Name: a protocol for decellularizing mouse cochleae for inner ear tissue engineering
Uploaded: 2018-01-01T14:00:00.000+00:00
Duration: 9 min 53 s
Description: Watch this Scientific Journal Video about A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering at JoVE.com

El proyecto fue financiado por la Universidad de Kansas Prueba de concepto de fondo. Nos gustaría agradecer al personal de enfermería en el KUMC (Kansas City, KS) para asistirnos en la obtención de cordones umbilicales humanos y David Jorgensen para ayudar con las culturas de cócleas.

Se realizaron todos los procedimientos, incluyendo la eutanasia animal, según el (ACUP #2014-2234) protocolo aprobado institucional cuidado Animal y el Comité uso (IACUC) en la Universidad de Kansas Medical Center (KUMC).
Nota: HWJCs fueron aislados de humanos los cordones umbilicales que fueron donados por los pacientes que el consentimiento informado y se utilizaron muestras según los protocolos aprobados por el Comité de temas humanos de Universidad de Kansas (KU-IRB #15402).
1. hueso Temporal cosecha y aislamiento de la cóclea
<ol>
	<li>Decapitar un ratón apropiadamente eutanasia, 15 semanas de edad por el corte entre el cráneo y primera vértebra cervical con un fuerte par de tijeras quirúrgicas, luego rocíe abajo de la cabeza con etanol al 70% para desinfectar (figura 1A).</li>
	<li>Atraviesan el cráneo en un plano sagital, utilizando el mismo par de afilado de tijeras quirúrgicas (figura 1B-C, las líneas punteadas).</li>
	<li>Retire el tejido cerebral del cráneo con un par de fórceps (figura 1, flecha).</li>
	<li>Identificar el hueso temporal a través de la presencia del auditorio y las raíces del nervio vestibular (Figura 1E-F, puntas de flecha) en el interior del cráneo y el conducto auditivo externo desde el exterior (Figura 1F, flecha). Corte cuidadosamente a través del cráneo para aislar el hueso temporal del resto del cráneo (figura 1). Nota: En algunos casos, puede ser posible para delicadamente los huesos circundantes del cráneo del hueso temporal sin necesidad de corte.</li>
	<li>Inserte el fórceps fino en la abertura del conducto auditivo aproximadamente 5 mm (figura 1 H), por lo que las puntas no hay riesgo de punción de la cóclea. Con cuidado romper el hueso de la ampolla (figura 1 H, área sombreada roja superior) para fracturas (figura 1I, rojo punteado de línea). Nota: Si es necesario, un microscopio de disección puede ayudar en este proceso para asegurar la exacta ubicación y manipulación de instrumentos.</li>
	<li>Con unas pinzas finas, haga palanca, el resto de la ampolla del hueso temporal, exponiendo el laberinto óseo de la cóclea (Figura 1J, soportes de rojo).</li>
	<li>Colocar una placa de Petri lo suficientemente grande como para sostener el hueso temporal (por ejemplo, 30 mm o mayor) por debajo de la disección alcance campo de visión y llenarlo con suficiente fosfato tampón salino (PBS) para cubrir el hueso temporal (p. ej., 0,5 a 1 mL).</li>
	<li>Sumergir el hueso temporal con la bulla en el PBS con las ventanas redondeos y ovales de la cóclea hacia arriba. Nota: El retiro del sistema vestibular de la cóclea no es necesario, como el sistema vestibular sirve bien como un mango para manipular y orientar las funciones de la cóclea (figura 1I y figura 2B).</li>
	<li>Con un ultra fino par de pinzas, quite la arteria stapedial, que pasa a través del estribo.</li>
	<li>Inserte una punta de las pinzas ultra finas por el arco del estribo donde la arteria pasa a través y delicadamente Levante el estribo. El estribo desarticulado debe levantar fuera de la ventana oval.</li>
	<li>Usando una punta de las pinzas ultra finas, el óvalo de la puntura y alrededor de windows.</li>
	<li>Usando un PBS 1 x relleno 28,5 calibre jeringa con un tubo (diámetro interno 0,28 mm, diámetro externo mm 0,61) Unido, coloque el tubo sobre la ventana oval (figura 2A -B). Nota: La apertura del tubo puede ser manipulada usando fórceps ultra fina, ayudando a garantizar una colocación exacta.<ol>
		<li>Utilice una nueva jeringa y tubo para cada cóclea.</li>
	</ol>
	</li>
	<li>Perfusión 2 mL de antibiótico-antimicótico (anti-anti) 10% y 10% penicilina-estreptomicina (pen-strep) en PBS a través de la cóclea más 5 minutos para quitar la perilinfa. Inunda demasiado rápido con demasiada presión puede dañar las estructuras finas dentro de la cóclea. Nota: Conducción manualmente de la jeringa con la mano para inundar el líquido a través de la cóclea es eficaz, aunque una bomba de perfusión podría utilizarse como alternativa.<ol>
		<li>Si inunda manualmente, utilice un par de multa, cierre pinza estabilizar la cóclea (figura 2A) durante la perfusión agarrando la porción vestibular del hueso temporal. Nota: Aunque no es necesario, esto deja ambas manos libres manejar los instrumentos; una mano puede conducir a la jeringa (figura 2A) mientras que el otro puede colocar el tubo correctamente (figura 2B).</li>
		<li>De este paso adelante, tenga cuidado para evitar exponer la cóclea innecesariamente al aire. Introducir burbujas de aire en los espacios líquidos bloqueará la circulación del líquido, y secar más dañará las bellas estructuras dentro de la cóclea.</li>
	</ol>
	</li>
	<li>Retire y deseche cualquier restantes fragmentos de tejido y hueso músculo con unas pinzas finas.</li>
	<li>Si es necesario, almacenar cócleas aislados a 4 ° C en anti-anti 10% y 10% solución de pen-strep en PBS durante siete días con cambios regulares de solución de antibiótico cada 48 h.</li>
</ol>
2. coclear procesamiento
<ol>
	<li>Descelularización
	<ol>
		<li>Para cada cóclea, llenar una cubeta de centelleo de vidrio de 20 mL con un 1% sodio dodecil sulfato en solución (SDS) en agua desionizada de (DI), que se utiliza para decellularize la cóclea.</li>
		<li>Cortar la punta de una plástico 7 mL pipeta usando una cuchilla de afeitar para que la abertura es suficientemente grande para que una cóclea pasar a través.</li>
		<li>Usando la pipeta de transferencia, suavemente elaborar la cóclea con la pipeta por lo que sólo pasa el borde de corte por unos milímetros.</li>
		<li>Expulsar a la cóclea en el vial de centelleo lleno de solución 1% SDS.</li>
		<li>Circular 2 mL de SDS 1% en agua desionizada (DI) a través de la cóclea en el vial de centelleo con una pipeta de transferencia.</li>
		<li>Coloque el frasco centelleo en un rotor y deje que el frasco centelleo girar a 10 rpm durante 72 h a temperatura ambiente.<ol>
			<li>Cambiar solución SDS al 1% cada 24 h durante un periodo de 72 h. Tenga cuidado cuando cambie de SDS para nunca exponer la cóclea al aire.</li>
		</ol>
		</li>
		<li>Lave la cóclea tres tiempos de 30 min con agua desionizada.</li></ol></li></ol>Realizar los lavados en el mismo vial de centelleo usado para los cargos de SDS; en esta etapa, la cóclea es decellularized.
	
