Damos las gracias a Erika Mileti para un excelente soporte técnico y el Dr. Antonio Di Sabatino para proporcionar las cámaras de oxígeno. Financiación: Este trabajo fue apoyado por becas de los programas marco de la UE séptimo (IBDase, ERC: Dendroworld) y Fondazione Cariplo a MR y el apoyo de una red Marie Curie de Formación Internacional Movilidad (Cross-Talk, del acuerdo de subvención n º: 21553-2) para KT.

1. Obtención y preparación del tejido <ol><li> Se obtiene de tejido (sano o EII mucosa) durante la cirugía. Una vez que la muestra se extirpa transferencia al laboratorio tan pronto como sea posible, manteniéndolo en Ca + + / Mg + + tampón HBSS suplementado con penicilina / estreptomicina a 4 º C o en hielo. </li></ol> * El tamaño de la muestra depende de la disponibilidad del tejido: la parte del tejido obtenido, es estrictamente lo que no se necesita para el diagnóstico y puede variar en gran medida entre sujetos sanos y EII. El tejido sano se escindió de los pacientes sometidos a cirugía por cáncer de colon. <ol start="2"><li> Lave el tejido suave y limpiarlo de todo el material mesentérica. Se separa la capa mucosa de la submucosa usando tijeras y pinzas estériles mientras se mantiene el tejido en tampón HBSS en una placa de Petri (no necesariamente sumergido). Toque la superficie de la mucosa con la pinza lo menos posible. </li><li> Cortar mucosa en las rayas, al menos, 1 cm de ancho y el mayor tiempo posible. Utilice dos bisturís estériles, tanto como si estuviera cortando los alimentos. </li><li> Lavar limpiar suavemente la mucosa en HBSS y se extienden sobre una placa de Petri con la parte apical hacia arriba. </li></ol> 2. Montaje del tejido <ol><li> Preparar medio fresco (tisDMEM). Utilizar DMEM suplementado con glutamina (2 mM) y NaPyr (1 mM), y añadir 15% de FBS-Na (suero bovino fetal - América del Norte), 1% SU-X y 200 ng / ml de EGF en el momento de uso. </li><li> Llenar una placa Petri de 10 cm con el resto de la FBS-Na (utilizar alrededor de 30 ml de manera que las rejillas de metal serán completamente sumergidos) y sumerja las rejillas de metal. Mantenga la placa de abrir y apoyar los cilindros de plástico de su grifo. </li></ol> * Las rejillas metálicas están hechas de hierro. La malla es de forma cuadrada con un lado de 0,5 mm. <ol start="3"><li> Tome 3 l de pegamento quirúrgico con un 10 o 20 l pipeta. Aplicar con cuidado a una de las fronteras de un cilindro de plástico mientras mantiene It fIRM con las pinzas. </li><li> Colocar suavemente el cilindro en el lado apical de la mucosa, lo más cercano a uno de los bordes de la tira como sea posible. </li><li> Para dar el tiempo de pegamento para unir firmemente el cilindro sobre el tejido, preparar la placa de cultivo de órganos de centro-así: Dispensar 850 l-1 ml de medio en el centro y así apoyar la rejilla de metal en los bordes del pocillo central de la placa. </li><li> Cortar el exceso de tejido de los bordes del cilindro con dos escalpelos estériles como antes. </li><li> Levante con cuidado el cilindro con el tejido adjunto y coloque el lado basolateral de la rejilla metálica en el centro de la placa. </li></ol> 3. Estímulo <ol><li> Utilice los estímulos de su elección en su concentración preferida. </li><li> Aplicar volumen apropiado en el centro del cilindro de plástico sin perturbar el cilindro o la superficie de la mucosa con la punta de la pipeta. Asegúrese de que el volumen elegido cubre uniformemente la superficie interior del cilindro. Envolúmenes dicated: Large cilindro de plástico: 200 l Medio cilindro de plástico: 100 l Pequeño cilindro de plástico: 50 l </li><li> Incubar durante un máximo de 3 horas en una incubadora convencional. </li></ol> * Si desea incubar puntos temporales más largos, asegúrese de usar un volumen mucho menor de los estímulos (10-20 l) y colocar directamente el tejido en una atmósfera de 100% de O 2 en la presión de 1 atm (ver 3.5) . <ol start="4"><li> Tome el medio con los estímulos de la superficie apical de los tejidos y no sustituyen con un nuevo medio, debido a que el tejido no recibe suficiente O 2 si una gruesa capa de soporte en la cara apical impide el intercambio gaseoso. Cubra el plato. </li><li> Coloque el tejido en una atmósfera de 100% de O 2 en la presión de 1 atm. </li></ol> 4. Cosecha <ol><li> Después de 24 horas de tiempo total de cultivo (± 2,5 h) retirar con cuidado el cilindro de thtejido de correo con la ayuda de un escalpelo pegamento raspando suavemente y almacenarlo en función del análisis deseado (fijar para inmunohistoquímica y / o ensayos de inmunofluorescencia, o congelamiento rápido en N2 líquido para la purificación de ARN y perfiles de expresión génica o la purificación de proteínas y ensayos de Western blot ). </li><li> Se recoge el medio basolateral para el perfil de secreción de citoquinas. Centrifugar a 8000 rpm durante 7 min a los desechos de pellets, transferir el sobrenadante en un tubo Eppendorf (alternativamente alícuota) y almacenar inmediatamente a -20 ° C. </li></ol> * Si desea mantener sobrenadantes durante más tiempo, guárdelas a -80 ° C.

