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

Nous remercions le Dr A. Jacobo, Dr J. Salvi et A. Petelski pour leurs contributions à la recherche originale sur laquelle se fonde le présent protocole. Nous remercions également J. lamas et W. Makmura pour l’assistance technique et de l’élevage. Nous reconnaissons subvention du NIDCD formation T32 DC009975, NIDCD accorder R01DC015530, Robertson thérapeutique Development Fund et la Fondation de la famille Caruso pour financement. Enfin, nous reconnaissons le soutien du Howard Hughes Medical Institute, dont le Dr Hudspeth est un enquêteur.

Toutes les méthodes décrites ici ont été approuvés par les comités de l’Université Rockefeller d’utilisation et animalier et de l’University of Southern California.
1. (facultative) préparation du collagène I Solution de Tendons de Mouse-tail
Remarque : Collagène I solutions sont disponibles dans le commerce. Suivez les instructions du fabricant pour la préparation du gel.
<ol><li>Euthanasier des souris jeunes adultes (âgés de ...

<ol>
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(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. 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Les signaux moléculaires qui médiat de la croissance et la différenciation dans l’oreille interne au cours du développement ont été étudiés intensivement5,6,7,8,9,10. Toutefois, les données obtenues dans le système utriculaire modèle suggèrent que signaux mécaniques, sentis à travers les jonctions cellulaires ...

L’étude de la plupart des aspects du développement des organes de mammifères a été facilitée par les systèmes in vitro . Deux méthodes principales sont maintenant utilisées pour la culture des organes sensoriels vestibulaires : flottant1 et adhérentes2 préparations. Les deux méthodes permettent l’étude des cellules ciliées vulnérabilités3 et régénération1,4 in vitro. En outre, le rôle du développement de l’encoche5,6, Wnt7,8et facteur de croissance épidermique receptor (EGFR)9,10 , signalisation des cascades dans l’oreille interne ont été établi, en partie, à l’aide de in vitro de cultures de l’épithélium sensoriel. Cependant, la différenciation et la croissance des cellules sont contrôlés, non seulement par le biais de signalisation par morphogènes, mais aussi par le biais de signaux physiques et mécaniques tels que des contacts intercellulaires, la rigidité de la matrice extracellulaire et mécaniques d’étirement ou constriction. Le rôle de ces stimuli mécaniques est difficile à étudier dans les pays en développement oreille interne in vivo. En outre, les méthodes actuelles de culture flottante et adhérentes ne conviennent pas pour ces études in vitro. Nous décrivons ici une méthode pour la culture organotypique tridimensionnelle en collagène I gelées de raideur variable. Cette méthode est en grande partie conserve l’architecture in vivo des organes sensoriels vestibulaires et cochléaires et permet d’étudier les effets de la force mécanique sur la croissance et la différenciation11.
Parce que les stimuli mécaniques sont connus pour activer les événements moléculaires en aval, tels que l’hippopotame signalisation voie12,13,14,15, il est important d’être capable de combiner la stimulation mécanique avec les manipulations génétiques et biochimiques. La méthode de culture décrite ici permet la livraison de gène induite par le virus et peut donc être utilisée pour étudier la signalisation mécanique et moléculaire au cours de l’oreille interne développement11.

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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>
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			<td height="21" style="height:21px;width:252px;">#5 Forceps</td>
			<td style="width:212px;">Dumont</td>
			<td style="width:119px;">11252-20</td>
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		<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>
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			<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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			<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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			<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>
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		<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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			<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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			<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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			<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>
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		<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>
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			<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>
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			<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>
		</tr>
		<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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			<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>
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			<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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			<td height="21" style="height:21px;">HBSS</td>
			<td style="width:212px;">Gibco</td>
			<td style="width:119px;">14025092</td>
			<td></td>
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			<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>
			<td></td>
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			<td height="21" style="height:21px;width:252px;">Insulin</td>
			<td style="width:212px;">Sigma</td>
			<td style="width:119px;">I3536</td>
			<td></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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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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			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Sodium bicarbonate</td>
			<td style="width:212px;">Sigma</td>
			<td style="width:119px;">S5761</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Sodium hydroxide</td>
			<td style="width:212px;">Sigma</td>
			<td style="width:119px;">S8045</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium selenite</td>
			<td style="width:212px;">Sigma</td>
			<td style="width:119px;">S5261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Tabletop vortex&#160;</td>
			<td style="width:212px;">VWR</td>
			<td style="width:119px;">97043-562</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Transferrin</td>
			<td style="width:212px;">Sigma</td>
			<td style="width:119px;">T8158</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan blue&#160;</td>
			<td style="width:212px;">Sigma</td>
			<td style="width:119px;">T6146</td>
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	</tbody>
</table>

