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.

Tutte le procedure descritte devono essere eseguite in conformità con le linee guida e regolamenti per l&#39;uso di animali vertebrati, tra cui l&#39;approvazione preliminare da parte del Comitato Istituzionale cura degli animali e l&#39;uso locale. 1. Preparazione di dissezione Strumenti e cultura dei media <ol><li> Prelavaggio tutti gli strumenti da utilizzare in dissezione con 3% di perossido di idrogeno e quindi in autoclave a 121 ° C per 20 min. Filtro mezzi BGJb con soluzione antibiotica e antimicotica usando un filtro pori di dimensioni di 0,22 micron con aspirazione di vuoto in una cappa. </li></ol> 2. Preparazione di apparecchiature Cultura <ol><li> Tagliare filtri a membrana di policarbonato che sosterranno gli organi palatali in piccoli triangoli, spruzzare con il 70% di alcol e lasciare asciugare. Preparare Centro-Well Culture Organ piatto (60x15 mm) inserendo una griglia pulito e sterilizzato equilatero triangolare fili (20 mm) sopra la piastra di coltura organo. Quindi riempire il pozzo con 1 ml di BGJB mezzi di coltura. NOTA: il materiale dovrebbe essere sufficiente a raggiungere il livello della griglia senza immergere il palato. Posizionare i filtri a membrana, lato lucido verso il basso, sulla parte superiore della griglia (3-4 membrane per griglia) .Depending all&#39;esperimento, trattamento (proteine, anticorpi o inibitori) possono essere aggiunti ai media </li></ol> 3. mouse embrioni Raccolta e preparazione per la Cultura <ol><li> Per ottenere wild type embrioni di topo, uso cronometrato 13.5 incinta CD-1 topo background genetico sforzo. Pulire l&#39;area dissezione con il 70% di etanolo. NOTA: Altri ceppi di topi possono essere usati. Preferiamo topi CD-1 a causa del numero più grande lettiera. </li><li> Euthanize topi in gravidanza a E13.5 DPC (giorni dopo il coito) con il 5% isoflurano seguita da dislocazione cervicale per assicurare la morte utilizzando protocolli approvati. </li><li> Spray etanolo al 70% sulla superficie ventrale addominale per evitare che il bastone capelli addominale agli strumenti. Quindi aprire la cavità addominale ventraleutilizzando un spigolo smussato forbici operativi e pinze micro-dissezione per individuare l&#39;utero. Utilizzando le pinze, sollevare l&#39;intero utero e separarlo dal corpo tagliando al corpo uterino e alle punte delle corna uterine con una luce forbici operativi. </li><li> Posizionare l&#39;utero su un tovagliolo di carta pulito sul ghiaccio (Secondo la guida protocollo per la cura e l&#39;uso di animali da laboratorio). Coprire l&#39;utero con un altro tovagliolo di carta pulito per evitare contaminazioni. Sterilizzare gli strumenti utilizzando etanolo al 70% spray prima di utilizzare ulteriormente. L&#39;utero e gli embrioni possono essere trasportati al laboratorio in ghiaccio. </li><li> In una cappa a flusso laminare aperto, fare attenzione una piccola apertura nel sacco vitellino con le forbici affilate operativi-smussato e pinze micro-dissezione. Rendere l&#39;apertura più ampia e quindi spingere delicatamente per esteriorizzare l&#39;embrione dal sacco vitellino. </li><li> Trasferire immediatamente gli embrioni di una capsula di Petri con freddo tampone fosfato Saline 1X (PBS) pH 7,4. Per assicurare la corretta fase, exammina dell&#39;embrione sotto un microscopio da dissezione per il corpo, dimensione della testa e degli arti morfologia. NOTA: Gli embrioni che non sono in fase di sviluppo destra non vengono utilizzati per l&#39;esperimento. </li></ol><img alt="Figura 2" src="/files/ftp_upload/53063/53063fig2.jpg" /> Figura 2. Arto Bud Morfologia. La parte anteriore e arti posteriori sono controllati per il grado di cinghia rientro tra le cifre. A E13.5, tutte le condensazioni cifre sono importanti, con il nastro e rientranze distale tra le cifre che appaiono molto distinto. Le cifre posteriori sono alle spalle da una mezza giornata 12 Riga superiore:. Zampa anteriore, inferiore fila: arti posteriori. 