	
	<li>Descalcificación
	<ol>
		<li>Quite el agua restante de la DI desde el vial de centelleo, dejando sólo lo suficiente para mantener la cóclea sumergido.</li>
		<li>Para empezar la descalcificación, añadir 5 mL de 10% de ácido etilendiaminotetracético (EDTA) (0.02 M) en agua desionizada para el vial de centelleo con la cóclea.</li>
		<li>Circular 2 mL de EDTA de 10% en agua desionizada a través de la cóclea en el vial de centelleo con una pipeta de transferencia.</li>
		<li>Vuelva a colocar el vial de centelleo en el rotor y deje que el frasco centelleo girar a 10 rpm durante 72 h a temperatura ambiente. Cambiar la solución de EDTA de 10% cada 24 h durante un periodo de 72 h. Tenga cuidado cuando cambie las soluciones para que la cóclea no está expuesta al aire.</li>
		<li>Enjuague la cóclea 3 veces durante 2 h cada uno con PBS 1 x; en esta etapa, la cóclea es descalcificada.</li>
	</ol>
	</li>
	<li>Almacenamiento de información
	<ol>
		<li>Almacenar las cócleas hasta 72 h en una solución 10% anti-anti, 10% pen-strep en PBS a 4 ° C. Nota: Períodos más largos de almacenamiento pueden ser posibles con cambios regulares de solución de antibiótico; sin embargo, almacenaje extendido de cócleas procesados no ha sido validado en el presente Protocolo.</li>
	</ol>
	</li>

