Sarah A. Alfaqeeh è finanziato dal tipo Saud University College of Dentistry, Ministero dell&#39;Istruzione Superiore, Regno dell&#39;Arabia Saudita.

1. Avviare <ol><li> Affinare gli strumenti di dissezione (utilizzando una pietra per affilare lubrificato con olio minerale) e sterilizzare prima di utilizzare per la cultura d&#39;organo con un spruzzo di etanolo al 70% e la sterilizzazione a calore secco. Contrasta gli aghi di dissezione mediante elettrolisi a 2 M di idrossido di sodio con una alimentazione a 12 V. </li><li> Preparare il terreno di coltura utilizzati per la dissezione e la cultura di organi embrionali, costituito Modified Eagle Medium F12 Advanced Dulbecco (DMEM F12) supplementato con 1% GlutaMAX e 1% di penicillina-streptomicina. </li></ol> 2. Embrione dissezione <ol><li> Abbattere una donna incinta a tempo accoppiato con un metodo programma uno come approvato dal Ministero degli Interni o di altro organo di governo. Sezionare via la pelle intorno al basso ventre, e tagliare aprire l&#39;addome con le forbici. Individuare i due corni uterini, che collegano insieme alla linea mediana inferiore. </li><li> Sezionare l&#39;utero e posto in un tubo riempito con il mezzo sul ghiaccio. When su ghiaccio, il tessuto può essere lasciato per diverse ore. </li><li> Rimuovere l&#39;utero e posto in una capsula di Petri in mezzo al microscopio. Ove possibile dissezioni embrionali dovrebbero essere eseguiti in una cappa a flusso laminare per minimizzare la contaminazione. </li><li> Sezionare gli embrioni dall&#39;utero e liberarli dai loro tessuti extraembrionali. </li><li> Decapitare gli embrioni usando un ago di tungsteno e trasferire le teste in un fresco piatto di mezzo. </li><li> Isolare le mandibole dalle teste inserendo un ago nella cavità orale e tagliare verso la parte posteriore della testa (Figure 1A e 1B). Isolato mandibole possono essere memorizzati temporaneamente in mezzo su ghiaccio durante l&#39;attesa per tritare. </li><li> Per intere fette mandibola stadi E11.5-E15.5 sono ideali per la cultura dente germe. Dopo E15.5 l&#39;osso nella mandibola di sviluppo è troppo difficile per tritare. Fette dente può ancora essere apportate ma l&#39;osso (dentario e formare l&#39;osso alveolare) devono prima essere rimossila dissezione prima tagliare il 9. Rimuovere accuratamente osseo con aghi di tungsteno e pinze. </li></ol> 3. Embrione affettare <ol><li> Preparare le fette di tessuto mandibola con un chopper tessuto tabella standard (Figura 1C). Utilizzare una lama fresca dopo circa 200 mandibole, e assicurarsi che si trova a filo con il disco di montaggio quando cade. </li><li> Pulire il disco di montaggio e lama con il 70% di etanolo prima dell&#39;uso. Bisogna fare attenzione quando si utilizza la macchina, in quanto la lama è molto tagliente. </li><li> Impostare il chopper ad una distanza di taglio tra 200-400 micron a seconda dell&#39;età del campione, ea seconda se è necessaria l&#39;intero germe dente. Impostare la pressione della lama al massimo. </li><li> Trasferire una mandibola al disco di montaggio in plastica con una pipetta o pinze. Disporre la mandibola sulla realizzazione del disco che l&#39;orientamento è ancora chiaro. La linguetta di sviluppo può essere utilizzato come punto di riferimento utile per determinare l&#39;orientamento. Per molari uso afRontal / orientamento trasversale (vedi piano di taglio in Figura 1B), mentre per incisivi utilizzare una fetta sagittale attraverso la mandibola per riflettere il diverso orientamento di questi denti crescono (vedi piano di taglio nella Figura 2A). </li><li> Rimuovere l&#39;eccesso di terreno da tutto il tessuto su carta da filtro in modo da asciugare leggermente la mandibola sul disco di montaggio. </li><li> Subito dopo l&#39;essiccazione, posizionare il disco sul tavolo in acciaio