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 os procedimentos descritos devem ser realizados de acordo com as diretrizes e regulamentos para o uso de animais vertebrados, incluindo a aprovação prévia pelo Comitê Animal Care Institucional e Use o local. 1. Preparação de Instrumentos dissecação e Meios de Cultura <ol><li> Pré-lavagem todos os instrumentos a serem usados ​​na dissecção com 3% de peróxido de hidrogénio e, em seguida, em autoclave a 121 ° C durante 20 min. Filtrar meios BGJb com solução de antibiótico e antimicótico usando um filtro de 0,22 um do tamanho de poros com sucção de vácuo em uma capa. </li></ol> 2. Preparação de Equipamentos Cultura <ol><li> Corte filtros de membrana de policarbonato que irá apoiar os órgãos palatais em pequenos triângulos, spray com 70% de álcool e deixe secar. Prepare prato Centro de Bem-Organ Culture (60x15 mm), colocando uma grade limpos e esterilizados de forma triangular equilátero fio (20 mm) sobre a placa de cultura de órgãos. Em seguida, encher o poço com 1 ml de BGJB meios de cultura de mídia. NOTA: A mídia deve ser suficiente para atingir o nível da rede sem submergir os paladares. Colocar os filtros de membrana, o lado brilhante para baixo, em cima da grelha (3-4 membranas por grade) .Depending na experiência, o tratamento (proteínas, anticorpos ou inibidores) podem ser adicionados aos meios </li></ol> 3. mouse Embryo Recolha e Preparação para a Cultura <ol><li> Para obter embriões tipo selvagem do rato, uso cronometrado 13,5 grávida CD-1 camundongo fundo genético tensão. Limpe a área de dissecção com etanol 70%. NOTA: Outras estirpes de ratos pode ser usada. Nós preferimos ratinhos CD-1 por causa do maior número de maca. </li><li> Euthanize camundongos grávidas em E13.5 DPC (dias pós-coito) com 5% de isoflurano seguido por deslocamento cervical para segurar a morte usando protocolos aprovados. </li><li> Spray de etanol a 70% sobre a superfície abdominal ventral para evitar que o pau de cabelo abdominal para os instrumentos. Em seguida, abra a cavidade abdominal ventralusando um forte franco-tesoura operacionais e micro-dissecando fórceps para localizar o útero. Usando fórceps, levantar todo o útero e separá-lo do corpo através do corte no corpo do útero e nas pontas dos cornos uterinos com uma luz de uma tesoura de funcionamento. </li><li> Coloque o útero sobre uma toalha de papel sobre o gelo (De acordo com o guia de protocolo para o cuidado e uso de animais de laboratório). Cubra o útero com outra toalha de papel limpo para evitar a contaminação. Esterilizar os instrumentos, utilizando 70% spray de etanol antes de usar ainda mais. O útero e os embriões podem ser transportadas para o laboratório em gelo. </li><li> Em uma câmara de fluxo laminar aberto, cuidadosamente fazer uma pequena abertura no saco vitelino com uma tesoura afiada-operacionais sem corte e micro-dissecando fórceps. Faça a maior abertura e, em seguida, empurre-o suavemente para exteriorizar o embrião a partir do saco vitelino. </li><li> Transferir os embriões imediatamente para uma placa de Petri com frio salino tamponado com fosfato 1X (PBS) pH 7,4. Para garantir o palco adequado, examina O embrião sob um microscópio de dissecação para o corpo, o tamanho da cabeça e membro morfologia. NOTA: Os embriões que não são na fase certa do desenvolvimento não são utilizados para o experimento. </li></ol><img alt="Figura 2" src="/files/ftp_upload/53063/53063fig2.jpg" /> Figura 2. Limb Bud Morfologia. O dianteiro e membros traseiros são verificados para o grau de recuo membrana entre os dígitos. No E13.5, todas as condensações dígitos são proeminentes, com o cinto e recortes distal entre os dígitos que aparece muito distinta. Os dígitos traseiras estão por trás por um meio-dia 12 de fileira superior:. Forelimb, fila inferior: pata traseira. 4. Palate Dissection <ol><li> Coloque um embrião de cada vez numa caixa de Petri com PBS sob o microscópio de dissecação. Usando as tesouras de dissecção pequenas pinças e separar a cabeça do corpo dos embriões. </li><li> Segurando cuidadosamente o crânio do mouse acima do nível do olho com uma pinça (evitandoo processo maxilar) fazer duas incisões em ambos os lados da linha dos lábios. Em seguida, retire a mandíbula e língua. Isolar a região processo maxilar, fazendo um corte ao nível dos olhos e remova o cérebro, olhos, septo nasal e tecido adjacente. </li></ol><img alt="Figura 3" src="/files/ftp_upload/53063/53063fig3.jpg" /> Figura 3. Palate tecido coletado de 13,5 embriões, quando as prateleiras palato tinha elevados, mas não fez contato. As lâminas palatinas foram colocados em cultura de órgão em um filtro na interface mídia de ar durante 72 horas no modelo estático. (A) Nível de dissecação em E13.5 embrião. (B) Blue estrelas demarcar áreas para segurar o tecido com uma pinça durante a dissecção. (C) Vista de lâminas palatinas dissecados colocados em uma membrana de filtro trapezoidal. (D) Vista lateral do prato de cultura após a colocação do paladares dissecados e adição de meio de cultura. <ol start="3"><li> Coloque o lado bucal palatal e remover o septo nasal eo tecido em torno dele, mantendo o palato primário anexado para ajudar a orientar no sentido antero-posterior. Use uma colher espátula e transfira com muito cuidado as lâminas palatinas (lado nasal para baixo) para os filtros preparados nas grades nas placas de cultura. NOTA: Ajustar o volume de mídia para permitir a adequada interface de mídia de ar. </li><li> Sob o microscópio estéreo, dissecar o palato primário e utilizar uma pinça para colocar as lâminas palatinas próximos uns dos outros. As lâminas palatinas não vai fundir se não forem colocados em contato. Cortar o filtro de membrana triangular em que a parte anterior do palato é localizado. A forma trapezoidal ajudará na localização da porção anterior do palato. </li><li> Cultura das lâminas palatinas individualmente ou com dois ou três na mesma placa de cultura de órgãos utilizando meios de cultura BGJb. </li></ol> 5. A cultura Prateleiras Rato Palatinas <ol><li> Incubar a tissues a 37 ° C numa mistura de gás humidificada (5% de CO 2 e 95% de ar) durante 72 horas e mudança do meio cada 24 horas. </li></ol> 6. Palate Processing para análise histológica <ol><li> Corrigir os paladares após 72 horas em cultura com 4% de formaldeído / tampão fosfato salina O / N a 4 ° C &#160; C. Em seguida, suavemente com uma pipeta de transferência para a lavagem do tecido na placa de cultura com PBS. Enrole os paladares em um pedaço de toalhetes de laboratório e lugar na incorporação de cassetes. NOTA: Envolvendo os paladares em toalhetes de laboratório evitar a sua perda durante o processo de desidratação. </li><li> Desidratar o tecido a seguir 70%, 80% e até 100% lavagens de etanol durante 1 hora, seguido pelo procedimento de parafina normal. Para a incorporação, coloque a parte anterior do palato de frente para a bandeja. </li><li> Corte de série 6 mm seções na orientação coronal de anterior para posterior. Um palato seção completa vai render 350-450 seçons. Corar as secções com hematoxilina e eosina (H &amp; E). 10 Pontuação cada 20 th seção usando o escore médio de fusão anteriormente descrito (MFS) escala (Tabela 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.