<ol>
	<li>Schuller, S., Lucas, M., Kaper, J. B., Giron, J. A., Phillips, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ex+vivo+response+of+human+intestinal+mucosa+to+enteropathogenic+Escherichia+coli+infection.">The ex vivo response of human intestinal mucosa to enteropathogenic Escherichia coli infection.</a> Cell. Microbiol. 11, 521-530 (2009).</li><li>Tsilingiri, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probiotic+and+postbiotic+activity+in+health+and+disease:+comparison+on+a+novel+polarised+ex-vivo+organ+culture+model.">Probiotic and postbiotic activity in health and disease: comparison on a novel polarised ex-vivo organ culture model.</a> Gut. , (2012).</li><li>Tsilingiri, K., Rescigno, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Should+probiotics+be+tested+on+ex+vivo+organ+culture+models?.">Should probiotics be tested on ex vivo organ culture models?.</a> Gut Microbes. , (2012).</li><li>Mileti, E., Matteoli, G., Iliev, I. D., Rescigno, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+immunomodulatory+properties+of+three+probiotic+strains+of+Lactobacilli+using+complex+culture+systems:+prediction+for+in+vivo+efficacy.">Comparison of the immunomodulatory properties of three probiotic strains of Lactobacilli using complex culture systems: prediction for in vivo efficacy.</a> PLoS One. 4, e7056 (2009).</li><li>Cencic, A., Langerholc, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+cell+models+of+the+gut+and+their+applications+in+food+microbiology--a+review.">Functional cell models of the gut and their applications in food microbiology--a review.</a> Int. J. Food Microbiol. 141, S4-S14 (2010).</li><li>te Velde, A. A., Verstege, M. A., Hommes, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+appraisal+of+the+current+practice+in+murine+TNBS-induced+colitis.">Critical appraisal of the current practice in murine TNBS-induced colitis.</a> Inflamm. Bowel Dis. 12, 995-999 (2006).</li><li>Senior, P. V., Pritchett, C. J., Sunter, J. P., Appleton, D. R., Watson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crypt+regeneration+in+adult+human+colonic+mucosa+during+prolonged+organ+culture.">Crypt regeneration in adult human colonic mucosa during prolonged organ culture.</a> J. Anat. 134, 459-469 (1982).</li><li>Browning, T. H., Trier, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+culture+of+mucosal+biopsies+of+human+small+intestine.">Organ culture of mucosal biopsies of human small intestine.</a> J. Clin. Invest. 48, 1423-1432 (1969).</li><li>Besselink, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probiotic+prophylaxis+in+predicted+severe+acute+pancreatitis:+a+randomised,+double-blind,+placebo-controlled+trial.">Probiotic prophylaxis in predicted severe acute pancreatitis: a randomised, double-blind, placebo-controlled trial.</a> Lancet. 371, 651-659 (2008).</li><li>Lakhdari, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+NF-kappaB+modulation+capabilities+within+human+intestinal+commensal+bacteria.">Identification of NF-kappaB modulation capabilities within human intestinal commensal bacteria.</a> J. Biomed. Biotechnol. 2011, 282356 (2011).</li></ol>

No hay conflictos de interés declarado.

Mucho tiempo se ha hecho hincapié en la necesidad de modelos fisiológicamente relevantes en los que los tratamientos para la mucosa intestinal humana se pueden probar válida. Muchos investigadores han identificado artefactos y defectos tanto en la célula 5 y 6 modelos de ratón utilizado hasta ahora para tal fin. En efecto, a pesar de que los datos preclínicos prometedores se obtienen a menudo, éstos rara vez se traducen en un beneficio clínico significativo. El ...

