Name: method of studying palatal fusion using static organ culture
Uploaded: 2015-09-19T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Method of Studying Palatal Fusion using Static Organ Culture at JoVE.com

The authors do not have any acknowledgements.

Todos los procedimientos descritos deben realizarse de acuerdo con las directrices y normas para el uso de los animales vertebrados, incluyendo la aprobación previa del Comité local de Animal Institucional de Cuidado y Uso. 1. Preparación de disección Instrumentos y Medios de Cultivo <ol><li> Prelavado todos los instrumentos que se utilizarán en la disección con peróxido de hidrógeno 3% y luego autoclave a 121 ° C durante 20 min. Filtra medios BGJb con solución antibiótica y antimicótica usando un filtro de 0,22 micras de poro mediano con succión al vacío en una campana. </li></ol> 2. Preparación del Equipo de Cultura <ol><li> Cortar filtros de membrana de policarbonato que apoyarán los órganos palatinas en pequeños triángulos, rocíe con un 70% de alcohol y dejar que se sequen. Preparar Center-Well Organ Culture Dish (60x15 mm) mediante la colocación de una rejilla limpio y esterilizado equilátero de forma triangular de alambre (20 mm) sobre la placa de cultivo de órganos. Luego llene el pozo con 1 ml de BGJB medios de cultivo medios. NOTA: Los medios de comunicación deberían ser suficientes para alcanzar el nivel de la red sin sumergir los paladares. Coloque los filtros de membrana, el lado brillante hacia abajo, sobre la parte superior de la rejilla (3-4 membranas por cuadrícula) .Depending en el experimento, de tratamiento (proteínas, anticuerpos o inhibidores) se pueden añadir a los medios de comunicación </li></ol> 3. embrión de ratón Recogida y preparación para la Cultura <ol><li> Para obtener salvaje embriones de ratón de tipo, el uso programado 13.5 embarazada CD-1 de ratón cepa antecedentes genéticos. Limpiar la zona de disección con etanol al 70%. NOTA: Otras cepas de ratones pueden ser utilizados. Preferimos ratones CD-1 debido a la cantidad de arena más grande. </li><li> La eutanasia a los ratones embarazadas en E13.5 dpc (días después del coito) con 5% isoflurano seguido por dislocación cervical para asegurar la muerte a través de protocolos aprobados. </li><li> Pulverizar etanol al 70% en la superficie abdominal ventral para evitar que el palillo del pelo abdominal de los instrumentos. A continuación, abra la cavidad abdominal ventralutilizando una de bordes romos tijeras de operación y pinzas de micro-disección para localizar el útero. Utilizando las pinzas, levantar todo el útero y separarlo del cuerpo mediante el corte en el cuerpo uterino y en las puntas de los cuernos uterinos con una luz tijeras de funcionamiento. </li><li> Coloque el útero sobre una toalla de papel limpia sobre hielo (De acuerdo con la guía de protocolo para el cuidado y uso de animales de laboratorio). Cubra el útero con otra toalla de papel limpia para evitar la contaminación. Esterilizar los instrumentos que utilizan el 70% de etanol aerosol antes de usar más. El útero y los embriones pueden ser transportados al laboratorio en hielo. </li><li> En una campana de flujo laminar abierta, hacer cuidadosamente una pequeña abertura en el saco vitelino con unas tijeras de funcionamiento de bordes romos y pinzas de micro-disección. Haga la mayor apertura y luego empuje suavemente a exteriorizar el embrión desde el saco vitelino. </li><li> La transferencia de los embriones inmediatamente a una placa Petri con fría tamponada con fosfato salino 1X (PBS) pH 7,4. Para asegurar el escenario adecuado, examina El embrión bajo un microscopio de disección para el cuerpo, tamaño de la cabeza y la integridad física morfología. NOTA: Los embriones que no son en la etapa correcta del desarrollo no se utilizan para el experimento. </li></ol><img alt="Figura 2" src="/files/ftp_upload/53063/53063fig2.jpg" /> Figura 2. Limb Bud Morfología. La proa y extremidades traseras se comprueba el grado de sangrado membrana entre los dígitos. En E13.5, todas las condensaciones dígitos son prominentes, con la cincha y hendiduras distales entre los dígitos que aparecen muy distinta. Los dígitos traseras están detrás de un medio-día 12 Fila superior:. Extremidad anterior, fila inferior: extremidad posterior. 