O protocolo neste artigo fornece um método de dissecação lâminas palatinas a partir de embriões no dia embrionário 13,5. Lâminas palatinas a partir de embriões de ratinho foram cultivadas num meio de cultura isento de soro, numa atmosfera de 95% de O2 5% de CO2 a 37 ° C. Dissecção palatino bem sucedido é criticamente dependente de vários factores, em cada passo durante o processo a partir do momento embrionário dos ratinhos sacrificados para a realização da cultura. Um dos factores ...

Fendas orofaciais são os defeitos de nascimento craniofaciais mais predominantes. Além disso, levando em consideração todos os possíveis defeitos craniofaciais, estes são a segunda anomalia nascimento mais comum em recém-nascidos 2. Fenda palatina ocorre em aproximadamente 1 em cada 700 nascimentos nos Estados Unidos (EUA) a cada ano, a incidência de fenda palatina é igual a 475 crianças nascidas com fissuras palatinas por mês ou 15 crianças com fissuras por dia 3. Cerca de 1% das crianças nasceram em todo o mundo a cada ano apresentam alguma forma de dysmorphology craniofacial. Fendas do palato e lábio precisa de um procedimento muito caro e complicado, com implicações ao longo da vida para os pacientes que têm essa anomalia. O custo estimado para cada paciente com fissura oral é de aproximadamente $ 100,000 4. O tratamento de um paciente com fissura labiopalatina requer uma equipe de médicos, incluindo cirurgiões craniofaciais, otorrinolaringologistas, geneticistas, anestesiologistas,fonoaudiológicas patologistas, nutricionistas, ortodontistas, prosthodontists, psicólogos, neurocirurgiões e oftalmologistas. Em palatogenesis, o palato secundário surge como conseqüências emparelhados, que inicialmente crescem verticalmente e se submetem a elevação prateleira palatal acima do dorso da língua. Após elevação, as lâminas palatinas emparelhados crescem em direcção da linha média (em E14.5 -E15 em ratinhos e em seres humanos semana 9). O epitélio borda medial (MEE), que cobre a ponta prateleira adere formar a costura linha média epitelial. Isto é seguido por epitelial para mesenquimal e / ou apoptose para permitir mesenquimais confluência. Adesão de se opor MEE é um evento essencial cuja alteração provoca fissura palatina. No entanto, poucos estudos investigaram a adesão inicial de 5 palatais prateleiras. As formas do palato duro através da diferenciação de células mesenquimais em osteoblastos. O desenvolvimento anormal do paladar pode produzir cleft paladares com ou sem envolvimento de lábio. As técnicas de cultura de órgãos paladar tinha sido amplamente utilizado para muitos laboratórios nos últimos 30 anos 6,7. Neste protocolo, descrever em detalhes um método de dissecção palatal e cultura de órgão estático. A vantagem de uma cultura de órgãos imóvel é que ele permite que lâminas palatinas para fundir. Esta técnica tinha sido usada com sucesso em nosso laboratório para muitos de fusão e de sinalização experimentos 8,9. No entanto, o âmbito da técnica é muito grande e pode ser utilizado sempre que sistema de cultura de órgão estático é necessário, incluindo a avaliação de respostas a agentes químicos exógenos, os efeitos de factores de crescimento em diferentes vias reguladoras e proteínas específicas. <img alt="figura 1" src="/files/ftp_upload/53063/53063fig1.jpg" /> Figura 1. Rato Palatogenesis. Estágios de desenvolvimento do palato camundongos. (BF) ELE Digitalizaçãomicrografias ctron (SEM) do palato secundário, por vezes, de desenvolvimento representativas. As setas vermelhas: mostrar a parte inicial da adesão palatal e fusão. Setas amarelas: Ponto para o espaço entre os paladares primárias e secundárias que irão desaparecer após a fusão (reproduzido a partir de Kaufman 11 com a permissão 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