<table border="1">
	<tbody>
		<tr>
			<td>Regeant</td>
			<td>Company</td>
			<td>Catalog No.</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Reagents</td>
		</tr>
		<tr>
			<td>10X HBSS</td>
			<td>Eurocalne</td>
			<td>ECM4006XL</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Peni/Strepto</td>
			<td>Lonza</td>
			<td>DE17602E</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>Gibco</td>
			<td>15750-037</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Lonza</td>
			<td>BE12614</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FBS-Na</td>
			<td>Gibco</td>
			<td>16000-044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>100X ITS-X</td>
			<td>Gibco</td>
			<td>51500-056</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Tebu-bio</td>
			<td>100-15</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Lonza</td>
			<td>BE17605E</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Non essential aminoacids 100X</td>
			<td>Gibco</td>
			<td>11140-035</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaPyr</td>
			<td>Gibco</td>
			<td>11360-039</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Materials</td>
		</tr>
		<tr>
			<td>Cloning cylinders 6x8 mm</td>
			<td>BellCo</td>
			<td>2090-00608</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cloning cylinders 8x8 mm</td>
			<td>BellCo</td>
			<td>2090-0080</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cloning cylinders 10x10 mm</td>
			<td>BellCo</td>
			<td>2090-01010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Metal grids</td>
			<td>Home made</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Centre well organ culture plate</td>
			<td>BD Falcon</td>
			<td>353037</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Surgical glue (Vetbond)</td>
			<td>3M</td>
			<td>1469SB</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Oxygen Jars</td>
			<td>Home made</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Oxygen tanks</td>
			<td>Air Liquid Sanit&agrave;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

a novel method for the culture and polarized stimulation of human intestinal mucosa explants

Pocos modelos existen actualmente para simular de forma realista micro-entorno del complejo del intestino humano, donde una variedad de interacciones tienen lugar. La homeostasis adecuada depende directamente en estas interacciones, que dan forma a una respuesta inmunológica toda la inducción de tolerancia contra antígenos de los alimentos, mientras que al mismo tiempo de montaje de las respuestas inmunes eficaces contra los microbios patógenos ingeridos accidentalmente con alimentos. La homeostasis intestinal se conserva también a través de diversas interacciones complejas entre la microbiota (incluyendo cepas bacterianas beneficiosas relacionados con los alimentos) y el anfitrión, que regulan la fijación / degradación de la mucosidad, la producción de péptidos antimicrobianos por la barrera epitelial, y la &quot;enseñanza&quot; de células epiteliales de que controla el fenotipo tolerogenic o inmunogénica de las células linfoides únicos, gut-residentes &quot;poblaciones. Estas interacciones han sido hasta ahora muy difícil de reproducir en los ensayos in vitroya sea utilizando líneas de células cultivadas o células mononucleares de sangre periférica. Además, los modelos de ratón difieren sustancialmente en los componentes de la mucosa intestinal (organización capa de moco, las bacterias comensales comunidad) con respecto a el intestino humano. Por lo tanto, los estudios de una variedad de tratamientos de cesación en las clínicas para las condiciones importantes relacionados con el estrés o patológicos tales como el síndrome del intestino irritable, enfermedad inflamatoria del intestino o cáncer colorrectal han sido difíciles de llevar a cabo. Para abordar estas cuestiones, hemos desarrollado un novedoso sistema que nos permite estimular explantes de mucosa intestinal humana que mantienen su in situ acondicionado por la microbiota de acogida y la respuesta inmune, de manera polarizada. Estimulación apical polarizado es de gran importancia para el resultado de la respuesta inmunitaria provocada. Se ha demostrado repetidamente que los mismos estímulos pueden producir completamente diferentes respuestas cuando se pasan por alto la cara apical de la epi intestinalthelium, estimulación de las células epiteliales hacia la membrana basolateral o que entren en contacto directo con los componentes de la lámina propia, cambiando el fenotipo de tolerógeno a inmunogénica y causando la inflamación innecesaria y excesiva en la zona. Hemos logrado la estimulación polarizada por un cilindro de encolado cueva que delimita la zona de estimulación en la cara apical de la mucosa como se describe en el protocolo. Se utilizó este modelo para examinar, entre otros, los efectos diferenciales de las tres cepas de lactobacilos diferentes. Se demuestra que este modelo de sistema es muy poderoso para evaluar las propiedades inmunomoduladoras de los probióticos en buenas condiciones de salud y la enfermedad.

Hemos tenido éxito en el mantenimiento de la mucosa intestinal humana sana y EII en cultivo durante al menos 24 horas preservar el bienestar de los tejidos. De acuerdo con observaciones anteriores 1, esto sólo era posible si la mayoría del tiempo de cultivo (al menos el 85% del total) se llevó a cabo en O 2, como el tejido no sobrevivió durante 24 horas en incubadoras convencionales (Figura 2). También hemos demostrado que las diferentes respuestas de los explantes pueden ser...