4. Boca Disección <ol><li> Coloque un embrión a la vez en una placa de Petri con PBS bajo el microscopio de disección. Usando pequeñas tijeras de disección y pinzas separar la cabeza del cuerpo de los embriones. </li><li> Sosteniendo con cuidado el cráneo del ratón sobre el nivel del ojo con pinzas (evitandoel proceso maxilar) hacer dos incisiones en ambos lados de la línea del labio. A continuación, retire la mandíbula y la lengua. Aislar la región maxilar proceso haciendo un corte a nivel del ojo y quitar el cerebro, los ojos, tabique nasal y el tejido adyacente. </li></ol><img alt="Figura 3" src="/files/ftp_upload/53063/53063fig3.jpg" /> Figura tejido 3. Boca obtiene de 13,5 embriones, cuando los estantes de paladar habían elevados pero no hizo contacto. Los palatinos se colocaron en cultivo de órganos en un filtro en la interfaz de medios aéreos durante 72 horas en el modelo estático. (A) Nivel de disección de embriones E13.5. (B) Azul estrellas demarcan áreas para mantener el tejido con pinzas durante la disección. (C) Vista de palatinos disecados colocados sobre una membrana de filtro trapezoidal. (D) Vista lateral de la placa de cultivo después de la colocación del paladares diseccionaron y adición de medios de cultivo. <ol start="3"><li> Coloque el lado oral, paladar y quitar el tabique nasal y el tejido que lo rodea, manteniendo el paladar primario unido para ayudar a orientar de anterior a posterior. Use una espátula de cuchara y transferir con mucho cuidado los procesos palatinos (lado nasal abajo) para los filtros preparados en las rejillas de las placas de cultivo. NOTA: Ajuste el volumen de los medios de comunicación para permitir adecuada interfaz aire-media. </li><li> Bajo el microscopio estereoscópico, diseccionar el paladar primario y utilizar pinzas para colocar los procesos palatinos cerca uno del otro. Los estantes de paladar no se fusionan si no se ponen en contacto. Cortar el filtro de membrana triangular donde se encuentra la parte anterior del paladar. La forma trapezoidal ayudará en la localización de la porción anterior del paladar. </li><li> Cultura de los procesos palatinos individual o con dos o tres en la misma placa de cultivo de órganos usando BGJb medios de cultivo. </li></ol> 5. Estantes cultivo ratón palatinos <ol><li> Incubar la tissues a 37 ° C en una mezcla de gas humidificado (5% de CO2 y 95% de aire) durante 72 horas, y cambian el medio cada 24 horas. </li></ol> 6. Paladar procesamiento para el análisis histológico <ol><li> Fijar los paladares después de 72 horas en cultivo con formaldehído al 4% / buffer fosfato salino O / N a los 4 ° &#160; C. Luego, suavemente con una pipeta de transferencia lavar el tejido en la placa de cultivo con PBS. Envuelva los paladares en un pedazo de toallitas de laboratorio y lugar en la incrustación de casetes. NOTA: Envolver los paladares en toallitas de laboratorio a prevenir su pérdida durante el proceso de deshidratación. </li><li> Deshidratar el tejido después de 70%, 80% y hasta el 100% lavados de etanol durante 1 hora, seguido por el procedimiento ordinario inclusión en parafina. Para incrustar, coloque la parte anterior del paladar hacia la bandeja. </li><li> Cortar serie 6 micras secciones en la orientación coronal de anterior a posterior. Una sección paladar completa producirá 350-450 sections. Teñir las secciones con hematoxilina y eosina (H &amp; E). Cada sección 20ª utilizando el descrito anteriormente Mean Fusión Puntuación (MFS) escala de 10 Score (Tabla 1). 8 </li></ol>