inox circolare del chopper. Accendere la macchina e premere start. La tabella attraversa automaticamente da sinistra a destra, mentre allo stesso tempo il braccio taglio portando la lama è sollevato e lasciato cadere fino a 200 colpi / min. </li><li> Spegnere la macchina una volta che il tessuto è stato completamente tranciato. Assicurarsi che il braccio lama è in posizione sollevata e rimuovere il disco. Non mettere le dita sotto il braccio lama o lasciare la macchina incustodita quando tagliare. </li><li> Prendete il disco di montaggio e aggiungere immediatamente medie to le fette sul disco al fine di evitare un eccessivo essiccamento. Rimuovere accuratamente il tessuto tranciato dal fondo del disco utilizzando un ago di tungsteno e poi pipetta in una piccola capsula di Petri di media (Figura 2A). </li><li> Separare le fette usando un ago e quindi selezionare le fette di tessuto contenenti il tessuto di interesse allo stereomicroscopio (Figure 1D, 1E, e 2B). Mettere le fette selezionati in piatti di coltura fresco con il mezzo. </li></ol> 4. Fetta Cultura <ol><li> Preparare organo coltura piatti centrali pozzetti pronti per cultura aggiungendo circa 2 ml di acqua autoclavato per pozzetto esterno per prevenire la disidratazione, e posizionando una griglia metallica attraverso il centro del piatto (Figure 3A e 3B). </li><li> Preparare le griglie metalliche tagliando strisce di circa 4 cm x 1.5 cm da fogli di maglia di acciaio inossidabile. Utilizzare un pugno buco per fare un buco al centro e piegarei lati per creare un fine sfalsata. La griglia dovrebbe adattarsi perfettamente sulle pozzetto centrale, situata parallelamente al fondo del piatto (Figure 3A e 3B). Autoclave le griglie per la sterilizzazione prima dell&#39;uso. </li><li> Tagliare un pezzo di polietilene tereftalato trasparente (PET) membrana (dimensione dei pori 0,4 micron) sufficiente a coprire il foro nella griglia. Posizionare la membrana tagliata nel piatto con la fetta selezionata. </li><li> Posizionare la fetta in cima alla membrana e poi sollevare con cura la membrana fuori del mezzo, con fetta appiattita nel centro. </li><li> Posizionare il filtro sulla griglia sopra il foro circolare al centro, e aggiungere circa 1 ml di terreno di coltura al pozzo, fino al livello della membrana. </li><li> Per manipolazioni aggiungere proteine ​​o piccole molecole inibitrici al mezzo (ad esempio 8). </li><li> Fotografare la fetta per un record di morfologia al giorno 0 della cultura. </li><li> Porre le capsule in atmosfera umidificata al 5% CO <sub&gt; 2 a 37 ° C in un incubatore. </li><li> Fotografare le culture a intervalli regolari per in media 7 giorni. Modificare il terreno di coltura ogni 48-72 ore. Per photography visualizzare le fette con una sorgente di luce da sotto (Figure 2C-E). </li></ol> 5. Lineage Tracing Se è necessario lignaggio tracing, coloranti lipofili come DII o DiO possono essere iniettati nel germe del dente prima del punto 4, della cultura fetta. <ol><li> Tirare aghi capillari di vetro con un estrattore ago. </li><li> Rompere la punta dell&#39;ago con pinze e inserire la punta verso il basso nella soluzione colorante. Lasciare agire per qualche minuto, mentre il colorante riempie la punta per azione capillare. </li><li> Posizionare l&#39;ago riempito in un supporto e collegare tramite tubi ad un iniettore o alla bocca di aspirazione. </li><li> Posizionare la punta dell&#39;ago nel mezzo di coltura di toccare la superficie della fetta a causa di etichettatura. Applicare una pressione dell&#39;aria di spostare una piccola quantità di colorante fuori un agoD sul tessuto. </li><li> Rimuovere l&#39;ago e controllare l&#39;etichettatura in fluorescenza. </li><li> Fette etichettati correttamente possono essere trasferiti ai filtri e coltivate come descritto nel passaggio 4. </li></ol>

