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 tackar Dr A. Jacobo, Dr. J. Salvi och A. Petelski för deras bidrag till den ursprungliga forskningen som detta protokoll baseras. Vi tackar också J. lamor och W. Makmura för tekniskt bistånd och djurhållning. Vi erkänner NIDCD utbildning grant T32 DC009975, NIDCD bevilja R01DC015530, Robertson terapeutiska utvecklingsfonden och stiftelsen Caruso familj för finansiering. Slutligen, vi erkänner stöd från Howard Hughes Medical Institute, som Dr Hudspeth är en utredare.

Alla metoderna som beskrivs här har godkänts av djur vård och användning kommittéer av Rockefeller University och vid University of Southern California.
1. (valfritt) beredning av kollagen jag lösning från Mouse-tail senor
Obs: Kollagen jag lösningar finns kommersiellt. Följ tillverkarens instruktioner för gel beredning.
<ol><li>Avliva 5-10 ung vuxen (3-5 veckor gamla) möss av någon vildtyp stam med koldioxid i enlighet med det protoko...

<ol>
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De molekylära signaler som medla tillväxt och differentiering i innerörat under utveckling har studerats ingående5,6,7,8,9,10. Bevisning som erhållits från utricular modell systemet tyder dock på att mekaniska ledtrådar, kände igenom cellen korsningar och aktivering av Hippo signalering, också spela en viktig roll i...

Studien av de flesta aspekter av däggdjur orgel utveckling har underlättats av in vitro- system. Två huvudsakliga metoder används nu för kulturen i vestibulära sensoriska organ: fritt flytande1 och vidhäftande2 preparat. Båda metoderna tillåta utredning av hår cell sårbarheter3 och förnyelse1,4 in vitro. De utvecklingsmässiga rollerna av Notch5,6, Wnt7,8, och epidermal tillväxtfaktor receptor (EGFR)9,10 signalering cascades i innerörat har dessutom fastställts, delvis med hjälp av in vitro- kulturer av sensorisk epitel. Dock är celltillväxt och differentiering kontrollerade, inte bara genom signalering av morphogens, men också genom fysiska och mekaniska signaler såsom intercellulära kontakter, styvheten hos extracellulärmatrix, och mekanisk stretching eller sammandragning. Rollen av sådana mekaniska stimuli är utmanande att undersöka i utvecklingsländer innerörat i vivo. Dessutom passar befintliga friflytande och vidhäftande kultur metoder inte sådana studier in vitro. Här beskriver vi en metod för tredimensionella organotypic kultur i kollagen geler jag varierande styvhet. Denna metod till stor del bevarar i vivo arkitekturen av de vestibulära och cochlear sinnesorgan och tillåter undersökning av effekterna av mekanisk kraft på tillväxt och differentiering11.
Eftersom mekaniska stimuli är känd för att aktivera nedströms molekylära händelser, såsom flodhästen signalering utbildningsavsnitt12,13,14,15, är det viktigt att kunna kombinera mekanisk stimulering med biokemiska och genetiska manipulationer. Metoden kultur beskrivs här tillåter virus-medierad gen leverans och kan därför användas för att studera både mekanisk och molekylär signalering under innerörat utveckling11.