three-dimensional organotypic cultures of vestibular and auditory sensory organs

Les organes sensoriels de l’oreille interne sont difficiles à étudier chez les mammifères en raison de leur inaccessibilité aux manipulations expérimentales et d’observation optique. En outre, bien que des techniques de culture existants permettent des perturbations biochimiques, ces méthodes ne fournissent pas un moyen d’étudier les effets de la force mécanique et la rigidité des tissus pendant le développement de l’oreille interne des organes sensoriels. Nous décrivons ici une méthode pour la culture organotypique tridimensionnelle de l’utricule murine intact et cochlée qui permet de surmonter ces limitations. La technique pour l’ajustement d’une rigidité de la matrice tridimensionnelle décrite ici permet la manipulation de la force élastique s’opposant à la croissance des tissus. Cette méthode peut donc être utilisée pour étudier le rôle des forces mécaniques au cours du développement de l’oreille interne. En outre, les cultures permettent la livraison de gène induite par le virus, qui peut être utilisée pour des expériences de gain - et perte de fonction. Cette méthode de culture préserve innée des cellules ciliées et des cellules et sert comme une alternative potentiellement supérieure à la culture traditionnelle à deux dimensions des organes sensoriels vestibulaires et auditives.

Organes sensoriels vestibulaires et auditives d’oreilles embryonnaires, cultivées en collagène 40-Pa j’ai gels imitant faible rigidité des conditions embryonnaires11, conserver les structures tridimensionnelles relativement normales (Figure 1) et l’entretien des cellules ciliées et soutenir des cellules (Figure 2 et Figure 3). Bien que soutenant la densité cellulair...

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

Cultures organotypique tridimensionnelle de l’utricule murin et la cochlée en défricher collagène j’ai gélifie morphologie tissulaire innée preserve optiquement, permettant une stimulation mécanique au moyen d’ajustement de la rigidité de la matrice et permettent la livraison de gène induite par le virus.

Culture organotypique en trois dimensions des organes sensoriels vestibulaires et auditives

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

Trancher les cultures organotypiques d&#39;études de neurogenèse postnatale

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

Recherche

Biologie du développement

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

Différenciation d&#39;un Neural Stem Cell humaine ligne sur Trois Cultures dimensionnelles, les gènes Analyse des microARN et Putative cibles

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

Quantification tridimensionnelle d&#39;épines dendritiques de Pyramidal neurones humains induits Dérivé de cellules souches pluripotentes

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

Utilisation<em&gt; Ex Vivo</em&gt; Cultures Upright gouttelettes de toute fœtus organes pour étudier les processus de développement au cours de la souris Organogenèse

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

Génération des cultures organotypiques de peau génétiquement modifiées en utilisant Dévitalisées derme humain

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

Transfert de gènes dans l&#39;organe de poulet Auditory par<em&gt; Dans Ovo</em&gt; Micro-électroporation

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

Protocoles pour Visualizing stéroïdogénique organes et leurs organes interactifs avec Immunostaining dans le Fruit Fly<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

L&#39;analyse des rétinoïque induite par l&#39;acide Neuro-Différenciation de cellules souches embryonnaires de souris en deux et trois dimensions corps embryoïdes

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

Horizontal Whole Mount: un protocole de traitement et d&#39;imagerie géniaux pour les sections transversales épaisses en tissu tridimensionnel

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

Une nouvelle utilisation d’échographie en trois dimensions de haute fréquence pour la caractérisation de grossesse précoce chez la souris

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 ...