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+culture+of+mature+organs+in+a+synthetic+medium.">The culture of mature organs in a synthetic medium.</a> Experimental cell research. 16, 118-147 (1959).</li><li>Matalova, E., Antonarakis, G. S., Sharpe, P. T., Tucker, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lineage+of+primary+and+secondary+enamel+knots.">Cell lineage of primary and secondary enamel knots.</a> Developmental dynamics : an official publication of the American Association of Anatomists. 233, 754-759 (2005).</li><li>Mitsiadis, T. A., Tucker, A. S., De Bari, C., Cobourne, M. T., Rice, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+regulatory+relationship+between+Tbx1+and+FGF+signaling+during+tooth+morphogenesis+and+ameloblast+lineage+determination.">A regulatory relationship between Tbx1 and FGF signaling during tooth morphogenesis and ameloblast lineage determination.</a> Developmental biology. 320, 39-48 (2008).</li><li>Diep, L., Matalova, E., Mitsiadis, T. A., Tucker, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+the+tooth+bud+mesenchyme+to+alveolar+bone.">Contribution of the tooth bud mesenchyme to alveolar bone.</a> Journal of experimental zoology. Part B, Molecular. 312B, 510-517 (2009).</li><li>Rothova, M., Feng, J., Sharpe, P. T., Peterkova, R., Tucker, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+mesoderm+to+the+developing+dental+papilla.">Contribution of mesoderm to the developing dental papilla.</a> The International journal of developmental biology. 55, 59-64 (2011).</li><li>Buchtova, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+and+patterning+of+the+snake+dentition+are+dependent+on+Sonic+hedgehog+signaling.">Initiation and patterning of the snake dentition are dependent on Sonic hedgehog signaling.</a> Developmental biology. 319, 132-145 (2008).</li><li>Buchtova, M., Stembirek, J., Glocova, K., Matalova, E., Tucker, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+regression+of+the+dental+lamina+underlies+the+development+of+diphyodont+dentitions.">Early regression of the dental lamina underlies the development of diphyodont dentitions.</a> Journal of dental research. 91, 491-498 (2012).</li><li>Cho, S. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+primary+enamel+knot+determines+the+position+of+the+first+buccal+cusp+in+developing+mice+molars.">The primary enamel knot determines the position of the first buccal cusp in developing mice molars.</a> Differentiation; research in biological diversity. 75, 441-451 (2007).</li><li>Sakano, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+dynamics+in+cervical+loop+epithelium+during+transition+from+crown+to+root:+implications+for+Hertwig's+epithelial+root+sheath+formation.">Cell dynamics in cervical loop epithelium during transition from crown to root: implications for Hertwig's epithelial root sheath formation.</a> Journal of periodontal research. , (2012).</li><li>Fisher, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+development+in+vitro+of+the+whole+and+halved+lower+molar+tooth+germ+of+the+mouse.">Morphological development in vitro of the whole and halved lower molar tooth germ of the mouse.</a> Archives of oral biology. 16, 1481-1496 (1971).</li><li>Coin, R., Schmitt, R., Lesot, H., Vonesch, J. L., Ruch, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+halved+embryonic+lower+first+mouse+molars:+correlation+with+the+distribution+pattern+of+non+dividing+IDE+cells,+the+putative+organizers+of+morphogenetic+units,+the+cusps.">Regeneration of halved embryonic lower first mouse molars: correlation with the distribution pattern of non dividing IDE cells, the putative organizers of morphogenetic units, the cusps.</a> The International journal of developmental biology. 44, 289-295 (2000).</li><li>Kavanagh, K. D., Evans, A. R., Jernvall, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+evolutionary+patterns+of+mammalian+teeth+from+development.">Predicting evolutionary patterns of mammalian teeth from development.</a> Nature. 449, 427-432 (2007).</li><li>Munne, P. M., Tummers, M., Jarvinen, E., Thesleff, I., Jernvall, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tinkering+with+the+inductive+mesenchyme:+Sostdc1+uncovers+the+role+of+dental+mesenchyme+in+limiting+tooth+induction.">Tinkering with the inductive mesenchyme: Sostdc1 uncovers the role of dental mesenchyme in limiting tooth induction.</a> Development. 136, 393-402 (2009).</li></ol>