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	<li>Nawshad, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palatal+seam+disintegration+to+die+or+not+to+die+that+is+no+longer+the+question.">Palatal seam disintegration to die or not to die that is no longer the question.</a> Developmental dynamics an official publication of the American Association of Anatomists. 237, 2643-2656 (2008).</li><li>Strong, E. B., Buckmiller, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+the+cleft+palate.">Management of the cleft palate.</a> Facial plastic surgery clinics of North America. 9, 15-25 (2001).</li><li>Witt, P. D., Marsh, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+assessing+outcome+of+surgical+repair+of+cleft+lip+and+cleft+palate.">Advances in assessing outcome of surgical repair of cleft lip and cleft palate.</a> Plastic and reconstructive surgery. 100, 1907-1917 (1997).</li><li>4miloro, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> principles of oral and maxillofafcial surgery. 209, 231-249 (1984).</li><li>Mima, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+epithelial+adhesion+molecule+CEACAM1+is+important+for+palate+formation.">Regulation of the epithelial adhesion molecule CEACAM1 is important for palate formation.</a> PloS one. 8, e61653 (2013).</li><li>Carette, M. J., Ferguson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fate+of+medial+edge+epithelial+cells+during+palatal+fusion+in+vitro+an+analysis+by+DiI+labelling+and+confocal+microscopy.">The fate of medial edge epithelial cells during palatal fusion in vitro an analysis by DiI labelling and confocal microscopy.</a> Development (Cambridge, England). 114, 379-388 (1992).</li><li>Ferguson, M. W., Honig, L. S., 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=Differentiation+of+cultured+palatal+shelves+from+alligator+chick+and+mouse+embryos.">Differentiation of cultured palatal shelves from alligator chick and mouse embryos.</a> The Anatomical record. 209, 231-249 (1984).</li><li>San Miguel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ephrin+reverse+signaling+controls+palate+fusion+via+a+PI3+kinase+dependent+mechanism.">Ephrin reverse signaling controls palate fusion via a PI3 kinase dependent mechanism.</a> Developmental dynamics an official publication of the American Association of Anatomists. 240, 357-364 (2011).</li><li>Kang, P., Svoboda, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PI+3+kinase+activity+is+required+for+epithelial+mesenchymal+transformation+during+palate+fusion.">PI 3 kinase activity is required for epithelial mesenchymal transformation during palate fusion.</a> Developmental dynamics an official publication of the American Association of Anatomists. 225, 316-322 (2002).</li><li>RD, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Histopathologic Technic and Practical Histochemistry. , (1965).</li><li>Kaufman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Atlas of Mouse Development. , (1992).</li><li>Taher, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+gene+expression+analysis+of+murine+limb+development.">Global gene expression analysis of murine limb development.</a> PloS one. 6, e28358 (2011).</li></ol>

The authors do not have any competing financial interests.

El protocolo en este artículo proporciona un método de disección palatinos de los embriones en el día embrionario 13.5. Palatinos de embriones de ratón se cultivaron en un medio de cultivo libre de suero en una atmósfera de 95% de O 2 5% de CO 2 a 37 ° C. Disección palatina éxito depende fundamentalmente de factores múltiples en cada paso durante el procedimiento desde el momento embrionario de los ratones sacrificados a la finalización de la cultura. Uno de los factores más importantes...