3. adquisición y ampliación de hWJCs
<ol>
	<li>HWJCs aislante según protocolos previamente publicados22. Brevemente, segmento de cordón umbilical en secciones de 3 cm.</li>
	<li>Cuidadosamente hacer una incisión superficial con un bisturí a lo largo de cada segmento de cordón umbilical para desenrollar y exponer la gelatina de Wharton del cordón umbilical. Use pinzas para sacar los vasos sanguíneos.</li>
	<li>Lave los segmentos cordón umbilical dos veces con suficiente PBS (p. ej., 40 mL en una placa de Petri de 60 mm) para sumergir el tejido. Pique finamente segmentos de tejido con un bisturí. Digerir el tejido en una placa de Petri con 50 mL de medio de digestión de 100 mm (colagenasa tipo 2 de 0.2%, 1% de penicilina-estreptomicina, baja glucosa en medio (DMEM modificado Eagle de Dulbecco)).</li>
	<li>Coloque el tejido en digerir medio en un agitador orbital a 50 rpm durante la noche en un 5% CO2, incubadora de 37 ° C. Al día siguiente, diluya medio de digestión en una proporción de 1:16 en el 2% antibiótico-antimicótico en PBS y de la pelotilla de las células por centrifugación a 500 x g a temperatura ambiente (~ 27 ° C) por 10 minutos descartar sobrenadante.</li>
	<li>Resuspender el pellet en células madre mesenquimales crecimiento medio (MSCGM) y combinar celdas. Las células de la placa a una densidad de 7 x 103 células/cm2 en cultivo de tejidos tratan frascos de T-75. HWJCs en MSCGM de la cultura y ampliar al paso 5 para experimentos. Nota: HWJCs puede cultivarse el en T-frascos más grandes (e.g., T-150, T-300) cuando pases para aumentar la producción de hWJCs para los experimentos.</li>
</ol>
4. infusión de hWJCs en cócleas Decellularized
<ol>
	<li>Preparar cócleas decellularized
	<ol>
		<li>Utilizando una técnica aséptica, lavar cócleas almacenados en PBS 1 x tres veces para 30 minutos.</li>
		<li>Transferencia de cócleas en pocillos distintos de una placa de 24 pocillos y agregar 1 mL de 37 º C previamente calentado MSCGM.</li>
		<li>Incubar cócleas en un 5% CO2, incubadora de cultura celular de 37 ° C durante un mínimo de 1 h antes de la infusión de células.</li>
	</ol>
	</li>
	<li>Se disocian hWJCs y resuspender
	<ol>
		<li>HWJCs de lavado con 37 º C previamente había calentado 1 x PBS dos veces.</li>
		<li>Añadir suficiente 37 º C previamente calentado tripsina con EDTA 0.5% para cubrir la superficie de la célula recipiente.</li>
		<li>Incubar hWJCs para hasta 5 min en un 5% CO2, incubadora de 37 ° C de la célula cultura.<ol>
			<li>Verificar que el 90% de las células han separado de la superficie de cultivo celular mediante la visualización de las células bajo un microscopio invertido. Nota: Las células que se mueven cuando la nave de cultura es golpeada suavemente, han destacado. Las células que permanecen inmóviles, no han separado.</li>
			<li>Golpee ligeramente los lados del frasco para desalojar más células adjuntas.</li>
		</ol>
		</li>
		<li>Utilizando una técnica aséptica, transferencia hWJCs del recipiente de cultivo a un tubo cónico de 50 mL que contiene un volumen equivalente de MSCGM en el volumen de la tripsina se utiliza para disociar las células.</li>
		<li>El hWJCs de pellets por centrifugación a 500 x g a temperatura ambiente (~ 27 ° C) durante 5 minutos.</li>
		<li>Aspirar el sobrenadante y resuspender el hWJCs en MSCGM en una concentración de 500.000 células/mL.</li>
	</ol>
	</li>
	<li>Perfusión de cócleas
	<ol>
		<li>Cócleas de transferencia a una nueva placa de 24 pocillos usando una pipeta de transferencia.</li>
		<li>Añadir una gota de MSCGM a cada cóclea para prevenir cualquier cóclea de la desecación.</li>
		<li>Oriente con delicadeza la cóclea usando fórceps ultra fina para que las ventanas ovales y redondeos son hacia arriba. Nota: Tenga mucho cuidado al manipular directamente cada cóclea como la cóclea se daña fácilmente.</li>
		<li>Utilizando una jeringa de insulina estéril de calibre 28,5 con tubería conectada, elaborar hWJCs 0,2 mL suspendida (100.000 células).</li>
		<li>Con unas pinzas finas esterilizadas, posición de la tubería sobre la ventana oval.</li>
		<li>Con delicadeza y lentamente perfusión la cóclea con 0.2 mL de hWJCs resuspendido utilizando una jeringa de insulina de 28,5 calibre conectada a la tubería de polietileno (diámetro externo: 0,61 mm, diámetro interior: mm 0,28) durante aproximadamente 5 minutos.</li>
		<li>Después de la perfusión, agregar 0,8 mL de 37 º C previamente calentado MSCGM a bien que contiene la cóclea para llevar el volumen total en el pozo a 1 mL.</li>
		<li>Repita los pasos 4.3.3-4.3.7 para cada cóclea, utilizando una jeringa nueva y cada vez que la tubería.</li>
		<li>Lugar de cócleas inundadas en un 5% CO2, incubadora de 37 ° C de la célula cultura y cambiar los medios de comunicación tres veces por semana.</li>
	</ol>
	</li>
</ol>
5. conservación y cosecha cóclea
Nota: Cócleas pueden ser cultivados y cosechan en cualquier momento perfusión posterior de hasta 30 días.
<ol>
	<li>Para conservar cócleas, fijar en paraformaldehído al 4% en PBS 1 x durante la noche a 4 ° C en una plataforma oscilante.</li>
	<li>Después de la fijación, lavado cócleas con PBS 1 x tres veces por 5 minutos cada uno.</li>
	<li>Poco a poco deshidratar cóclea con etanol y claro con un solvente hidrocarburo alifático antes de inclusión en parafina.</li>
	<li>Sección muestras a un grosor de 10 μm utilizando un micrótomo y montar en portaobjetos de vidrio. Nota: Las muestras están ahora listas para histológica o immunohistochemical usando protocolos estándar.</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroCT+versus+sTSLIM+3D+imaging+of+the+mouse+cochlea.">MicroCT versus sTSLIM 3D imaging of the mouse cochlea.</a> J Histochem Cytochem. 61 (5), 382-395 (2013).</li><li>Nonoyama, H., 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=Investigation+of+the+ototoxicity+of+gadoteridol+(ProHance)+and+gadodiamide+(Omniscan)+in+mice.">Investigation of the ototoxicity of gadoteridol (ProHance) and gadodiamide (Omniscan) in mice.</a> Acta Otolaryngol. 136 (11), 1091-1096 (2016).</li><li>Dai, 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=Rhesus+Cochlear+and+Vestibular+Functions+Are+Preserved+After+Inner+Ear+Injection+of+Saline+Volume+Sufficient+for+Gene+Therapy+Delivery.">Rhesus Cochlear and Vestibular Functions Are Preserved After Inner Ear Injection of Saline Volume Sufficient for Gene Therapy Delivery.</a> J Assoc Res Otolaryngol. , (2017).</li><li>Sutherland, A. J., Converse, G. L., Hopkins, R. A., Detamore, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bioactivity+of+cartilage+extracellular+matrix+in+articular+cartilage+regeneration.">The bioactivity of cartilage extracellular matrix in articular cartilage regeneration.</a> Adv Healthc Mater. 4 (1), 29-39 (2015).</li></ol>