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			<td height="21" style="height:21px;width:252px;">#10 Surgical Blades</td>
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			<td height="21" style="height:21px;width:252px;">100 mm Petri dish</td>
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			<td height="21" style="height:21px;">4-Hydroxytamoxifen&#160;</td>
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			<td height="21" style="height:21px;">60&#160;mm Petri dish</td>
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			<td height="21" style="height:21px;width:252px;">Ad-GFP</td>
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			<td height="21" style="height:21px;width:252px;">Anti-GFP, chicken IgY fraction</td>
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			<td height="21" style="height:21px;width:252px;">Anti-Myo7A</td>
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			<td height="21" style="height:21px;">Bicinchoninic acid assay</td>
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			<td height="42" style="height:42px;width:252px;">Click-iT EdU Alexa Fluor 647 Imaging Kit</td>
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			<td style="width:119px;">C10340</td>
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			<td height="21" style="height:21px;">Collagenase I</td>
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			<td height="21" style="height:21px;">D-glucose</td>
			<td style="width:212px;">Sigma</td>
			<td style="width:119px;">G8270</td>
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			<td height="21" style="height:21px;">Epidermal growth factor</td>
			<td style="width:212px;">Sigma</td>
			<td style="width:119px;">E9644</td>
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			<td height="21" style="height:21px;width:252px;">Fetal Bovine Serum (FBS)</td>
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			<td style="width:119px;">16140063</td>
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			<td height="21" style="height:21px;">Fibroblast growth factor</td>
			<td style="width:212px;">Sigma</td>
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			<td height="21" style="height:21px;width:252px;">Hemocytometer&#160;</td>
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			<td height="21" style="height:21px;width:252px;">PBS (10X), pH 7.4</td>
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			<td height="21" style="height:21px;width:252px;">Phenol Red pH indicator&#160;</td>
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three-dimensional organotypic cultures of vestibular and auditory sensory organs

De sensoriska organen i innerörat är utmanande för att studera hos däggdjur deras otillgänglighet till experimentella manipulation och optisk observation. Dessutom även om befintliga kultur tekniker tillåter biokemiska störningar, ger dessa metoder inte ett sätt att studera effekterna av mekanisk kraft och vävnad stelhet under utvecklingen av innerörat sensoriska organ. Här beskriver vi en metod för tredimensionella organotypic kultur intakt murina utricle och snäckan som övervinner dessa begränsningar. Tekniken för justering av en tredimensionell matris stelhet beskrivs här tillåter manipulering av elastisk kraft motsatta vävnadstillväxt. Denna metod kan därför användas för att studera rollen av mekaniska krafter under innerörat utveckling. Dessutom, ger kulturerna virus-medierad gen leverans, som kan användas för vinst - och förlust-av-funktion experiment. Denna kultur metod bevarar medfödda hårcellerna och stöder celler och fungerar som ett potentiellt överlägset alternativ till den traditionella tvådimensionella kulturen av vestibulära och auditiv sinnesorgan.

Vestibulära och auditiv sinnesorgan från embryonala öron, odlade i 40-Pa kollagen geler härma låg styvhet embryonala villkor11, behålla relativt normala tredimensionella strukturer (figur 1) och underhålla hårcellerna och stödja celler (figur 2 och figur 3). Även om stödja cell densiteten minskar med över 30% (Students t -test: n = 4, p < 0,004) o...

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

Tredimensionella organotypic kulturer av murina utricle och snäckan i optiskt klart kollagen jag geler bevara medfödda vävnad morfologi, möjliggöra mekanisk stimulering genom justering av matrix stelhet och tillåta virus-medierad gen leverans.

Tredimensionella Organotypic kulturer av vestibulära och auditiv sinnesorgan

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

Organotypic Slice kulturer för Studier av Födsel 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

Differentiering av en mänsklig Neural Stem Cell Line den Tredimensionell kulturer, Analys av MicroRNA och Förmodade målgenerna

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

Tredimensionell Kvantifiering av Dendritutskotten från Pyramidal nervceller Härstammar från Human inducerade pluripotenta 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

Använda<em&gt; Ex vivo</em&gt; Upright Dropp Kulturer av hela organ hos fostren att studera utvecklingsprocesser under Mouse Organogenesis

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

Generation av genetiskt modifierade Organotypiska Skin kulturer Använda devitaliserad Human 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

Genöverföring till kyckling hörselorganet genom<em&gt; I Ovo</em&gt; Micro-elektroporering

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

Protokoll för Visualisering steroidogena organ och deras interaktiva organ med immunfärgning i fruktflugan<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

Analys av retinsyra-inducerade Neural differentiering av Mouse embryonala stamceller i två och tre-dimensionella Embryoid organ

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

Horisontell helmontering: Ett nytt bearbetnings- och bildprotokoll för tjocka, tredimensionella vävnads tvärsnitt 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

En ny användning av tredimensionella högfrekvent ultraljud för tidig graviditet karakterisering i 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 ...