Hendiduras orofaciales son los defectos congénitos craneofaciales más predominantes. Además, tomando en consideración todos los posibles defectos craneofaciales, se trata de la segunda anomalía de nacimiento más común en los recién nacidos 2. Paladar hendido ocurren en aproximadamente 1 de cada 700 nacimientos en los Estados Unidos (US) todos los años, la incidencia de paladar hendido es igual a 475 niños nacidos con paladar hendido al mes o 15 niños con hendiduras por día 3. Aproximadamente el 1% de los niños nacidos en todo el mundo cada año presentan algún tipo de dismorfología craneofacial. Las hendiduras del paladar y el labio necesitan un procedimiento muy costoso y complicado con implicaciones para toda la vida para los pacientes con esta anomalía. El costo estimado para cada paciente con fisura oral es de aproximadamente $ 100.000 4. El tratamiento de un paciente con labio leporino y paladar hendido requiere un equipo de médicos, incluyendo craneofaciales cirujanos, otorrinolaringólogos, genetistas, anestesiólogos,patólogos del habla-lenguaje, nutricionistas, ortodoncistas, protesistas, psicólogos, neurocirujanos y oftalmólogos. En palatogénesis, paladar secundario surge como excrecencias pareadas, que inicialmente crecen verticalmente y se someten a la elevación estante palatina sobre el dorso de la lengua. A raíz de la elevación, los palatinos pareadas crecen hacia la línea media (en E14.5 -E15 en ratones y la semana 9 en los seres humanos). El epitelio borde medial (MEE) que cubre la punta estante se adhiere formando la costura epitelial línea media. Esto es seguido por epitelial a mesenquimal transición y / o apoptosis para permitir la confluencia mesenquimales. Adhesión de oponerse MEE es un evento esencial cuya alteración provoca paladar hendido. Sin embargo, sólo unos pocos estudios investigaron la adhesión inicial de palatinos 5. Las formas de paladar duro por la diferenciación de las células mesenquimales en osteoblastos. El desarrollo anormal del paladar puede producir cleft paladares con o sin la participación del labio. Técnicas de cultivo de órganos Paladar habían utilizado ampliamente durante muchos laboratorios en los últimos 30 años 6,7. En este protocolo se describe en detalle un método de la disección del paladar y de la cultura de órganos estática. La ventaja de un cultivo de órganos inmóvil es que permite palatinos se fusionen. Esta técnica se ha utilizado con éxito en nuestro laboratorio para muchos de fusión y de señalización experimentos 8,9. Sin embargo, el ámbito de aplicación de la técnica es muy amplia y se puede utilizar siempre que se requiera el sistema de cultivo de órganos estática, incluyendo la evaluación de las respuestas a los agentes químicos exógenos, los efectos de los factores de crecimiento de regulación en diferentes vías y proteínas específicas. <img alt="Figura 1" src="/files/ftp_upload/53063/53063fig1.jpg" /> Figura 1. Ratón palatogénesis. Etapas de desarrollo del paladar ratones. (BF) ele Escaneomicrografías ctron (SEM) del paladar secundario en tiempos de desarrollo representativas. Flechas rojas: muestran la parte inicial de la adhesión del paladar y la fusión. Flechas amarillas: punto al espacio entre los paladares de primaria y secundaria que desaparecerán después de la fusión (Tomado de Kaufman 11 con permiso de PLoS ONE).

<table><tbody><tr><td>BGJ&lt;sub&gt;b&lt;/sub&gt;</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Lonza Inc.</td><td>17-602E</td><td>100X</td></tr><tr><td>Petri dishes</td><td>VWR</td><td>25384-088</td><td>100 x15 mm</td></tr><tr><td>Center-well organ culture dish</td><td>Falcon&amp;nbsp;</td><td>#353037</td><td>60 x15 mm</td></tr><tr><td>Wire triangular grid</td><td>Custom made with stainless steel wire mesh to fit the organ culture dish well.&amp;nbsp;&amp;nbsp;</td><td></td><td></td></tr><tr><td>Polycarbonate filter</td><td>GE&amp;amp; Water and Process Technologies</td><td>K04BP04700</td><td>Black 0.4 micron, 47 mm</td></tr><tr><td>Stereo microscope</td><td>ZEISS&amp;nbsp; Stemi SR</td><td></td><td></td></tr><tr><td>Culture hood</td><td>NUAIRE Laminar Flow</td><td></td><td></td></tr><tr><td>Microdissecting forceps</td><td>Dumont Medical&amp;nbsp;</td><td></td><td>#5</td></tr><tr><td>Microdissecting scissors</td><td>Kent Scientific Corporation</td><td>INS14003-G</td><td>Vannas Scissors, straight, 8 cm long, 0.1mm tips, 5 mm blades, German made</td></tr><tr><td>Microdissecting scissors</td><td>Kent Scientific Corporation</td><td>INS500086</td><td>Vannas Scissors, straight, 8.5 cm long, 0.025 mm x 0.015 mm tips, 7 mm blades</td></tr><tr><td>Microdissecting scissors</td><td>Kent Scientific Corporation</td><td>INS14127-G</td><td>Spring scissors curved curved, 10.5 cm long, 8 mm blades, German made</td></tr><tr><td>Surgical currete (spoon spatula)</td><td>Hu-friedy</td><td></td><td>CM 2/4</td></tr><tr><td>Protector laboratory hood</td><td>Labconco</td><td></td><td></td></tr><tr><td>Incubator</td><td>Thermo Scientific</td><td></td><td>Farma</td></tr><tr><td>Series11 water Jacket</td><td></td><td></td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td></td><td></td><td></td></tr><tr><td>PBS&amp;nbsp;</td><td>GIBCO</td><td>10010-023&amp;nbsp;</td><td>1X</td></tr><tr><td>Fiber optic Light source</td><td>Fiber-light&amp;nbsp; Dolan-Jenner</td><td>PL-750</td><td></td></tr><tr><td>Embedding cassette&amp;nbsp;</td><td>Statlab</td><td>EC301</td><td></td></tr><tr><td>Kim Wipes</td><td>VWR</td><td>470173-504</td><td></td></tr></tbody></table>