Los autores no tienen nada que revelar.

Con éxito hemos demostrado que las células cocleares nativas pueden extraerse de la cóclea a través de un proceso de descelularización, que permite el uso de la cóclea como un andamio de tejido intrincado, tridimensional. Santi et al. 15 desarrolló el método inicial para cócleas decellularizing y con precisión se han estimado los volúmenes de muchas estructuras cocleares a través con la ayuda de microscopia de luz hoja23. Estos primeros trabajos sirvier...

La cóclea es una estructura intrincada espiral encontrada en el hueso temporal. Se compone de un exterior laberinto óseo y laberinto membranoso concéntricos, interior1. El laberinto membranoso consta de tres espacios fluidos: vestibuli de Scala, Scala tympani de Scala1y medios. Los medios del scala alberga el epitelio sensorial, que se compone de una multitud de tipos celulares, pero las células de pelo sensoriales (HC), que transduce la energía mecánica de las ondas sonoras a impulsos nerviosos2, son de particular interés. Exposición a trauma acústico3,4,5, medicamento6, enfermedad7,8y9 de envejecimiento puede resultar en deterioro de la función auditivo por medio de la muerte HC. Pérdida de células ciliadas en los mamíferos es permanente, a diferencia de HCs aviares, que pueden regenerar después de lesión10.
Una variedad de esfuerzos de investigación han intentado restablecer perdidos HCs, aunque varían los enfoques experimentales específicos. Manipulación de expresión génica en el epitelio sensorial y la implantación de células diferenciadas fuera del cuerpo son los enfoques dominantes en el campo, aunque han sido métodos de inducción que buscan diferenciar células madre en organoides coclear trató de11,12,13. Cada uno de estos enfoques es dependiente directamente de las células madre o las claves del desarrollo utilizadas por las células de vástago; sin embargo, un segundo compartido y potencialmente crítico, elemento es el ECM de la cóclea se14,15.
El ECM no sólo proporciona soporte físico para las células y tejidos, que incluye una superficie para la adhesión celular, proliferación, supervivencia y migración, pero también juega un papel crítico en el desarrollo de HCs y la espiral del ganglio15,16 ,17. Naturalmente ECM que ofrece señales inductivas que pueden guiar la determinación de fenotipo celular o de adhesión, proliferación y supervivencia de la célula18. En consecuencia, el uso de la cóclea decellularized en combinación con hWJCs cultivadas ofrecen una oportunidad única para explorar el papel de la regeneración de ECM y HC. HWJCs son un tipo de disponible, no controvertida de la célula aislado de humanos los cordones umbilicales que se comportan como células madre mesenquimales19. HWJCs han demostrado la capacidad de diferenciarse hacia abajo célula neurosensorial linajes20,21. Así, el protocolo actual detalla el aislamiento, la descelularización y perfusión de cócleas de canales de ratón C57BL con hWJCs para ingeniería de tejidos del oído interno.

