Name: three-dimensional organotypic cultures of vestibular and auditory sensory organs
Uploaded: 2018-06-01T14:00:00
Description: Watch this Scientific Journal Video about Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs at JoVE.com

Vi takker Dr. A. Jacobo, Dr. J. Salvi og A. Petelski for deres bidrag til den opprinnelige forskningen som er basert på denne protokollen. Vi takker også J. lamaer og W. Makmura for kundestøtte og husdyrhold. Vi erkjenner NIDCD trening grant T32 DC009975, NIDCD gi R01DC015530 Robertson terapeutiske Development Fund og Caruso familien grunnlaget for finansiering. Til slutt, vi erkjenner støtte fra Howard Hughes Medical Institute, som Dr. Hudspeth er en etterforsker.

Alle metodene som er beskrevet her er godkjent av Animal Care og bruk komiteer av Rockefeller University og ved University of Southern California.
1. (valgfritt) utarbeidelse av kollagen jeg løsningen fra Mouse-tail sener
Merk: Kollagen jeg løsninger er tilgjengelige kommersielt. Følg produsentens instruksjoner for gel forberedelse.
<ol><li>Euthanize 5-10 unge voksne (3-5 uker gamle) mus i vill type belastning med karbondioksid i samsvar med pr...

<ol>
	<li>Oesterle, E. C., Tsue, T. T., Reh, T. A., Rubel, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hair-cell+regeneration+in+organ+cultures+of+the+postnatal+chicken+inner+ear.">Hair-cell regeneration in organ cultures of the postnatal chicken inner ear.</a> Hear Res. 70 (1), 85-108 (1993).</li><li>Meyers, J. R., Corwin, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shape+change+controls+supporting+cell+proliferation+in+lesioned+mammalian+balance+epithelium.">Shape change controls supporting cell proliferation in lesioned mammalian balance epithelium.</a> J Neurosci Off J Soc Neurosci. 27 (16), 4313-4325 (2007).</li><li>Cunningham, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adult+mouse+utricle+as+an+in+vitro+preparation+for+studies+of+ototoxic-drug-induced+sensory+hair+cell+death.">The adult mouse utricle as an in vitro preparation for studies of ototoxic-drug-induced sensory hair cell death.</a> Brain Res. 1091 (1), 277-281 (2006).</li><li>Warchol, M. E., Lambert, P. R., Goldstein, B. J., Forge, A., Corwin, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+proliferation+in+inner+ear+sensory+epithelia+from+adult+guinea+pigs+and+humans.">Regenerative proliferation in inner ear sensory epithelia from adult guinea pigs and humans.</a> Science. 259 (5101), 1619-1622 (1993).</li><li>Lin, V., Golub, J. S., Nguyen, T. B., Hume, C. R., Oesterle, E. C., Stone, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+Notch+activity+promotes+nonmitotic+regeneration+of+hair+cells+in+the+adult+mouse+utricles.">Inhibition of Notch activity promotes nonmitotic regeneration of hair cells in the adult mouse utricles.</a> J Neurosci Off J Soc Neurosci. 31 (43), 15329-15339 (2011).</li><li>Wu, J., 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=Co-regulation+of+the+Notch+and+Wnt+signaling+pathways+promotes+supporting+cell+proliferation+and+hair+cell+regeneration+in+mouse+utricles.">Co-regulation of the Notch and Wnt signaling pathways promotes supporting cell proliferation and hair cell regeneration in mouse utricles.</a> Sci Rep. 6, 29418 (2016).</li><li>Chai, R., 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=Wnt+signaling+induces+proliferation+of+sensory+precursors+in+the+postnatal+mouse+cochlea.">Wnt signaling induces proliferation of sensory precursors in the postnatal mouse cochlea.</a> Proc Natl Acad Sci U S A. 109 (21), 8167-8172 (2012).</li><li>Wang, T., 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=Lgr5++cells+regenerate+hair+cells+via+proliferation+and+direct+transdifferentiation+in+damaged+neonatal+mouse+utricle.">Lgr5+ cells regenerate hair cells via proliferation and direct transdifferentiation in damaged neonatal mouse utricle.</a> Nat Commun. 6, 6613 (2015).</li><li>Doetzlhofer, A., White, P. M., Johnson, J. E., Segil, N., Groves, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+growth+and+differentiation+of+mammalian+sensory+hair+cell+progenitors:+a+requirement+for+EGF+and+periotic+mesenchyme.">In vitro growth and differentiation of mammalian sensory hair cell progenitors: a requirement for EGF and periotic mesenchyme.</a> Dev Biol. 272 (2), 432-447 (2004).</li><li>White, P. M., Stone, J. S., Groves, A. K., Segil, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EGFR+signaling+is+required+for+regenerative+proliferation+in+the+cochlea:+conservation+in+birds+and+mammals.">EGFR signaling is required for regenerative proliferation in the cochlea: conservation in birds and mammals.</a> Dev Biol. 363 (1), 191-200 (2012).