Watch this Scientific Journal Video about A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants at JoVE.com

A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants

Se introduce un nuevo método para el mantenimiento de la mucosa intestinal humana en la cultura y el seguimiento de la respuesta a diversos tipos de estímulos durante al menos 24 horas. Con nuestro método, se mantiene la polaridad del tejido, lo que permite una estimulación fisiológica a través de la ruta apical.

Un nuevo método para la Cultura y polarizado estimulación de explantes mucosa intestinal humana

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper ...

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Biology

13.0K vistas.  European Institute of Oncology. Se introduce un nuevo método para el mantenimiento de la mucosa intestinal humana en la cultura y el seguimiento de la respuesta a diversos tipos de estímulos durante al menos 24 horas. Con nuestro método, se mantiene la polaridad del tejido, lo que permite una estimulación fisiológica a través de la ruta apical.

Video: A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

Método para la Cultura de primeros embriones del polluelo ex vivo (Nueva Cultura)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Investigación

Biología

Preparation and Culture of Rat Lens Epithelial Explants for Studying Terminal Differentiation

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  Following division, these cells migrate posteriorly, where FGF diffusing from the retina induces them to differentiate into a posterior array of elongated lens fiber cells, which compose the bulk of the lens.  Differentiation of lens epithelial cells into lens fibers can be induced in vitro by culturing explants of the central region of the anterior epithelium in the presence of FGF-2.  Explants are prepared from lenses of neonatal rats by removing the lens from the eye and grasping the lens capsule on the posterior side with dissecting tweezers.  The posterior capsule is then gently torn open and pressed down into the plastic bottom of a tissue culture dish. The peripheral regions of the explant are removed with a scalpel and the central area is then cultured in the presence of 100ng/ml FGF-2 for as long as 2-3 weeks, depending on the parameters to be studied.  Since epithelial cells in cultured explants differentiate in approximate synchrony over a period of days to weeks, the time course of signaling and gene expression can be determined using molecular, biochemical, and pharmacological techniques.  Immunofluorescence microscopy is a powerful adjunct to these methods as it demonstrates the subcellular localization of proteins of interest and can reveal the physiological consequences of experimental manipulations of signaling pathways.

Explants of the central region of rat lens epithelia differentiate synchronously when cultured in the presence of FGF-2. Immunofluorescence microscopy of such cultures can provides novel information about gene expression and signaling events associated with terminal differentiation.

Preparación y Cultura de la lente explantes Rata epiteliales para el estudio de la diferenciación terminal

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  ...

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development and growth of mammalian embryos are critically dependent on the function of the placenta, WEC technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages during embryonic day (E) 6.5-E12.5 in the mouse or E8.5-E14.5 in the rat 2, 3, 4. In WEC, we can directly target desired areas of embryos using fine glass capillaries because embryos can be manipulated under the microscope. Therefore, rodent WEC is very useful technique when we want to study dynamic developmental processes of postimplanted mammalian embryos. Up to date, several types of WEC systems have been developed 1. Among those, the rotator-type bottle culture system is most popular and suitable for long-term culture of embryos at midgestation, i.e., after E9.5 and E11.5 in the mouse and rat, respectively 1. In this video protocol, we demonstrate our standard procedures of rat WEC after E12.5 using a refined model of the original rotator system, which was designed by New and Cockroft 5, 6, and introduce various applications of WEC technique for studies in mammalian developmental biology.

Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.

El método de cultivo de embriones de roedores enteros utilizando el sistema de tipo de botella rotador Cultura

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development ...

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 ...

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.

El método de cultivo de la rebanada por Siguiendo desarrollo de gérmenes de dientes en Explante Cultura

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental ...

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 ...

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

Un protocolo para la de Lentiviral Transducción y Análisis de Aguas abajo de Intestinal organoides

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

Un método para la selección de aptámeros Estructura de conmutación Aplicado a una de nanopartículas de oro ensayo colorimétrico

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The colonic epithelium is dynamically turned over and epithelial cells are generated in the stem cell containing crypts of Lieberk&#252;hn. Progenitor cells produced in the crypt-bases migrate toward the luminal surface, undergoing a process of cellular differentiation before being shed into the gut lumen. In order to study these processes at the molecular level, we have developed a simple method for the microdissection of two spatially distinct regions of the colonic mucosa; the proliferative crypt zone, and the differentiated surface epithelial cells. Our objective is to isolate specific crypt and surface epithelial cell populations from mouse colonic mucosa for the isolation of RNA and protein.

Here we describe a simple method for the isolation of spatially distinct murine colonic epithelial surface cells from the underlying crypt-base cells.

Método cryosectioning para microdisección de murino mucosa colónica

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The ...