<table><tbody><tr><td>Allegra X-14R Centrifuge</td><td>Beckman-Coulter</td><td>B08861</td><td></td></tr><tr><td>Intramedic Semi-Rigid Tubing</td><td>Becton Dickinson</td><td>427401</td><td></td></tr><tr><td>New Brunswick Innova 2000 Orbital Shaler</td><td>Eppendorf</td><td>M1190-0002</td><td></td></tr><tr><td>Surgical Scissors</td><td>Fine Science Tools</td><td>14060-10</td><td></td></tr><tr><td>Fine Forceps</td><td>Fine Science Tools</td><td>11370-40</td><td></td></tr><tr><td>Ultra-Fine Forceps</td><td>Fine Science Tools</td><td>18155-13</td><td></td></tr><tr><td>50-mL Conical Tubes</td><td>Fisher Scientific</td><td>12565271</td><td></td></tr><tr><td>Petri Dish</td><td>Fisher Scientific</td><td>FB087579B</td><td></td></tr><tr><td>U-100 Insulin Syringe</td><td>Fisher Scientific</td><td>14-829-1B</td><td></td></tr><tr><td>Scintillation Vial</td><td>Fisher Scientific</td><td>03-341-73</td><td></td></tr><tr><td>Rotator</td><td>Fisher Scientific</td><td>88-861-049</td><td></td></tr><tr><td>Transfer Pipette</td><td>Fisher Scientific</td><td>22-170-404</td><td></td></tr><tr><td>Razor Blade</td><td>Fisher Scientific</td><td>12-640</td><td></td></tr><tr><td>Antibiotic-Antimycotic</td><td>Fisher Scientific</td><td>15-240-062</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Fisher Scientific</td><td>15-140-122</td><td></td></tr><tr><td>24-Well Plate</td><td>Fisher Scientific</td><td>07-200-84</td><td></td></tr><tr><td>SuperFrost PLUS Glass Microscope Slides</td><td>Fisher Scientific</td><td>12-550-15</td><td></td></tr><tr><td>Transfer Pipette</td><td>Fisher Scientific</td><td>22-170-404</td><td></td></tr><tr><td>ProLong Gold Antifade Mountant with DAPI</td><td>Fisher Scientific</td><td>P36935</td><td></td></tr><tr><td>Clear-Rite 3</td><td>Fisher Scientific</td><td>22-046341</td><td></td></tr><tr><td>Thermo Scientific Forma Series II 3110 Water-Jacekted CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Fisher Scientific</td><td>13-998-078</td><td></td></tr><tr><td>Mesenchymal Stem Cell Growth Medium</td><td>Lonza</td><td>PT-3001</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Lonza</td><td>CC-3232</td><td></td></tr><tr><td>TPP T-75 Culture Flask</td><td>MidSci</td><td>TP90076</td><td></td></tr><tr><td>TPP T-150 Culture Flask</td><td>MidSci</td><td>TP90151</td><td></td></tr><tr><td>TPP T-300 Culture Flask</td><td>MidSci</td><td>TP90301</td><td></td></tr><tr><td>Dissection Microscope</td><td>Nikon Instruments</td><td>SMZ800</td><td></td></tr><tr><td>Nikon Eclipse Ts2R-FL Inverted Microscope</td><td>Nikon Instruments</td><td>MFA51010</td><td></td></tr><tr><td>NuAire Class II, Type A2 Biosafety Cabinet</td><td>NuAire</td><td>NU-425-600</td><td></td></tr><tr><td>1X PBS</td><td>Sigma-Aldrich</td><td>P5368-10PAK</td><td></td></tr><tr><td>1% SDS Solution</td><td>Sigma-Aldrich</td><td>436143-100G</td><td></td></tr><tr><td>10% EDTA</td><td>Sigma-Aldrich</td><td>E9884-100G</td><td></td></tr></tbody></table>

a protocol for decellularizing mouse cochleae for inner ear tissue engineering

En los mamíferos, las células de pelo de mechanosensory que facilitan la falta de audiencia la capacidad de regenerar, que ha limitado los tratamientos para la pérdida auditiva. Estrategias actuales de la medicina regenerativa se han centrado en el trasplante de células madre o la manipulación genética de células de apoyo en el oído interno para estimular la sustitución de células dañadas para corregir la pérdida de la audición de alrededor. Sin embargo, la matriz extracelular (ECM) puede jugar un papel vital en inducir y mantener la función de las células de pelo y no ha sido bien investigada. Usando el coclear ECM como un andamio para cultivar células madre adultas puede proporcionar perspectivas únicas en cómo la composición y la arquitectura del entorno extracelular ayuda a las células en el mantenimiento de la función auditiva. Aquí presentamos un método para aislar y decellularizing cócleas de ratones para usar como andamios aceptar perfusión células madre adultas. En el actual protocolo, cócleas son aislados de ratones eutanasia, decellularized y descalcificada. Después, celdas de jalea de Wharton humana (hWJCs) que fueron aisladas de cordón umbilical fueron perfundidas con cuidado en cada cóclea. Las cócleas fueron utilizados como Biorreactores, y las células fueron cultivadas durante 30 días antes de someterse a tratamiento para el análisis. Decellularized cócleas conservan estructuras extracelulares identificables, pero no reveló la presencia de células o notables fragmentos de ADN. Perfundidos en la cóclea las células invadieron la mayor parte del interior y el exterior de la cóclea y crecieron sin incidentes con una duración de 30 días. Así, el método actual que puede utilizarse para estudiar cómo coclear ECM afecta desarrollo de las células y comportamiento.

