Name: the slice culture method for following development of tooth germs in explant culture
Uploaded: 2013-11-13T20:30:00
Description: Watch this Scientific Journal Video about The Slice Culture Method for Following Development of Tooth Germs In Explant Culture at JoVE.com

Sarah A. Alfaqeeh er finansieret af Kind Saud University College of Dentistry, Ministeriet for Videregående Uddannelser, Kongeriget Saudi-Arabien.

1.. Set Up <ol><li> Slib Dissektionsinstrumenter (ved hjælp af en slibesten smurt med mineralsk olie) og sterilisere før brug for orgel kultur ved hjælp af en 70% ethanol spray og tør-varme sterilisation. Skærp dissektion nåle ved hjælp af elektrolyse i 2 M natriumhydroxid ved hjælp af en 12 V forsyning. </li><li> Forbered dyrkningsmediet anvendes til dissektion og dyrkning af embryonale organer bestående af Advanced Dulbeccos Modified Eagle Medium F12 (DMEM F12) suppleret med 1% Glutamax og 1% penicillin-str...

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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. 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Denne metode til tand kultur har den fordel, at få adgang til tanden kim er fremragende, så nøjagtig afstamning sporing og placering af perler i epitel eller mesenchyme. Definerede regioner i at udvikle tand kim kan derfor målrettet. Under dyrkning kan følges skiftende morfologi af tanden kim, og virkningen af ​​manipulationer hurtigt vurderes. Metoden er dog kun egnet til unge tand bakterier før væsentlig dannelse af hårde væv, såsom dentin og emalje, da disse ikke kan hakkes ...

For en række forsøg er det vigtigt at være i stand til kultur væv ex vivo til at følge udviklingen. Kultur udvikle væv giver adgang ved definerede perioder af udvikling og tillader manipulation af gener ved tilsætning af faktorer til dyrkningsmediet, eller indlæst perler, og ved anvendelse af transfektion og elektroporation 1. For mange forsøg er det vigtigt at være i stand til at visualisere det væv, som det vokser, for eksempel for at følge skæbnen for afstamning mærkede celler som vævet undergår morfogenese. Dette kan være særligt problematisk for væv, der udvikler dybt i embryonet, som ikke er indlysende, når en blok af væv fra embryonet dyrkes. Tænder er gode eksempler på dette, som de udvikler i underkæben, overkæben og frontal nasal proces. Når hele underkæben dyrkes de overfladiske strukturer i tanden kan ses, men ændringer i morfologi kan kun analyseret efter sektionering af fast væv <sop&gt; 2.. Vi har tilpasset en live skive kultur teknik giver os mulighed for at følge kim udvikling tand og give adgang til de forskellige dele af tanden under udviklingen. Teknikken kulturer tand kim skiver på gas-væske-grænseflade, ved hjælp af en modificeret Trowell metode 3. Disse skivekulturer har været meget nyttige i direkte efter morfogenese af tanden, og tillader slægt sporing af forskellige komponenter, såsom emalje knude og dental papilla og follikelstimulerende 1,4-7. Teknikken er ikke begrænset til musefostre og har med succes været anvendt til dyrkning af levende udsnit af svin og slange dentalt væv 8,9. Ud over den fordel af at være i stand til at visualisere tandudvikling, udsnittet metode har også den fordel, at de tynde skiver af væv har øget adgang til næringsstoffer fra mediet og luft fra inkubatoren. Dette resulterer i en forbedret vækst af tand bakterier, som matcher udvikling in vivo og viser invasion af endotelceller i papilla 7. I modsætning hertil tand bakterier i hele underkæbe kulturer udvikle langsommere end in vivo og midten af kulturen er ofte nekrotisk i langvarige kulturer. I skive kultur, tanden udvikler sig inden for en skive af kæben, og kan overvåges dens samspil med det omgivende udvikle knogler og andre væv. I vores metode vævet hakket lige efter dissektion med noget behov for at integrere i en støtte medium 10,11 og intet behov for et system til at fastgøre væv til huggeblokken. Metoden er derfor noninvasive og hurtig, så mange kæber, der skal skæres i en session.

<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

Eksplantation kultur muliggør manipulation at udvikle organer på bestemte tidspunkter, og er derfor en vigtig metode til udviklingsmæssige biolog. For mange organer er det vanskeligt at få adgang til at udvikle væv for at muliggøre overvågning under ex vivo-kultur. Den skive dyrkningsmetode giver adgang til væv, så morfogenetiske bevægelser kan følges og specifikke cellepopulationer kan målrettes til manipulation eller afstamning sporing. I denne artikel beskriver vi en metode til skive kultur, der har været meget vellykket for kultur af tand bakterier i en række arter. Metoden giver fremragende adgang til tand bakterier, der udvikler sig ved en lignende sats, der blev observeret in vivo, omgivet af de andre kæbe væv. Dette giver vævsinteraktioner mellem tanden og det omgivende væv, der skal overvåges. Selv om dette dokument koncentrerer sig om tand bakterier, kan den samme protokol skal anvendes til at følge udviklingen af ​​en rækkeandre organer, såsom spytkirtler, Meckel brusk, nasale kirtler, tunge og øre.

For at følge bevægelsen af ​​den første kindtand tand follikel var 250 um frontal udsnit taget gennem en underkæbe ved hjælp af den ovenfor beskrevne metode. Den dentale follikel er laget af mesenkym der omgiver den ydre emalje epitel (OEE) i udviklingslandene tand, og er tidligere blevet vist at deltage i dannelsen af væv periodontium 6. Kindbakkerne blev dissekeret på E14.5, hætten fase af tand udvikling. I den skive var klart omridset af dental epitel og kondenserende dental mesenchyme kunne id...

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 (Video) | JoVE

Her har vi detalje en metode til kultur tand bakterier i underkæbe skiver ved hjælp af en vævshakker. Denne metode giver enestående adgang til tanden under udviklingen, hvilket giver god mulighed for manipulation og afstamning opsporing, ikke er til rådighed ved hjælp af mere traditionelle dyrkningsmetoder.

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

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