method of studying palatal fusion using static organ culture

El labio leporino y el paladar se encuentran entre los más comunes de todos los defectos congénitos. Las formas de paladar secundario de estantes mesenquimales cubiertas de epitelio que se adhiere al formar la costura epiteliales de la línea media (MES). Las teorías sugieren que las células MES siguen un epitelio de transición mesenquimal (EMT), la apoptosis y la migración, haciendo un paladar fusionado 1. La desintegración completa de la economía de mercado es la fase esencial definitiva de confluencia del paladar y la zona células mesenquimales. Ofrecemos un método para cultivo de órganos paladar. La desarrollado protocolo in vitro permite el estudio de los procesos biológicos y moleculares durante la fusión. Las aplicaciones de esta técnica son numerosas, incluyendo la evaluación de las respuestas a los agentes químicos exógenos, efectos de los factores de regulación y de crecimiento y proteínas específicas. Cultivo de órganos Palatal tiene una serie de ventajas, incluyendo la manipulación en diferentes etapas de desarrollo que no es posible utilizando los estudios in vivo.

La técnica se puede aplicar a diferentes tipos de experiments.The siguiente estudio fue diseñado para probar el efecto teratogénico de la nicotina in vitro en la fusión palatal. Dos cepas de ratones fueron utilizados para este experimento, CD1 y el tejido C57.Palatal se trató con concentraciones mM de hemisulfato de nicotina 0,06 y 6. Se observó que los patrones de fusión palatinas y el momento eran diferentes en las dos cepas diferentes. En ratones CD1, estantes palatales del grupo de control continuaro...

Watch this Scientific Journal Video about Method of Studying Palatal Fusion using Static Organ Culture at JoVE.com

Method of Studying Palatal Fusion using Static Organ Culture

Los estudios sobre el desarrollo del paladar están motivados por la incidencia de paladar hendido, un defecto de nacimiento que impone una carga de salud tremenda y puede dejar la desfiguración permanente. Se demuestra aquí una técnica para estantes cultura palatinas que se pueden utilizar para estudiar diferentes vías de señalización implicadas en el desarrollo de paladar y la fusión.

Método de estudio palatina Fusión usando estático Organ Culture

Cleft lip and palate are among the most common of all birth defects. The secondary palate forms from mesenchymal shelves covered with epithelium that ...

method-of-studying-palatal-fusion-using-static-organ-culture

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

8.7K vistas.  Texas A&M University Baylor College of Dentistry.  Los estudios sobre el desarrollo del paladar están motivados por la incidencia de paladar hendido, un defecto de nacimiento que impone una carga de salud tremenda y puede dejar la desfiguración permanente. Se demuestra aquí una técnica para estantes cultura palatinas que se pueden utilizar para estudiar diferentes vías de señalización implicadas en el desarrollo de paladar y la fusión. 