Utilizando los métodos presentados aquí, éxito descelularización de cócleas se evaluó mediante el examen de la presencia o ausencia de ADN a través de 4', 6-diamidino-2-phenylindole (DAPI) tinción. Cócleas eran considerados totalmente decellularized si ADN no fue identificado dentro de la cóclea decellularized. Una cóclea nativa de un experimento anterior que no experimentaron descelularización o descalcificación se utilizó como control positivo para ilustrar las estructuras...

Watch this Scientific Journal Video about A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering at JoVE.com

A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering

Un protocolo para Decellularizing ratón cócleas para ingeniería de tejidos del oído interno

El objetivo de este protocolo es un método eficaz para decellularize y descalcificar cócleas de ratón para utilización como andamios para aplicaciones de ingeniería de tejidos.

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current ...

a-protocol-for-decellularizing-mouse-cochleae-for-inner-ear-tissue

Research

JoVE Journal

Bioengineering

10.7K vistas.  University of Kansas Medical Center. El objetivo de este protocolo es un método eficaz para decellularize y descalcificar cócleas de ratón para utilización como andamios para aplicaciones de ingeniería de tejidos.

Video: A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

Cultura de embriones de ratón explantes cocleares y Gene Transfer por electroporación

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

Investigación

Biología del Desarrollo

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

A largo plazo Lapso de tiempo de imágenes de Ratón cocleares explantes

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

Neurociencias

The Mouse Round-window Approach for Ototoxic Agent Delivery: A Rapid and Reliable Technique for Inducing Cochlear Cell Degeneration

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research involving the cochlea and its associated neural pathways entails exposure of model cochleae to a variety of ototoxic agents. This allows investigators to study the effects of targeted damage to cochlear structures, and in some cases, the self-repair or regeneration of those structures. Various techniques exist for delivery of ototoxic agents to the cochlea. When selecting a particular technique, investigators must consider a number of factors, including the induction of inadvertent systemic toxicity, the amount of cochlear damage produced by the surgical procedure itself, the type of lesion desired, animal survivability, and reproducibility/reliability of results. Currently established techniques include parenteral injection, intra-peritoneal injection, trans-tympanic injection, endolymphatic sac injection, and cochleostomy with perilymphatic perfusion. Each of these methods has been successfully utilized and is well described in the literature; yet, each has various shortcomings. Here, we present a technique for topical application of ototoxic agents directly to the round window niche. This technique is non-invasive to inner ear structures, produces rapid onset of reliably targeted lesions, avoids systemic toxicity, and allows for an intra-animal control (the contra-lateral ear). Results stemming from this approach have helped deeper understanding of auditory pathophysiology, cochlear cell degeneration, and regenerative capacity in response to an acute injury. Future investigations may use this method to conduct interventional studies involving gene therapy and stem cell transplantation to combat hearing loss.

Various methods exist for introducing ototoxic agents to the cochleae of animal models. Presented is a surgical protocol for delivery of ototoxic agents to the round window niche. The procedure is reliable, creates targeted intra-cochlear lesions, and avoids mechanical damage to the microarchitecture. Examination of cochlear self-repair/regeneration is possible.

El Enfoque del ratón Ronda-ventana para ototóxicos Agente de entrega: Una rápida y técnica fiable para la inducción de degeneración de las células cocleares

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research ...

Medicina

Evaluation of Planar-Cell-Polarity Phenotypes in Ciliopathy Mouse Mutant Cochlea

In recent years, primary cilia have emerged as key regulators in development and disease by influencing numerous signaling pathways. One of the earliest signaling pathways shown to be associated with ciliary function was the non-canonical Wnt signaling pathway, also referred to as planar cell polarity (PCP) signaling. One of the best places in which to study the effects of planar cell polarity (PCP) signaling during vertebrate development is the mammalian cochlea. PCP signaling disruption in the mouse cochlea disrupts cochlear outgrowth, cellular patterning and hair cell orientation, all of which are affected by cilia dysfunction. The goal of this protocol is to describe the analysis of PCP signaling in the developing mammalian cochlea via phenotypic analysis, immunohistochemistry and scanning electron microscopy. Defects in convergence and extension are manifested as a shortening of the cochlear duct and/or changes in cellular patterning, which can be quantified following dissection from developing mouse mutants. Changes in stereociliary bundle orientation and kinocilia length or positioning can be observed and quantitated using either immunofluorescence or scanning electron microscopy (SEM). A deeper insight into the role of ciliary proteins in cellular signaling pathways and other biological phenomena is crucial for our understanding of cellular and developmental biology, as well as for the development of targeted treatment strategies.

Primary cilia influence various signaling pathways. The mammalian cochlea is ideal for examining planar cell polarity (PCP) signaling. Cilia dysfunction affects cochlear outgrowth, cellular patterning and hair cell orientation, readouts of PCP. Our goal is to analyze PCP signaling in mouse cochlea via phenotypic analysis, immunohistochemistry and scanning electron microscopy.