A fissura labiopalatina estão entre os mais comuns de todos os defeitos de nascimento. As formas palato secundário de prateleiras mesenquimais cobertas com epitélio que adere para formar a costura linha média epitelial (MES). As teorias sugerem que as células MES seguir um epitelial para mesenquimal (EMT), apoptose e migração, fazendo um paladar fundido 1. Desintegração completa dos MES é a fase essencial final de palatal confluência com áreas células mesenquimais. Nós fornecemos um método de cultura de órgãos paladar. O protocolo desenvolvido em vitro permite o estudo de processos biológicos e moleculares durante a fusão. As aplicações desta técnica são numerosos, incluindo avaliando respostas aos agentes químicos exógenos, efeitos de fatores de regulação e de crescimento e proteínas específicas. Palatal cultura de órgãos tem um número de vantagens, incluindo a manipulação em diferentes fases de desenvolvimento que não é possível, utilizando estudos in vivo.

A técnica pode ser aplicada a diferentes tipos de experiments.The seguinte estudo foi concebido para testar o efeito teratogénico de nicotina in vitro em fusão palatino. Duas estirpes de ratos foram utilizados para esta experiência, CD1 e tecido C57.Palatal foi tratada com 0,06 e 6 mM concentrações de hemissulfato de nicotina. Observou-se que os padrões de fusão palatal e tempo foram diferentes nas duas estirpes diferentes. Em ratinhos CD1, lâminas palatinas do grupo controle continuou fusão no tempo ...

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

Estudos de desenvolvimento palato são motivadas pela incidência de fenda palatina, um defeito de nascença que impõe uma carga tremenda de saúde e pode deixar desfiguração duradoura. Nós demonstramos aqui uma técnica para prateleiras palatinas cultura que podem ser utilizados para estudar diferentes vias de sinalização envolvidos no desenvolvimento do palato e de fusão.

Método de Estudar Palatal Fusão usando estático Órgão Cultura

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

Research

JoVE Journal

Developmental Biology

8.7K visualizações.  Texas A&M University Baylor College of Dentistry.  Estudos de desenvolvimento palato são motivadas pela incidência de fenda palatina, um defeito de nascença que impõe uma carga tremenda de saúde e pode deixar desfiguração duradoura. Nós demonstramos aqui uma técnica para prateleiras palatinas cultura que podem ser utilizados para estudar diferentes vias de sinalização envolvidos no desenvolvimento do palato e de fusão. 

Vídeo: 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 Cultura Vivo de Pharyngeal Arcos para estudo do coração e do músculo Progenitores e seu 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 ...

Biologia do Desenvolvimento

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.

Análise de biofilme oral de Palatal Expansores por fluorescência de hibridização in situ e microscopia confocal a laser Scanning

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.

Isolamento e Cultura de Células Papila fungiforme Humanos Taste

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

Neurociência

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.

Imagem ao vivo do mouse Fusão do palato secundário

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 primária e Eletroporação Plasmid do Órgão 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 ...

Biologia

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.

Isolamento e imagem de lapso de tempo das células mesenquima palatinas embrionárias do camundongo primário para analisar atributos de movimento coletivo

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 da cultura de órgãos do tipo Trowell para estudar a regulação de células-tronco dentárias

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.

Colorindo, Visualização e Análise de Papilas de Fungos, Circunvalolados e 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.

Cultura e Imagem de Organoides Epiteliais Nasais 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.

Isolamento e Cultura de Queratinócitos Orais Primários do Paladar do Rato Adulto

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