Gli autori dichiarano di non avere interessi finanziari in competizione.

Questo metodo di coltura dente ha il vantaggio che l&#39;accesso al germe dente è eccellente, consentendo lignaggio accurato tracciamento e posizionamento di perline all&#39;interno dell&#39;epitelio o mesenchima. Regioni definite del dente di sviluppo germe possono quindi essere specificamente mirati. Durante la coltura morfologia mutevole del germe dente può essere seguito, e l&#39;effetto di manipolazioni valutato rapidamente. Il metodo, tuttavia, è adatto solo per giovani germi dente ...

Per una serie di esperimenti è importante essere in grado di coltura di tessuti ex vivo per seguire lo sviluppo. Cultura di sviluppo del tessuto fornisce l&#39;accesso a determinati periodi di sviluppo e permette la manipolazione dei geni con l&#39;aggiunta di fattori al terreno di coltura, o microsfere caricate, e con l&#39;uso di trasfezione e elettroporazione 1. Per molti esperimenti è importante essere in grado di visualizzare il tessuto come cresce, per esempio, per seguire il destino di cellule della linea etichettato come il tessuto subisce morfogenesi. Questo può essere particolarmente problematico per i tessuti che si sviluppano in profondità all&#39;interno dell&#39;embrione, che non sono evidenti quando un blocco di tessuto dall&#39;embrione è coltivato. I denti sono buoni esempi di questo, come si sviluppano all&#39;interno del processo nasale della mandibola, mascellare e frontale. Quando tutta la mandibola viene coltivato le strutture superficiali del dente possono essere visualizzati ma cambiamenti nella morfologia possono essere analizzati solo dopo il sezionamento di tessuto fissato <sup&gt; 2. Abbiamo adattato una fetta tecnica di coltura in diretta che ci permette di seguire dente sviluppo germe e l&#39;accesso alle varie parti del dente durante lo sviluppo. Le culture tecnica le fette dente germinali all&#39;interfaccia gas-liquido, usando un metodo Trowell modificato 3. Queste culture fetta sono state molto utili nelle seguenti direttamente morfogenesi del dente, e permettendo lineage tracing di componenti distinti, quali il nodo smalto e la papilla dentale e follicolo 1,4-7. La tecnica non è limitata agli embrioni di topo ed è stato usato con successo per coltura fette diretta di maiale e serpente 8,9 tessuto dentale. Oltre al vantaggio di essere in grado di visualizzare lo sviluppo dei denti, il metodo slice ha anche il vantaggio che le fettine di tessuto sono aumentati accesso ai nutrienti dal mezzo e aria dall&#39;incubatore. Ciò si traduce in un miglioramento della crescita dei germi dei denti, che corrispondono sviluppo in vivo e spettacoli invasione delle cellule endoteliali nella papilla 7. Al contrario, germi da denti in intere culture mandibola sviluppano più lenti di quelli in vivo e il centro della cultura è spesso necrotico in colture a lungo termine. Nella cultura fetta, il dente si sviluppa all&#39;interno di una fetta della mandibola, e la sua interazione con l&#39;osso circostante e sviluppare altri tessuti può essere monitorato. Nel nostro metodo il tessuto viene tagliato subito dopo dissezione senza necessità di incorporare in un mezzo di supporto 10,11 e non necessita di alcun sistema per attaccare il tessuto per il taglio di blocco. Il metodo è quindi non invasivo e rapido, consentendo molti mandibole da sezionare in una sessione.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>VWR</td>
			<td>101077Y</td>
			<td>100% ethanol was diluted in distilled H2O to 70%.</td>
		</tr>
		<tr>
			<td>DMEM F12</td>
			<td>Gibco</td>
			<td>12634-010</td>
			<td>Advanced Dulbecco&#39;s Modified Eagle Medium F12</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Sigma</td>
			<td>P0781</td>
			<td></td>
		</tr>
		<tr>
			<td>DiI (Molecular probes)</td>
			<td>Vybrant</td>
			<td>V-22885</td>
			<td>Cell-labeling solution</td>
		</tr>
		<tr>
			<td></td>
			<td>Invitrogen</td>
			<td></td>
			<td>Cell tracker CM-DiI, C-7000</td>
		</tr>
		<tr>
			<td>DiO (Molecular probes)</td>
			<td>Vybrant</td>
			<td>V22886</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Geminator 500</td>
			<td>Thomas</td>
			<td>Thomas No. 3885A20</td>
			<td>Dry-heat sterilization</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>McIlwain tissue chopper</td>
			<td>Ted Pella, Inc.</td>
			<td>10180</td>
			<td>Standard table</td>
		</tr>
		<tr>
			<td>Organ culture dish (Center-Well Organ Culture Dish)</td>
			<td>Falcon</td>
			<td>353037</td>
			<td></td>
		</tr>
		<tr>
			<td>Membranes (Cell Culture Inserts, 0.4 &#956;m pore size)</td>
			<td>BD Falcon</td>
			<td>353090</td>
			<td>PET track-etched membrane, 6-well format</td>
		</tr>
		<tr>
			<td>Metal grids (Stainless Steel - AISI 304 - Mesh)</td>
			<td>Goodfellow</td>
			<td>FE228710</td>
			<td>(Fe/Cr18/Ni10)</td>
		</tr>
		<tr>
			<td>AutoFlow Direct Heat CO2 Incubator</td>
			<td>Nuaire</td>
			<td>NU-5500</td>
			<td></td>
		</tr>
		<tr>
			<td>Picospritzer III</td>
			<td>Intracel Ltd</td>
			<td>051-0500-900 0-100 psi</td>
			<td>Single channel picospritzer III</td>
		</tr>
		<tr>
			<td>Glass capillary with filament 1 mm</td>
			<td>WPI</td>
			<td>TW100F-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Tungsten wire 0.1 mm</td>
			<td>Goodfellow</td>
			<td>W005138</td>
			<td></td>
		</tr>
		<tr>
			<td>Tungsten wire 0.38 mm</td>
			<td>Goodfellow</td>
			<td>W005155</td>
			<td></td>
		</tr>
		<tr>
			<td>Aspirator tubes</td>
			<td>Sigma</td>
			<td>A5177</td>
			<td>Used for mouth aspiration lineage tracers</td>
		</tr>
	</tbody>
</table>

