, F. G., Tefft, D., Shi, W., Keshet, E., Bellusci, S., Warburton, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF-A+signaling+through+Flk-1+is+a+critical+facilitator+of+early+embryonic+lung+epithelial+to+endothelial+crosstalk+and+branching+morphogenesis.">VEGF-A signaling through Flk-1 is a critical facilitator of early embryonic lung epithelial to endothelial crosstalk and branching morphogenesis.</a> Dev Biol. 290, 177-188 (2006).</li><li>Jaskoll, T. F., Don-Wheeler, G., Johnson, R., Slavkin, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+mouse+lung+morphogenesis+and+type+II+cytodifferentiation+in+serumless,+chemically+defined+medium+using+prolonged+in+vitro+cultures.">Embryonic mouse lung morphogenesis and type II cytodifferentiation in serumless, chemically defined medium using prolonged in vitro cultures.</a> Cell Differ. 24, 105-117 (1988).</li><li>Seth, R., Shum, L., Wu, F., Wuenschell, C., Hall, F. L., Slavkin, H. C., Warburton, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+epidermal+growth+factor+expression+in+early+mouse+embryo+lung+branching+morphogenesis+in+culture:+antisense+oligodeoxynucleotide+inhibitory+strategy.">Role of epidermal growth factor expression in early mouse embryo lung branching morphogenesis in culture: antisense oligodeoxynucleotide inhibitory strategy.</a> Dev Biol. 158, 555-559 (1993).</li></ol>

mouse embryonic lung culture, a system to evaluate the molecular mechanisms of branching

Especificación de pulmón primordial, así como la morfogénesis de ramificación, y la formación de varios linajes de células pulmonares requiere una interacción específica del endodermo de pulmón con su mesénquima circundante y mesotelio. Mesénquima pulmonar ha demostrado ser la fuente de señales inductivas para la morfogénesis de pulmón ramificación. Epitelio-mesénquima-mesoteliales interacciones también son esenciales para la morfogénesis de pulmón embrionario. Principios de la cultura de órganos embrionarios de pulmón es un sistema muy útil para estudiar las interacciones epitelio-mesenquimal. Epiteliales y mesenquimales procede morfogénesis en condiciones específicas que pueden ser fácilmente manipulados en este sistema (en ausencia de la influencia de la madre y el flujo de sangre). Más importante que esta técnica puede ser fácilmente hecho en un medio de comunicación serumless, la cultura de constitución química definida. Ganancia y pérdida de la función se puede lograr utilizando las proteínas expresadas, vectores virales recombinantes y / o análisis de las cepas de ratones transgénicos, el ARN antisentido, así como la caída en la interferencia del ARN del gen.

Watch this Scientific Journal Video about Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching at JoVE.com

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Principios de la cultura de órganos embrionarios de pulmón es un sistema muy útil para estudiar las interacciones epitelio-mesenquimal. Epiteliales y mesenquimales procede morfogénesis en condiciones específicas que pueden ser fácilmente manipulados en este sistema.

Cultura de embriones de ratón de pulmón, un sistema para evaluar los mecanismos moleculares de la ramificación

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of ...

mouse-embryonic-lung-culture-system-to-evaluate-molecular-mechanisms

Research

JoVE Journal

Biology

18.4K vistas.  Childrens Hospital Los Angeles. Principios de la cultura de órganos embrionarios de pulmón es un sistema muy útil para estudiar las interacciones epitelio-mesenquimal. Epiteliales y mesenquimales procede morfogénesis en condiciones específicas que pueden ser fácilmente manipulados en este sistema.

Video: Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

Aislamiento y cultivo de células de la zona nefrogénica del riñón de embriones de ratón

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Investigación

Biología

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

Todo el montaje El análisis inmunohistoquímico de la piel del miembro Vasculatura embrionarias: un sistema modelo para estudiar la morfogénesis vascular ramificación en embriones

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility4-6. Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer7.
Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development8. Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages2,9. Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology5,6,10,11. While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression12, antibody staining13, and electron microscopy1. Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures14. Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail15. We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.

La visualización de Caenorhabditis Elegans Estructuras de la cutícula uso del colorante lipofílico Vital, DII

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi de detección para identificar fenotipos postembrionarios en C. elegans</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Un sistema para la<em&gt; Ex vivo</em&gt; Cultivo de páncreas embrionario

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

Los explantes blastómeras para la Prueba de Compromiso destino celular durante el desarrollo embrionario

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

A Novel<em&gt; Ex vivo</em&gt; Método de la cultura para el corazón de embriones de ratón

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

Embriones de ratón con el desarrollo en todo un sistema de cultivo de embriones sin suero

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

Aislamiento y cultivo primario de células endoteliales aórticas de ratón

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Procedure to Evaluate the Efficiency of Flocculants for the Removal of Dispersed Particles from Plant Extracts

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used as manufacturing platforms for added-value products such as biopharmaceuticals. In both cases, liquid plant extracts may need to be clarified to remove particulates. Optimal clarification reduces the costs of filtration and centrifugation by increasing capacity and longevity. This can be achieved by introducing charged polymers known as flocculants, which cross-link dispersed particles to facilitate solid-liquid separation. There are no mechanistic flocculation models for complex mixtures such as plant extracts so empirical models are used instead. Here a design-of-experiments procedure is described that allows the rapid screening of different flocculants, optimizing the clarification of plant extracts and significantly reducing turbidity. The resulting predictive models allow the identification of robust process conditions and sets of polymers with complementary properties, e.g. effective flocculation in extracts with specific conductivities. The results presented for tobacco leaf extracts can easily be adapted to other plant species or tissues and will thus facilitate the development of more cost-effective downstream processes for commodities and plant-derived pharmaceuticals.

The design-of-experiments procedure presented here allows the evaluation of different flocculants in terms of their ability to aggregate dispersed particles in plant extracts, thus reducing turbidity and the costs of downstream processing.

Procedimiento para evaluar la eficacia de floculantes para la eliminación de partículas dispersas en extractos de plantas

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used ...