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 oprindelige forskning som denne protokol er baseret. Vi takker også J. lamaer og W. Makmura for teknisk bistand og husdyrhold. Vi anerkender NIDCD uddannelse grant T32 DC009975, NIDCD give R01DC015530, Robertson terapeutiske Udviklingsfond og Caruso Family Foundation for finansiering. Endelig, vi anerkender støtte fra Howard Hughes Medical Institute, som Dr. Hudspeth er en Investigator.

Alle metoder beskrevet her er blevet godkendt af Animal Care og brug udvalg af Rockefeller University og af University of Southern California.
1. (valgfrit) forberedelse af kollagen jeg løsning fra Mouse-tail sener
Bemærk: Kollagen jeg løsninger findes i handelen. Følg producentens anvisninger for gel forberedelse.
<ol><li>Aflive 5-10 unge voksne (3-5 uger gamle) mus af enhver vildtype stamme med kuldioxid i overensstemmelse med den protokol, ...

<ol>
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De molekylære signaler, der mægle vækst og differentiering i det indre øre under udvikling er blevet studeret grundigt5,6,7,8,9,10. Dog tyder fremstillet af utricular modelsystem på, at mekanisk stikord, fornemmede gennem celle vejkryds og aktivering af Hippo signalering, også spille en vigtig rolle i disse processer<su...

Studiet af de fleste aspekter af pattedyr orgel udvikling er blevet fremmet af in vitro- systemer. To vigtigste metoder bruges nu til kulturen af vestibulære sanseorganer: frit svævende1 og vedhængende2 præparater. Begge metoder tillade undersøgelsen af hår celle sårbarheder3 og regenerering1,4 in vitro. Notch5,6, Wnt7,8og epidermal vækstfaktor receptor (EGFR)9,10 signalering cascades i det indre øre udviklingsmæssige roller har derudover blevet etableret, dels ved hjælp af in vitro- kulturer af sensoriske epitheler. Dog er cellevækst og differentiering kontrolleret, ikke kun gennem signalering af morphogens, men også gennem fysiske og mekaniske stikord såsom intercellulære kontakter, stivhed af ekstracellulære matrix, og mekanisk strække eller konstriktion. Rollen som sådan mekaniske stimuli er udfordrende at undersøge i den tredje indre øre in vivo. Derudover er eksisterende frit svævende og vedhængende kultur metoder ikke egnet til sådanne undersøgelser in vitro. Her vi beskrive en metode for tre-dimensionelle organotypic kultur i kollagen jeg geler af varierende stivhed. Denne metode i høj grad bevarer de vestibulære og cochlear sanseorganer i vivo arkitektur og giver mulighed for undersøgelse af virkningerne af mekaniske kraft på vækst og differentiering11.
Fordi mekaniske stimuli er kendt for at aktivere downstream molekylære begivenheder, såsom Hippo signalering vej12,13,14,15, er det vigtigt at være i stand til at kombinere mekaniske stimulation med biokemiske og genetiske manipulationer. Kultur metoden her tillader virus-medieret gen levering og kan derfor bruges til at studere både mekanisk og molekylær signalering under indre øre udvikling11.

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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;">#5 Forceps</td>
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			<td height="21" style="height:21px;width:252px;">100 mm Petri dish</td>
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			<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>
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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>
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		<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>
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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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			<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>
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		<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>
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			<td height="21" style="height:21px;">Bicinchoninic acid assay</td>
			<td style="width:212px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">23225</td>
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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>
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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>
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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;">DMEM/F12&#160;</td>
			<td style="width:212px;">Gibco</td>
			<td style="width:119px;">11320033</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>
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		<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>
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			<td height="21" style="height:21px;">Fibroblast growth factor</td>
			<td style="width:212px;">Sigma</td>
			<td style="width:119px;">F5392</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>
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			<td height="21" style="height:21px;">HBSS</td>
			<td style="width:212px;">Gibco</td>
			<td style="width:119px;">14025092</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;">Microinjector</td>
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			<td height="21" style="height:21px;">Nicotinamide</td>
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			<td style="width:119px;">N0636</td>
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			<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>
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		<tr height="21">
			<td height="21" style="height:21px;">PBS (1X), pH 7.4</td>
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			<td style="width:119px;">10010023</td>
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			<td height="21" style="height:21px;width:252px;">Phenol Red pH indicator&#160;</td>
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			<td height="21" style="height:21px;width:252px;">RFP antibody</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

Sanseorganer i det indre øre er udfordrende for at studere i pattedyr på grund af deres manglende adgang til eksperimentelle manipulation og optisk observation. Desuden, selvom eksisterende kultur teknikker tillader biokemiske forstyrrelser, disse metoder ikke giver et middel til at studere virkningerne af mekanisk kraft og væv stivhed i udviklingen af de indre øre sanseorganer. Her beskriver vi en metode for tre-dimensionelle organotypic kultur intakt murine utricle og cochlea, der overvinder disse begrænsninger. Teknik til justering af en tre-dimensionel matrix stivhed beskrevet her tillader manipulation af elastisk kraft imod vækst af væv. Denne metode kan derfor bruges til at studere rollen af mekaniske styrker under indre øre udvikling. Derudover tillader kulturer virus-medieret gen levering, som kan bruges til eksperimenter med gevinst og tab-af-funktion. Denne kultur metode bevarer medfødte hårceller og støtte celler og fungerer som en potentielt bedre alternativ til den traditionelle to-dimensionelle kultur af vestibulære og auditive sanseorganer.

Vestibulære og auditive sanseorganer fra embryonale ører, kulturperler i 40-Pa kollagen geler efterligne lav stivhed embryonale betingelser11, bevare relativt normale tredimensionale strukturer (figur 1) og vedligeholde hårceller og støtte celler (figur 2 og figur 3). Selv om støtte celle tæthed mindskes med over 30% (Students t -test: n = 4, p < 0,004)...

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

Tre-dimensionelle organotypic kulturer af murine utricle og cochlea i optisk klare kollagen jeg geler Bevar medfødte væv morfologi, tillader mekanisk stimulering gennem tilpasning af matrix stivhed, og tillade virus-medieret gen levering.

Tre-dimensionelle Organotypic kulturer af vestibulære og auditive 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 skivekulturer for Studier af Fødselsdepression Neurogenese

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 af en human neural stamcelle Line på tredimensionelle kulturer, Analyse af MicroRNA og formodede 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

Tre-dimensionel Kvantificering af dendritiske Spines fra pyramideneuroner Afledt af menneskeskabte pluripotente 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

Brug<em&gt; Ex vivo</em&gt; Upright dråbedistribution Kulturer af Whole Fetal organer til at studere udviklingsprocesser under Mus 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

Generering af genetisk modificerede Organotypiske Skin Cultures Brug devitaliseret 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 til kylling Auditory Organ med<em&gt; I Ovo</em&gt; Micro-elektroporation

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 til visualisering steroidogeniske organer og deres interaktive organer med Immunfarvning i bananfluen<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 af retinoidsyre-induceret Neural Differentiation af muse embryonale stamceller i to og tre-dimensionelle embryoide legemer

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 Hel Mount: En Novel Processing and Imaging Protocol for Tykke, Tredimensionale Tissue Tværsnit af Skin

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 roman brug af tre-dimensionelle høj frekvens ultralyd for tidlig graviditet karakterisering i mus

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 ...