Evaluación de planar-Cell-polaridad fenotipos en ciliopatía ratón mutante cóclea

In recent years, primary cilia have emerged as key regulators in development and disease by influencing numerous signaling pathways. One of the earliest ...

Cryosectioning and Immunostaining Mouse Inner Ear Tissue: From Embryonic to Adult Stages

This protocol details general histology methods for preparing inner ear samples from embryonic, neonatal, and adult mice. The purpose of this protocol is to provide a straightforward and standardized method for inner ear tissue processing for researchers, possibly new to the field, interested in working with the mouse cochlea. Included here are protocols for dissection, fixation, embedding, cryosectioning, and immunostaining. Section immunostaining is one of the best methods for examining individual cells within the context of the entire cochlea.
Key steps include dissecting the inner ear away from the skull, fixing the tissue using 4% paraformaldehyde, embedding with Optimal Cutting Temperature compound, cryosectioning, and immunostaining with antibodies targeting specific proteins expressed by the cochlea.
Included in our methods are special considerations made for the cochlea given its 
shape and structure.&#160;Reasonably good focus and dissection skills are needed, as tissue damage can affect the quality of immunostaining and consistency among samples. However, with the procedures we outline, most users can be trained in a few weeks to make beautiful preparations. Overall, this protocol offers a valuable way to promote research to understand auditory development, function, and disease in mouse models.

This protocol, intended for novice users of mouse inner ear tissue, comprehensively details steps for processing mouse inner ears at different developmental stages for section immunostaining.

Criosección e inmunotinción de tejido del oído interno de ratón: desde la etapa embrionaria hasta la adulta

This protocol details general histology methods for preparing inner ear samples from embryonic, neonatal, and adult mice. The purpose of this protocol is ...

Dextran Labeling and Uptake in Live and Functional Murine Cochlear Hair Cells

The hair cell mechanotransduction (MET) channel plays an important role in hearing. However, the molecular identity and structural information of MET remain unknown. Electrophysiological studies of hair cells revealed that the MET channel has a large conductance and is permeable to relatively large fluorescent cationic molecules, including some styryl dyes and Texas Red-labeled aminoglycoside antibiotics. In this protocol, we describe a method to visualize and evaluate the uptake of fluorescent dextrans in hair cells of the organ of Corti explants that can be used to assay for functional MET channels. We found that 3 kDa Texas Red-labeled dextran specifically labels functional auditory hair cells after 1&#8211;2 h incubation. In particular, 3 kDa dextran labels the two shorter stereocilia rows and accumulates in the cell body in a diffuse pattern when functional MET channels are present. An additional vesicle-like pattern of labeling was observed in the cell body of hair cells and surrounding supporting cells. Our data suggest that 3 kDa Texas-Red dextran can be used to visualize and study two pathways for cellular dye uptake; a hair cell-specific entry route through functional MET channels and endocytosis, a pattern also available to larger dextran.

Here, we present a method for visualizing the uptake of 3 kDa Texas Red-labeled dextran in auditory hair cells with functional mechanotransduction channels. In addition, dextrans of 3&#8211;10 kDa can be used to study endocytosis in hair and supporting cells of the organ of Corti.

Etiquetado y toma de Dextran en células de cabello cocleares de murine en vivo y funcionales

The hair cell mechanotransduction (MET) channel plays an important role in hearing. However, the molecular identity and structural information of MET ...

Bioingeniería

Cochlear Surface Preparation in the Adult Mouse

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous etiologies such as exposure to excessive noise, the use of ototoxic medications, bacterial or viral ear infections, head injuries, and the aging process. Loss of sensory hair cells is a common pathological feature of the varieties of acquired hearing loss. Additionally, the inner hair cell synapse can be damaged by mild insults. Therefore, surface preparations of cochlear epithelia, in combination with immunolabeling techniques and confocal imagery, are a very useful tool for the investigation of cochlear pathologies, including losses of ribbon synapses and sensory hair cells, changes in protein levels in hair cells and supporting cells, hair cell regeneration, and determination of report gene expression (i.e., GFP) for verification of successful transduction and identification of transduced cell types. The cochlea, a bony spiral-shaped structure in the inner ear, holds the auditory sensory end organ, the organ of Corti (OC). Sensory hair cells and surrounding supporting cells in the OC are contained in the cochlear duct and rest on the basilar membrane, organized in a tonotopic fashion with high-frequency detection occurring in the base and low-frequency in the apex. With the availability of molecular and genetic information and the ability to manipulate genes by knockout and knock-in techniques, mice have been widely used in biological research, including in hearing science. However, the adult mouse cochlea is miniscule, and the cochlear epithelium is encapsulated in a bony labyrinth, making microdissection difficult. Although dissection techniques have been developed and used in many laboratories, this modified microdissection method using cell and tissue adhesive is easier and more convenient. It can be used in all types of adult mouse cochleae following decalcification.