4. Palate Dissection <ol><li> Collocare un embrione alla volta in una capsula di Petri con PBS al microscopio da dissezione. Utilizzo di forbicine e pinzette dissezione separare la testa dal corpo degli embrioni. </li><li> Tenendo attentamente il cranio del mouse sopra il livello degli occhi con una pinza (evitandoil processo mascellare) fare due incisioni su entrambi i lati della linea del labbro. Quindi rimuovere la mandibola e la lingua. Isolare la regione mascellare processo effettuando un taglio a livello degli occhi e rimuovere il cervello, gli occhi, setto nasale e del tessuto adiacente. </li></ol><img alt="Figura 3" src="/files/ftp_upload/53063/53063fig3.jpg" /> Figura 3. Palato tessuti raccolti da 13,5 embrioni, quando gli scaffali palato erano elevati, ma non in contatto. I ripiani palatali sono stati messi in coltura d&#39;organo su un filtro a livello di interfaccia multimediale ad aria per 72 ore nel modello statico. (A) Livello di dissezione su E13.5 embrione. (B) Blu stelle delimitare aree di tenere il tessuto con una pinza durante la dissezione. (C) Veduta di scaffali palatali sezionato posti su una membrana filtrante trapezoidale. (D) Vista laterale del piatto cultura dopo il posizionamento della palati sezionato e l&#39;aggiunta di coltura. <ol start="3"><li> Posizionare il lato orale palatina e rimuovere il setto nasale e il tessuto circostante, mantenendo il palato primario collegato per aiutarlo a orientarsi da anteriore a posteriore. Utilizzare una spatola cucchiaio e trasferire con molta attenzione gli scaffali palatali (lato nasale giù) per i filtri preparate sulle griglie nei piatti di coltura. NOTA: Regolare il volume dei mezzi di comunicazione per consentire una corretta interfaccia aria-media. </li><li> Al microscopio stereo, sezionare il palato primario e utilizzare una pinzetta per posizionare gli scaffali palatali vicini l&#39;uno all&#39;altro. Gli scaffali palatali non fondere se non vengono posti a contatto. Tagliare il filtro a membrana triangolare in cui si trova la parte anteriore del palato. La forma trapezoidale sarà di aiuto nel localizzare la porzione anteriore del palato. </li><li> Cultura scaffali singolarmente o con due o tre nello stesso piatto cultura organistica utilizzando BGJb coltura palatali. </li></ol> 5. Ripiani Coltura mouse palatali <ol><li> Incubare la tissues a 37 ° C in una miscela di gas umidificato (5% di CO 2 e il 95% di aria) per 72 ore, e cambiare il mezzo di ogni 24 ore. </li></ol> 6. Palate di elaborazione per l&#39;analisi istologica <ol><li> Fissare i palati dopo 72 ore di cultura con il 4% di formaldeide / tampone fosfato salino O / N a 4 ° &#160; C. Poi, delicatamente con una pipetta di trasferimento lavare il tessuto nel piatto cultura con PBS. Avvolgere i palati in un pezzo di salviette di laboratorio e luogo di incorporamento cassette. NOTA: Spostamento i palati di salviettine laboratorio prevenire la loro perdita durante il processo di disidratazione. </li><li> Disidratare il tessuto seguente 70%, 80% e fino al 100% lavaggi etanolo per 1 ora, seguito dalla procedura paraffina regolare. Per incorporare, posizionare la parte anteriore del palato rivolto verso il vassoio. </li><li> Tagliare seriali 6 sezioni micron con l&#39;orientamento coronale da anteriore a posteriore. Una sezione palato completa sarà resa 350-450 sections. Macchiare le sezioni con ematossilina eosina (H &amp; E). 10 Nota ogni 20 ° sezione utilizzando il già descritto medio Fusion Score (MFS) Scala (Tabella 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.