</li><li>Gnedeva, K., Jacobo, A., Salvi, J. D., Petelski, A. A., Hudspeth, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastic+force+restricts+growth+of+the+murine+utricle.">Elastic force restricts growth of the murine utricle.</a> eLife. 6, (2017).</li><li>Aragona, 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=A+mechanical+checkpoint+controls+multicellular+growth+through+YAP/TAZ+regulation+by+actin-processing+factors.">A mechanical checkpoint controls multicellular growth through YAP/TAZ regulation by actin-processing factors.</a> Cell. 154 (5), 1047-1059 (2013).</li><li>Dong, J., 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=Elucidation+of+a+universal+size-control+mechanism+in+Drosophila+and+mammals.">Elucidation of a universal size-control mechanism in Drosophila and mammals.</a> Cell. 130 (6), 1120-1133 (2007).</li><li>Low, B. C., Pan, C. Q., Shivashankar, G. V., Bershadsky, A., Sudol, M., Sheetz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=YAP/TAZ+as+mechanosensors+and+mechanotransducers+in+regulating+organ+size+and+tumor+growth.">YAP/TAZ as mechanosensors and mechanotransducers in regulating organ size and tumor growth.</a> FEBS Lett. 588 (16), 2663-2670 (2014).</li><li>Zhao, B., 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=Inactivation+of+YAP+oncoprotein+by+the+Hippo+pathway+is+involved+in+cell+contact+inhibition+and+tissue+growth+control.">Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control.</a> Genes Dev. 21 (21), 2747-2761 (2007).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> AVMA Guidelines for the Euthanasia of Animals: 2013 Edition. , (2013).</li><li>Semerci, F., 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=Lunatic+fringe-mediated+Notch+signaling+regulates+adult+hippocampal+neural+stem+cell+maintenance.">Lunatic fringe-mediated Notch signaling regulates adult hippocampal neural stem cell maintenance.</a> eLife. 6, (2017).</li><li>Tuan, R. S., Lo, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+biology+protocols.">Developmental biology protocols.</a> Methods in molecular biology. , 137 (2000).</li><li>Brandon, C. S., Voelkel-Johnson, C., May, L. A., Cunningham, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+adult+mouse+utricle+and+adenovirus-mediated+supporting-cell+infection.">Dissection of adult mouse utricle and adenovirus-mediated supporting-cell infection.</a> J Vis Exp JoVE. (61), (2012).</li><li>Gosset, M., Berenbaum, F., Thirion, S., Jacques, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+and+phenotyping+of+murine+chondrocytes.">Primary culture and phenotyping of murine chondrocytes.</a> Nat Protoc. 3 (8), 1253-1260 (2008).</li><li>Landegger, L. D., 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=A+synthetic+AAV+vector+enables+safe+and+efficient+gene+transfer+to+the+mammalian+inner+ear.">A synthetic AAV vector enables safe and efficient gene transfer to the mammalian inner ear.</a> Nat Biotechnol. 35 (3), 280-284 (2017).</li><li>Burns, J. C., 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=Reinforcement+of+cell+junctions+correlates+with+the+absence+of+hair+cell+regeneration+in+mammals+and+its+occurrence+in+birds.">Reinforcement of cell junctions correlates with the absence of hair cell regeneration in mammals and its occurrence in birds.</a> J Comp Neurol. 511 (3), 396-414 (2008).</li><li>Wang, J., 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=Regulation+of+polarized+extension+and+planar+cell+polarity+in+the+cochlea+by+the+vertebrate+PCP+pathway.">Regulation of polarized extension and planar cell polarity in the cochlea by the vertebrate PCP pathway.</a> Nat Genet. 37 (9), 980-985 (2005).</li><li>Chacon-Heszele, M. F., Ren, D., Reynolds, A. B., Chi, F., Chen, P. <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+cochlear+convergent+extension+by+the+vertebrate+planar+cell+polarity+pathway+is+dependent+on+p120-catenin.">Regulation of cochlear convergent extension by the vertebrate planar cell polarity pathway is dependent on p120-catenin.</a> Dev Camb Engl. 139 (5), 968-978 (2012).</li><li>Yamamoto, N., Okano, T., Ma, X., Adelstein, R. S., Kelley, 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=Myosin+II+regulates+extension,+growth+and+patterning+in+the+mammalian+cochlear+duct.">Myosin II regulates extension, growth and patterning in the mammalian cochlear duct.</a> Dev Camb Engl. 136 (12), 1977-1986 (2009).</li><li>Tada, M., Heisenberg, C. -. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convergent+extension:+using+collective+cell+migration+and+cell+intercalation+to+shape+embryos.">Convergent extension: using collective cell migration and cell intercalation to shape embryos.</a> Dev Camb Engl. 139 (21), 3897-3904 (2012).</li></ol>