the slice culture method for following development of tooth germs in explant culture

Cultura espianto permette la manipolazione di sviluppare organi in punti temporali specifici ed è quindi un metodo importante per il biologo dello sviluppo. Per molti organi è difficile accedere tessuto sviluppo per consentire il controllo nelle ex vivo cultura. Il metodo di coltura fetta permette l&#39;accesso al tessuto in modo che i movimenti morfogenetici possono essere seguiti e specifiche popolazioni cellulari possono essere oggetto di manipolazione o lineage tracing. In questo articolo descriviamo un metodo di cultura fetta che ha avuto molto successo per la cultura di germi denti in una vasta gamma di specie. Il metodo fornisce un eccellente accesso ai germi dei denti, che si sviluppano a un ritmo simile a quello osservato in vivo, circondato da altri tessuti della mascella. Questo permette interazioni tessuto tra il dente e del tessuto circostante da monitorare. Sebbene questo documento si concentra sulla germi dei denti, lo stesso protocollo può essere applicato a seguire lo sviluppo di un numerodi altri organi, come ad esempio le ghiandole salivari, cartilagine di Meckel, ghiandole nasali, lingua e orecchie.

Al fine di seguire il movimento del primo follicolo dentale molare, 250 micron fette frontali sono stati prelevati attraverso una mandibola con il metodo sopra descritto. Il follicolo dentale è lo strato di mesenchima che circonda lo smalto epitelio esterno (OEE) del dente di sviluppo, ed è stato già dimostrato di partecipare alla formazione di tessuto del periodonto 6. Mandibole sono stati sezionati a E14.5, nella fase tappo dello sviluppo dei denti. Nella fetta il contorno dell&#39;epitelio dentale era c...

Watch this Scientific Journal Video about The Slice Culture Method for Following Development of Tooth Germs In Explant Culture at JoVE.com

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

Qui ci dettaglio un metodo per cultura germi denti in fette mandibola con un chopper tessuto. Questo metodo consente l&#39;accesso esclusivo al dente durante lo sviluppo, fornendo un&#39;ottima opportunità per la manipolazione e lignaggio tracing, non disponibile utilizzando metodi di coltura più tradizionali.

Il Metodo Culture Slice per seguire lo sviluppo dei germi dente Espianto Cultura

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

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Research

JoVE Journal

Biology

14.0K Views.  King's College London.  Qui ci dettaglio un metodo per cultura germi denti in fette mandibola con un chopper tessuto. Questo metodo consente l'accesso esclusivo al dente durante lo sviluppo, fornendo un'ottima opportunità per la manipolazione e lignaggio tracing, non disponibile utilizzando metodi di coltura più tradizionali. 

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

Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed protocol for biolistic transfection of rat hippocampal slices, from the initial preparation of DNA coated bullets to the final shooting of the organotypic slice cultures using a gene gun. Gene gun transfection is an efficient and easy means of transfecting neurons and is especially useful for fluorescently labeling a small subset of cells in tissue slice. In this video, we first outline the steps required to coat gold particles with DNA. We next demonstrate how to line the inside of plastic tubing with the gold/DNA bullets, and how to cut this tubing to obtain the plastic cartridges for loading into the gene gun. Finally, we perform biolistic transfection of rat hippocampal slice cultures, demonstrating handling of the Bio-Rad Helios gene gun, and offering trouble shooting advice to obtain healthy and optimally transfected tissue slices.  