Il protocollo in questo articolo fornisce un metodo di dissezione scaffali palatali da embrioni al giorno embrionale 13.5. Ripiani palatali da embrioni di topo sono state coltivate in una coltura privo di siero in una atmosfera di 95% O 2 5% CO 2 a 37 ° C. Dissezione palatale di successo è criticamente dipendente da molteplici fattori ad ogni passo durante la procedura dal momento embrionali dei topi sacrificati al completamento della cultura. Uno dei fattori più importanti che influenzano palatale fusione...

Schisi oro-facciali sono i difetti di nascita cranio-facciali più predominanti. Inoltre, prendendo in considerazione tutti i possibili difetti cranio-facciali, questi sono i seconda anomalia nascita più comune nei neonati 2. Labbro leporino si verificano in circa 1 ogni 700 nascite negli Stati Uniti (US) ogni anno, l&#39;incidenza di palatoschisi è pari a 475 bambini nati con labbro leporino al mese o 15 bambini con crepacci al giorno 3. Circa l&#39;1% dei bambini nati in tutto il mondo ogni anno mostra una qualche forma di dismorfologia cranio-facciale. Clefts del palato e il labbro bisogno di una procedura molto costosa e complicata, con implicazioni permanente per i pazienti che hanno questa anomalia. Il costo stimato per ogni paziente con schisi orale è di circa 100.000 $ 4. Il trattamento di un paziente con labiopalatoschisi richiede un team di medici, tra cui cranio-facciali, chirurghi otorinolaringoiatri, genetisti, anestesisti,discorso in lingua patologi, nutrizionisti, ortodontisti, protesisti, psicologi, neurochirurghi, e oftalmologi. Nel palatogenesis, il palato secondario si pone come escrescenze accoppiati, che inizialmente si sviluppano verticalmente e subiscono elevazione mensola palatale sopra il dorso della lingua. A seguito di elevazione, gli scaffali palatali appaiati crescono verso la linea mediana (a E14.5 -E15 nei topi e negli esseri umani settimana 9). L&#39;epitelio bordo mediale (MEE), che copre la punta mensola aderisce formando la cucitura linea mediana epiteliale. Segue la transizione epitelio mesenchimale e / o apoptosi consentire mesenchimali confluenza. Adesione di opporsi MEE è un evento essenziale la cui alterazione provoca palatoschisi. Tuttavia, solo pochi studi hanno esaminato l&#39;adesione iniziale dei ripiani palatali 5. Le forme palato duro di differenziazione delle cellule mesenchimali in osteoblasti. Lo sviluppo anormale del palato in grado di produrre cleft palati con o senza coinvolgimento del labbro. Tecniche di coltura di organi palato erano stati utilizzati ampiamente per molti laboratori nel corso degli ultimi 30 anni 6,7. In questo protocollo si descrive in dettaglio un metodo di dissezione palatale e della cultura d&#39;organo statica. Il vantaggio di una cultura organo immobile è che permette scaffali palatali di fondersi. Questa tecnica è stata utilizzata con successo nel nostro laboratorio per molti di fusione e di segnalazione esperimenti 8,9. Tuttavia l&#39;applicazione della tecnica è vasto e può essere utilizzato ogni volta che è richiesto sistema di coltura organo statico, compresa la valutazione della risposta agli agenti chimici esogeni, gli effetti dei fattori di crescita regolamentazioni di vari percorsi e proteine ​​specifiche. <img alt="Figura 1" src="/files/ftp_upload/53063/53063fig1.jpg" /> Figura 1. Topo Palatogenesis. Stadi di sviluppo del palato topi. (BF) ele Scansionemicrografie CTRON (SEM) del palato secondario, a volte sviluppo rappresentative. Le frecce rosse indicano: la parte iniziale di palatale adesione e fusione. Frecce gialle: punto per lo spazio tra i palati primarie e secondarie che spariranno dopo la fusione (ristampato da Kaufman 11 con il permesso di 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