Molekylær signaler som megle vekst og differensiering i det indre øret under utvikling har vært studert5,,6,,7,,8,,9,,10. Bevis fra utricular modell systemet antyder imidlertid at mekanisk signaler, følt gjennom cellen veikryss og aktivering av Hippo signalnettverk, også spille en viktig rolle i disse prosessene<sup clas...

Studiet av de fleste aspekter av pattedyr orgel utvikling har blitt lettere ved in vitro -systemer. To viktigste metoder blir brukt til kultur vestibular Sanseorganer: frittflytende1 og tilhenger2 forberedelser. Begge metodene tillater etterforskningen av hår celle sikkerhetsproblemer3 og regeneration1,4 i vitro. I tillegg har utviklingsmessige rollene hakk5,6, Wnt7,8og epidermal vekstfaktor reseptor (EGFR)9,10 signalering kaskader i det indre øret opprettet en delvis gjennom bruk av i vitro kulturer av sensoriske epithelia. Imidlertid cellevekst og differensiering kontrolleres, ikke bare gjennom signalering av morphogens, men også gjennom fysisk og mekanisk signaler som intercellulære kontakter, stivhet av ekstracellulær matrix, og mekanisk strekkes eller innsnevring. Rollen av slike mekaniske stimuli er utfordrende å undersøke i utvikle indre øret i vivo. Videre er eksisterende frittflytende og tilhenger kultur metoder ikke egnet for slike studier i vitro. Her vi beskriver en metode for tredimensjonale organotypic kultur i kollagen jeg gels av varierende stivhet. Denne metoden hovedsakelig bevarer arkitektur i vivo vestibular og cochlear sensoriske organer og tillater undersøkelse av effekten av mekanisk kraft på vekst og differensiering11.
Fordi mekanisk stimuli er kjent for å aktivere nedstrøms molekylære hendelsene Hippo signalering veien12,13,14,15, er det viktig å kunne kombinere mekanisk stimulering med biokjemiske og genetiske manipulasjoner. Kultur metoden beskrevet her tillater virus-mediert gen levering og kan derfor brukes til å studere både mekanisk og molekylære signalering under indre øret utvikling11.