We describe a method for preparing DNA coated gold bullets and demonstrate the use of such bullets to biolistically transfect neurons in cultured hippocampal slices.

Preparazione di proiettili pistola Gene e Biolistic Transfection dei neuroni in cultura Slice

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed ...

Ricerca

Biologia

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

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

Method for Culture of Early Chick Embryos ex vivo New Culture

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

Metodo per la Cultura di Chick primi embrioni ex vivo (Nuova Cultura)

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

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

Un neuronale e astrociti Co-Cultura Assay per l&#39;analisi dei contenuti elevati di neurotossicità

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

Cultura e manutenzione di cellule staminali embrionali umane

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

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 ...

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

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper homeostasis directly depends on these interactions, as they shape an entire immunological response inducing tolerance against food antigens while at the same time mounting effective immune responses against pathogenic microbes accidentally ingested with food.
Intestinal homeostasis is preserved also through various complex interactions between the microbiota (including food-associated beneficial bacterial strains) and the host, that regulate the attachment/degradation of mucus, the production of antimicrobial peptides by the epithelial barrier, and the "education" of epithelial cells' that controls the tolerogenic or immunogenic phenotype of unique, gut-resident lymphoid cells' populations. These interactions have been so far very difficult to reproduce with in vitro assays using either cultured cell lines or peripheral blood mononuclear cells. In addition, mouse models differ substantially in components of the intestinal mucosa (mucus layer organization, commensal bacteria community) with respect to the human gut. Thus, studies of a variety of treatments to be brought in the clinics for important stress-related or pathological conditions such as irritable bowel syndrome, inflammatory bowel disease or colorectal cancer have been difficult to carry out.
To address these issues, we developed a novel system that enables us to stimulate explants of human intestinal mucosa that retain their in situ conditioning by the host microbiota and immune response, in a polarized fashion. Polarized apical stimulation is of great importance for the outcome of the elicited immune response. It has been repeatedly shown that the same stimuli can produce completely different responses when they bypass the apical face of the intestinal epithelium, stimulating epithelial cells basolaterally or coming into direct contact with lamina propria components, switching the phenotype from tolerogenic to immunogenic and causing unnecessary and excessive inflammation in the area.
We achieved polarized stimulation by gluing a cave cylinder which delimited the area of stimulation on the apical face of the mucosa as will be described in the protocol. We used this model to examine, among others, differential effects of three different Lactobacilli strains. We show that this model system is very powerful to assess the immunomodulatory properties of probiotics in healthy and disease conditions.

We introduce a novel method for the maintenance of human intestinal mucosa in culture and monitoring of the response to various types of stimuli over at least 24 hrs. With our method, the polarity of the tissue is maintained, allowing for a physiological stimulation via the apical route.

Un nuovo metodo per la Cultura e polarizzata stimolazione umani espianti mucosa intestinale

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

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

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

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

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

A Novel<em&gt; Ex vivo</em&gt; Metodo di Cultura per il mouse embrionale del cuore

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

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

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

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

Embrionali di topo di sviluppo in un embrione di tutta la cultura di sistema privo di siero

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

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of neurulation, migrates extensively towards various organs, and differentiates into many types of derivatives (neurons, glia, cartilage and bone, pigmented and endocrine cells). In this protocol, we describe how to dissect the premigratory cranial NC from Xenopus laevis embryos, in order to study NC development in vivo and in vitro. The frog model offers many advantages to study early development; abundant batches are available, embryos develop rapidly, in vivo gain and loss of function strategies allow manipulation of gene expression prior to NC dissection in donor and/or host embryos. The NC explants can be plated on fibronectin and used for in vitro studies. They can be cultured for several days in a serum-free defined medium. We also describe how to graft NC explants back into host embryos for studying NC migration and differentiation in vivo.

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

This protocol describes how to dissect premigratory cranial neural crest (NC) from Xenopus laevis neurulas. These explants can be plated on fibronectin and cultured in vitro, or grafted back into host embryos. This technique allows studying the mechanisms of NC epithelium-to-mesenchyme transition, migration, and differentiation.

Dissezione dei<em&gt; Xenopus laevis</em&gt; Cresta neurale per<em&gt; In vitro</em&gt; Espianto Cultura o<em&gt; In vivo</em&gt; Trapianto

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of ...

Establishment of an Extracellular Acidic pH Culture System

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported.
Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

Istituzione di un pH extracellulare acido sistema cultura

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...