Labio-palatoschisi sono tra i più comuni di tutti i difetti di nascita. Le forme secondarie palato dagli scaffali mesenchimali coperti di epitelio che aderisce a formare la linea di giunzione linea mediana epiteliale (MES). Le teorie suggeriscono che le cellule MES seguono una epiteliale di transizione mesenchimale (EMT), l&#39;apoptosi e la migrazione, per un palato fuso 1. Completa disintegrazione delle MES è la fase finale di essenziale palatale confluenza con circostante cellule mesenchimali. Forniamo un metodo per la cultura organo palato. Il protocollo sviluppato in vitro permette lo studio dei processi biologici e molecolari durante la fusione. Le applicazioni di questa tecnica sono numerosi, incluse le risposte di valutazione agli agenti chimici esogeni, effetti di fattori di regolazione e di crescita e proteine ​​specifiche. Cultura organo palatale ha una serie di vantaggi tra cui manipolazione a diversi stadi di sviluppo che non è possibile utilizzare in studi in vivo.

La tecnica può essere applicata a diversi tipi di experiments.The seguente studio è stato progettato per testare l&#39;effetto teratogeno della nicotina in vitro su palatale fusione. Due ceppi di topi sono stati utilizzati per questo esperimento, CD1 e tessuti C57.Palatal stata trattata con concentrazioni di nicotina emisolfato 0,06 e 6 mm. È stato osservato che i modelli di fusione palatali e tempi erano differenti nei due ceppi diversi. Nei topi CD1, mensole palatale del gruppo di controllo hanno continuat...

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

Gli studi di sviluppo palato sono motivate dalla incidenza di palatoschisi, un difetto di nascita che impone un onere sanitario enorme e può lasciare deturpazione duratura. Si dimostra qui una tecnica per scaffali cultura palatali che possono essere utilizzati per studiare diverse vie di segnalazione coinvolti nello sviluppo del palato e fusione.

Metodo di studio palatale Fusion utilizzando Statico Cultura Organo

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 Views.  Texas A&M University Baylor College of Dentistry.  Gli studi di sviluppo palato sono motivate dalla incidenza di palatoschisi, un difetto di nascita che impone un onere sanitario enorme e può lasciare deturpazione duratura. Si dimostra qui una tecnica per scaffali cultura palatali che possono essere utilizzati per studiare diverse vie di segnalazione coinvolti nello sviluppo del palato e fusione. 

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 Cultura Vivo del faringea Archi allo Studio cuore e muscoli Progenitori e la loro nicchia

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

Ricerca

Biologia dello sviluppo

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.

Analisi biofilm orale di espansori palatali da Fluorescence In Situ-ibridazione e microscopia confocale a scansione laser

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 la coltura di cellule umane Gusto papille fungiformi

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

Neuroscienze

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.

Imaging Live di Mouse Seconda 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 ...

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 e elettroporazione plasmidi dell&#39;Organo murini di 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 ...

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 imaging time-lapse di cellule mesenchimiche palatali embrionali di topo primarie per analizzare gli attributi del movimento collettivo

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 della coltura di organi di tipo Trowell per studiare la regolazione delle cellule staminali dentali

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.

Colorazione, visualizzazione e analisi di papille gustative fungiformi, circumvallate e palato

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.

Coltura e imaging di organoidi epiteliali nasali umani

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 coltura di cheratinociti orali primari dal palato del topo adulto

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