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			<td height="21" style="height:21px;width:252px;">#10 Surgical Blades</td>
			<td style="width:212px;">Miltex</td>
			<td style="width:119px;">4-110</td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:252px;">#5 Forceps</td>
			<td style="width:212px;">Dumont</td>
			<td style="width:119px;">11252-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">100 mm Petri dish</td>
			<td style="width:212px;">Sigma</td>
			<td style="width:119px;">P5856-500EA</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:252px;">250 uL large orifice pipette tips</td>
			<td style="width:212px;">USA Scientific</td>
			<td style="width:119px;">1011-8406</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:252px;">30 mm glass-bottom Petri dish</td>
			<td style="width:212px;">Matsunami Glass USA Corporation</td>
			<td style="width:119px;">D35-14-1.5-U</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:252px;">4 well plate</td>
			<td style="width:212px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">176740</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-Hydroxytamoxifen&#160;</td>
			<td style="width:212px;">Sigma</td>
			<td style="width:119px;">H7904</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60&#160;mm Petri dish</td>
			<td style="width:212px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">123TS1</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Acetic acid&#160;</td>
			<td style="width:212px;">Sigma</td>
			<td style="width:119px;">537020</td>
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			<td height="21" style="height:21px;width:252px;">Ad-GFP</td>
			<td style="width:212px;">Vector Biolabs</td>
			<td style="width:119px;">1060</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Anti-GFP, chicken IgY fraction</td>
			<td style="width:212px;">Invitrogen</td>
			<td style="width:119px;">A10262&#160;</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Anti-Myo7A</td>
			<td style="width:212px;">Proteus Biosciences</td>
			<td style="width:119px;">25-6790</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Anti-Sox2 Antibody (Y-17)</td>
			<td style="width:212px;">Santa Cruz</td>
			<td style="width:119px;">sc-17320</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bicinchoninic acid assay</td>
			<td style="width:212px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">23225</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:252px;">Click-iT EdU Alexa Fluor 647 Imaging Kit</td>
			<td style="width:212px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">C10340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase I</td>
			<td style="width:212px;">Gibco</td>
			<td style="width:119px;">17100017</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-glucose</td>
			<td style="width:212px;">Sigma</td>
			<td style="width:119px;">G8270</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F12&#160;</td>
			<td style="width:212px;">Gibco</td>
			<td style="width:119px;">11320033</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Epidermal growth factor</td>
			<td style="width:212px;">Sigma</td>
			<td style="width:119px;">E9644</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:212px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">16140063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fibroblast growth factor</td>
			<td style="width:212px;">Sigma</td>
			<td style="width:119px;">F5392</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Flaming/Brown Micropipette Puller</td>
			<td style="width:212px;">Sutter Instrument</td>
			<td style="width:119px;">P-97</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Glutamine</td>
			<td style="width:212px;">Sigma</td>
			<td style="width:119px;">G8540</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">HBSS</td>
			<td style="width:212px;">Gibco</td>
			<td style="width:119px;">14025092</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Hemocytometer&#160;</td>
			<td style="width:212px;">Daigger</td>
			<td style="width:119px;">EF16034F</td>
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			<td height="21" style="height:21px;width:252px;">HEPES</td>
			<td style="width:212px;">Sigma</td>
			<td style="width:119px;">H4034</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Insulin</td>
			<td style="width:212px;">Sigma</td>
			<td style="width:119px;">I3536</td>
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			<td height="21" style="height:21px;">Iridectomy scissors&#160;</td>
			<td style="width:212px;">Zepf Medical Instruments</td>
			<td style="width:119px;">08-1201-10&#160;&#160;</td>
			<td></td>
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			<td height="21" style="height:21px;width:252px;">Microinjector</td>
			<td style="width:212px;">Narishige</td>
			<td style="width:119px;">IM-6</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Nicotinamide</td>
			<td style="width:212px;">Sigma</td>
			<td style="width:119px;">N0636</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:252px;">PBS (10X), pH 7.4</td>
			<td style="width:212px;">Gibco</td>
			<td style="width:119px;">70011044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS (1X), pH 7.4</td>
			<td style="width:212px;">Gibco</td>
			<td style="width:119px;">10010023</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Phenol Red pH indicator&#160;</td>
			<td style="width:212px;">Sigma</td>
			<td style="width:119px;">P4633&#160;</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Pure Ethanol, 200 Proof</td>
			<td style="width:212px;">Decon Labs&#160;</td>
			<td style="width:119px;">2716</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:252px;">RFP antibody</td>
			<td style="width:212px;">ChromoTek</td>
			<td style="width:119px;">&#160;5F8</td>
			<td></td>
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			<td height="21" style="height:21px;width:252px;">Sodium bicarbonate</td>
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			<td height="21" style="height:21px;">Sodium selenite</td>
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			<td height="21" style="height:21px;width:252px;">Transferrin</td>
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</table>