Video: Method of Studying Palatal Fusion using Static Organ Culture

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head and heart progenitor cell development with pharyngeal arches (also known as branchial arches) ex vivo. Using this method, we have recently described that the second pharyngeal arch contains self-renewing heart progenitors and serves as a microenvironment for expansion of the progenitors during mouse heart development. The progenitor cells remain undifferentiated and expansive inside the arch, but quickly become functional cardiomyocytes as they migrate out of the arch. We also reported that first pharyngeal arch contains muscle progenitors giving rise to myotubes after leaving the arch. Here, we demonstrate the procedure for the dissection and ex vivo culture of first and second pharyngeal arches from developing mouse embryos. The method enables one to study head and heart progenitor/muscle development, including cardiomyocyte and myotube formation in detail ex vivo.

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Here, we present a protocol to culture pharyngeal arches to study the biology of heart and muscle progenitor cells and their microenvironment.

Ex Vivo Cultura de faríngea Arcos para el Estudio de corazón y músculo Progenitores y su nicho

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head ...

Investigación

Biología del Desarrollo

Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of them being fluorescence in situ hybridization (FISH).1,2 We performed a pilot study in which oral biofilm samples collected from fixed orthodontic appliances (palatal expanders) were stained by FISH, the objective being to assess the three-dimensional organization of natural biofilm and plaque accumulation.3,4 FISH creates an opportunity to stain cells in their native biofilm environment by the use of fluorescently labeled 16S rRNA-targeting probes.4-7,19 Compared to alternative techniques like immunofluorescent labeling, this is an inexpensive, precise and straightforward labeling technique to investigate different bacterial groups in mixed biofilm consortia.18,20 General probes were used that bind to Eubacteria (EUB338 + EUB338II + EUB338III; hereafter EUBmix),8-10 Firmicutes (LGC354 A-C; hereafter LGCmix),9,10 and Bacteroidetes (Bac303).11 In addition, specific probes binding to Streptococcus mutans (MUT590)12,13 and Porphyromonas gingivalis (POGI)13,14 were used. The extreme hardness of the surface materials involved (stainless steel and acrylic resin) compelled us to find new ways of preparing the biofilm. As these surface materials could not be readily cut with a cryotome, various sampling methods were explored to obtain intact oral biofilm. The most workable of these approaches is presented in this communication. Small flakes of the biofilm-carrying acrylic resin were scraped off with a sterile scalpel, taking care not to damage the biofilm structure. Forceps were used to collect biofilm from the steel surfaces. Once collected, the samples were fixed and placed directly on polysine coated glass slides. FISH was performed directly on these slides with the probes mentioned above. Various FISH protocols were combined and modified to create a new protocol that was easy to handle.5,10,14,15 Subsequently the samples were analyzed by confocal laser scanning microscopy. Well-known configurations3,4,16,17 could be visualized, including mushroom-style formations and clusters of coccoid bacteria pervaded by channels. In addition, the bacterial composition of these typical biofilm structures were analyzed and 2D and 3D images created.

We present a protocol for structural and compositional analysis of natural oral biofilm from orthodontic appliances with in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Oral biofilm samples were collected from palatal expanders, scraping acrylic-resin flakes off their surface and referring them for molecular processing.

Análisis de biofilm oral del paladar Expansores por fluorescencia de hibridación in situ y microscopía de escaneo láser confocal

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of ...