three-dimensional organotypic cultures of vestibular and auditory sensory organs

Sensoriske organer i det indre øret er utfordrende for å studere i pattedyr på grunn av utilgjengelighet deres eksperimentelle manipulasjon og optisk observasjon. Videre, selv om eksisterende kultur teknikker tillater biokjemiske forstyrrelser, gir ikke disse metodene å studere virkningene av mekanisk kraft og vev stivhet under utviklingen av indre øret sensoriske organer. Her beskriver vi en metode for tredimensjonale organotypic kultur intakt murine utricle og sneglehuset som overvinner disse begrensningene. Teknikken for justering av en tredimensjonal matrix stivhet beskrevet her tillater manipulering av elastisk force motsatte vevvekst. Denne metoden kan derfor brukes til å studere rollen til mekaniske krefter under utviklingen av indre øret. I tillegg tillater kulturer virus-mediert gen levering, som kan brukes for gevinst og tap-av-funksjon eksperimenter. Denne kultur metoden bevarer medfødte hårcellene og støtte celler og fungerer som et potensielt bedre alternativ til tradisjonelle todimensjonal kultur vestibular og auditiv Sanseorganer.

Vestibular og auditiv Sanseorganer fra embryonale ører, kultivert i 40-Pa kollagen jeg gels mimicking lav stivhet embryonale forhold11, beholde relativt normalt tredimensjonale strukturer (figur 1) og vedlikeholde hårcellene og støtter celler (figur 2 og Figur 3). Selv om støtte celle tettheten reduseres med over 30% (Student t -test: n = 4, p < 0.004) og...

Watch this Scientific Journal Video about Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs at JoVE.com

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

Tredimensjonale organotypic kulturer murine utricle og sneglehuset i optisk fjerner kollagen jeg gels bevare medfødte vev morfologi, tillater mekanisk stimulering gjennom justering av matrix stivhet, og tillater virus-mediert gen levering.

Tredimensjonale Organotypic kulturer av Vestibular og auditiv Sanseorganer

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. ...

Research

JoVE Journal

Developmental Biology

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Organotypiske Slice kulturer for Studier av Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes

Differensiering av Human Neural Stem Cell Line on tredimensjonal kulturer, Analyse av mikroRNA og Antatte målgener

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local ...

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Tredimensjonal Kvantifisering av dendrittutløperne fra Pyramideformet Nevroner Avledet fra menneskeskapte Pluripotent stamceller

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Bruke<em&gt; Ex Vivo</em&gt; Upright Droplet Kulturer av Whole føtale organer å studere utviklingsprosesser i løpet Mouse organogenesen

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop ...

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Generering av genmodifiserte organotypiske Skin kulturer Bruke devitalized Menneskelig Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Gene Transfer inn i Chicken hørbar Organ av<em&gt; I Ovo</em&gt; Micro-electroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

Protokoller for Visualisering Steroidogenic organer og deres Interaktiv organer med Farging i bananflue<em&gt; Drosophila melanogaster</em

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response ...

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Analyse av retinsyre-indusert Neural Differensiering av mus embryonale stamceller i to og tre-dimensjonale embryoidlegemer

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Horisontal helmontering: En romanbehandlings- og bildeprotokoll for tykke, tredimensjonale vev Tverrsnitt av huden

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

Romanen bruk av tredimensjonal høyfrekvente Ultrasonography for tidlig Graviditet karakterisering musen

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...