Medicina

Isolation and Culture of Human Fungiform Taste Papillae Cells

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple stimuli and complex chemical molecules contained in foods. In human, individual fungiform papillae contain from zero to as many as 20 taste buds. There is no established protocol for culturing human taste cells, although the ability to maintain taste papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Earlier studies of taste cells have been done using freshly isolated cells in primary culture, explant cultures from rodents, or semi-intact taste buds in tissue slices1,2,3,4. Although each of these preparations has advantages, the development of long-term cultures would have provided significant benefits, particularly for studies of taste cell proliferation and differentiation. Several groups, including ours, have been interested in the development and establishment of taste cell culture models. Most attempts to culture taste cells have reported limited viability, with cells typically not lasting beyond 3-5 d5,6,7,8. We recently reported on a successful method for the extended culture of rodent taste cells9. We here report for the first time the establishment of an in vitro culture system for isolated human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Cells displayed many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C &beta;2, (PLC-&beta;2) mRNA were detected in many cells by reverse transcriptase-polymerase chain reaction and confirmed by sequencing. Immunocytochemistry analysis demonstrated the presence of gustducin and PLC-&beta;2 expression in cultured taste cells. Cultured human fungiform cells also exhibited increases in intracellular calcium in response to appropriate concentrations of several taste stimuli indicating that taste receptors and at least some of the signalling pathways were present. These results sufficient indicate that taste cells from adult humans can be generated and maintained for at least eight passages. Many of the cells retain physiological and biochemical characteristics of acutely isolated cells from the adult taste epithelium to support their use as a model taste system. This system will enable further studies of the processes involved in proliferation, differentiation and function of mammalian taste receptor cells in an in vitro preparation.
Human fungiform taste papillae used for establishing human fungiform cell culture were donated for research following proper informed consent under research protocols that were reviewed and approved by the IRB committee. The protocol (#0934) was approved by Schulman Associates Institutional Review Board Inc., Cincinnati, OH. Written protocol below is based on published parameters reported by Ozdener et al. 201110.

We aimed to develop a reproducible protocol for isolating and maintaining long-term cultures of human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability.

Aislamiento y cultivo de células humanas las papilas fungiformes gusto

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple ...

Neurociencias

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

Imágenes en vivo de la fusión secundaria del paladar del ratón

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

Cultura primaria y electroporación plásmido del órgano de Corti murino.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Biología

Isolation and Time-Lapse Imaging of Primary Mouse Embryonic Palatal Mesenchyme Cells to Analyze Collective Movement Attributes

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and fusion above the tongue. Defects in this process lead to cleft palate, a common birth defect. Recent studies have shown that palatal shelf elevation involves a remodeling process that transforms the orientation of the shelf from a vertical to a horizontal one. The role of the palatal shelf mesenchymal cells in this dynamic remodeling has been difficult to study. Time-lapse-imaging-based quantitative analysis has been recently used to show that primary mouse embryonic palatal mesenchymal (MEPM) cells can self-organize into a collective movement. Quantitative analyses could identify differences in mutant MEPM cells from a mouse model with palate elevation defects. This paper describes methods to isolate and culture MEPM cells from E13.5 embryos-specifically for time-lapse imaging-and to determine various cellular attributes of collective movement, including measures for stream formation, shape alignment, and persistence of direction. It posits that MEPM cells can serve as a proxy model for studying the role of palatal shelf mesenchyme during the dynamic process of elevation. These quantitative methods will allow investigators in the craniofacial field to assess and compare collective movement attributes in control and mutant cells, which will augment the understanding of mesenchymal remodeling during palatal shelf elevation. Furthermore, MEPM cells provide a rare mesenchymal cell model for investigation of collective cell movement in general.

We present a protocol for isolation and culture of primary mouse embryonic palatal mesenchymal cells for time-lapse imaging of two-dimensional (2D) growth and wound-repair assays. We also provide the methodology for analysis of the time-lapse imaging data to determine cell-stream formation and directional motility.

Aislamiento e imágenes de lapso de tiempo de células mesenquimas palatinas embrionarias primarias de ratón para analizar los atributos del movimiento colectivo

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and ...

Use of Trowell-Type Organ Culture to Study Regulation of Dental Stem Cells

Organ development, function, and regeneration depend on stem cells, which reside within discrete anatomical spaces called stem cell niches. The continuously growing mouse incisor provides an excellent model to study tissue-specific stem cells. The epithelial tissue-specific stem cells of the incisor are located at the proximal end of the tooth in a niche called the cervical loop. They provide a continuous influx of cells to counterbalance the constant abrasion of the self-sharpening tip of the tooth. Presented here is a detailed protocol for the isolation and culture of the proximal end of the mouse incisor that houses stem cells and their niche. This is a modified Trowell-type organ culture protocol that enables in vitro culture of tissue pieces (explants), as well as the thick tissue slices at the liquid/air interface on a filter supported by a metal grid. The organ culture protocol described here enables tissue manipulations not feasible in vivo, and when combined with the use of a fluorescent reporter(s), it provides a platform for the identification and tracking of discrete cell populations in live tissues over time, including stem cells. Various regulatory molecules and pharmacological compounds can be tested in this system for their effect on stem cells and their niches. This ultimately provides a valuable tool to study stem cell regulation and maintenance.

The Trowell-type organ culture method has been used to unravel complex signaling networks that govern tooth development and, more recently, for studying regulation involved in stem cells of the continuously growing mouse incisor. Fluorescent-reporter animal models and live-imaging methods facilitate in-depth analyses of dental stem cells and their specific niche microenvironment.

Uso del cultivo de órganos tipo Trowell para estudiar la regulación de las células madre dentales

Organ development, function, and regeneration depend on stem cells, which reside within discrete anatomical spaces called stem cell niches. The ...

Whole-Mount Staining, Visualization, and Analysis of Fungiform, Circumvallate, and Palate Taste Buds

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells communicate with nerve fibers that carry this information to the brain. Because taste-transducing cells continuously die and are replaced throughout adulthood, the taste-bud environment is both complex and dynamic, requiring detailed analyses of its cell types, their locations, and any physical relationships between them. Detailed analyses have been limited by tongue-tissue heterogeneity and density that have significantly reduced antibody permeability. These obstacles require sectioning protocols that result in splitting taste buds across sections so that measurements are only approximated, and cell relationships are lost. To overcome these challenges, the methods described herein involve collecting, imaging, and analyzing whole taste buds and individual terminal arbors from three taste regions: fungiform papillae, circumvallate papillae, and the palate. Collecting whole taste buds reduces bias and technical variability and can be used to report absolute numbers for features including taste-bud volume, total taste-bud innervation, transducing-cell counts, and the morphology of individual terminal arbors. To demonstrate the advantages of this method, this paper provides comparisons of taste bud and innervation volumes between fungiform and circumvallate taste buds using a general taste-bud marker and a label for all taste fibers. A workflow for the use of sparse-cell genetic labeling of taste neurons (with labeled subsets of taste-transducing cells) is also provided. This workflow analyzes the structures of individual taste-nerve arbors, cell type numbers, and the physical relationships between cells using image analysis software. Together, these workflows provide a novel approach for tissue preparation and analysis of both whole taste buds and the complete morphology of their innervating arbors.

This paper describes methods for tissue preparation, staining, and analysis of whole fungiform, circumvallate, and palate taste buds that consistently yield whole and intact taste buds (including the nerve fibers that innervate them) and maintain the relationships between structures within taste buds and the surrounding papilla.

Tinción, visualización y análisis de papilas gustativas fungiformes, circunvaladas y del paladar

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells ...

Culture and Imaging of Human Nasal Epithelial Organoids

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis Transmembrane conductance Regulator (CFTR) activity and restoration from small molecule compounds. Our group recently focused on establishing a well-differentiated organoid model directly derived from primary human nasal epithelial cells (HNE). Histology of sectioned organoids, whole-mount immunofluorescent staining, and imaging (using confocal microscopy, immunofluorescent microscopy, and bright field) are essential to characterize organoids and confirm epithelial differentiation in preparation for functional assays. Furthermore, HNE organoids produce lumens of varying sizes that correlate with CFTR activity, distinguishing between CF and non-CF organoids. In this manuscript, the methodology for culturing HNE organoids are described in detail, focusing on the assessment of differentiation using the imaging modalities, including the measurement of baseline lumen area (a method of CFTR activity measurement in organoids that any laboratory with a microscope can employ) as well as the developed automated approach to a functional assay (which requires more specialized equipment).

A detailed protocol is presented here to describe an in vitro organoid model from human nasal epithelial cells. The protocol has options for measurements requiring standard laboratory equipment, with additional possibilities for specialized equipment and software.

Cultivo e imágenes de organoides epiteliales nasales humanos

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

Aislamiento y cultivo de queratinocitos orales primarios del paladar del ratón adulto

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...