This article presents a modified cochlear surface preparation method that requires decalcification and use of a cell and tissue adhesive to adhere the pieces of cochlear epithelia to 10 mm round cover slips for immunohistochemistry in adult mouse cochleae.

Preparación de la superficie coclear en el ratón adulto

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous ...

Whole Neonatal Cochlear Explants as an In vitro Model

Untreated hearing loss imposes significant costs on the global healthcare system and impairs individuals' quality of life. Sensorineural hearing loss is characterized by the cumulative and irreversible loss of sensory hair cells and auditory nerves in the cochlea. Entire and vital cochlear explants are one of the fundamental tools in hearing research to detect hair cell loss and to characterize the molecular mechanisms of the inner ear cells. Many years ago, a protocol for neonatal cochlear isolation was developed, and although it has been modified over time, it still holds potential for improvement. 
This paper presents an optimized protocol for isolating and culturing whole neonatal cochlear explants in multi-well culture chambers that enables the study of hair cells and spiral ganglion neuron cells along the entire length of the cochlea. The protocol was tested using cochlear explants from mice and rats. Healthy cochlear explants were obtained to study the interaction between hair cells, spiral ganglion neuron cells, and the surrounding supporting cells. 
One of the main advantages of this method is that it simplifies the organ culture steps without compromising the quality of the explants. All three turns of the organ of Corti are attached to the bottom of the chamber, which facilitates in vitro experiments and the comprehensive analysis of the explants. We provide some examples of cochlear images from different experiments with live and fixed explants, demonstrating that the explants retain their structure despite exposure to ototoxic drugs. This optimized protocol can be widely used for the integrative analysis of the mammalian cochlea.

Whole Neonatal Cochlear Explants as an In vitro Model

The current protocol updates previous protocols and incorporates relatively straightforward approaches for culturing high-quality cochlear explants. This provides reliable data acquisition and high-resolution imaging in live and fixed cells. This protocol supports the ongoing trend of studying inner ear cells.

Explantes cocleares neonatales enteros como modelo in vitro

Untreated hearing loss imposes significant costs on the global healthcare system and impairs individuals' quality of life. Sensorineural hearing loss is ...

A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection

We present two minimally invasive microsurgical techniques in rodents for specific drug delivery into the middle ear so that it may reach the inner ear. The first procedure consists of perforation of the tympanic bulla, termed bullostomy; the second one is a transtympanic injection. Both emulate human clinical intratympanic procedures.
Chitosan-glycerophosphate (CGP) and Ringer&#180;s Lactate buffer (RL) were used as biocompatible vehicles for local drug delivery. CGP is a nontoxic biodegradable polymer widely used in pharmaceutical applications. It is a viscous liquid at RT&#160;but it congeals to a semi solid phase at body temperature. RL is an isotonic solution used for intravenous administrations in humans. A small volume of this vehicle is precisely placed on the Round Window (RW) niche by means of a bullostomy. A transtympanic injection fills the middle ear and allows less control but broader access to the inner ear.
The safety profiles of both techniques were studied and compared by using functional and morphological tests. Hearing was evaluated by registering the Auditory Brainstem Response (ABR) before and several times after microsurgery. The cytoarchitecture and preservation level of cochlear structures were studied by conventional histological techniques in paraformaldehyde-fixed and decalcified cochlear samples. In parallel, unfixed cochlear samples were taken and immediately frozen to analyze gene expression profiles of inflammatory markers by quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR).
Both procedures are suitable as drug delivery methods into the mouse middle ear, although transtympanic injection proved to be less invasive compared to bullostomy.

A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection

Two microsurgery approaches for local drug delivery to the inner ear are described here and compared in terms of impact on hearing parameters, cochlear cytoarchitecture and expression of inflammatory markers.

Estudio comparativo de los métodos de entrega de fármacos dirigidos a la oreja del ratón interno: Bullostomy<em&gt; vs.</em&gt; Inyección transtimpánicos

We present two minimally invasive microsurgical techniques in rodents for specific drug delivery into the middle ear so that it may reach the inner ear. ...

Biología

Extracting the Cochlea from a Human Temporal Bone: A Cadaveric Protocol

The extraction of the cochlea from a cadaver human temporal bone may be required for different studies of the inner ear. For histological evaluations, the inner ear must be extracted from the temporal bone to facilitate histologic processing; likewise, some micro-computed tomography devices are too small to accommodate the complete temporal bones; additionally, the image quality can be enhanced when the cochlea is isolated.
The inner ear is located within the petrous part of the temporal bone. The inner ear can be divided into the osseous labyrinth or otic capsule and the membranous labyrinth inside the otic capsule. Furthermore, the inner ear can be divided into the vestibular system (the semicircular canals and the vestibule) and the cochlea. The appreciation of the location and orientation of the cochlea within the temporal bone is difficult, as it is embedded within bony structures and thus cannot be directly visualized. Nevertheless, there are distinct anatomical structures that can help guide the process to allow a reliable drill-out of the cochlea. The landmarks in the posterior parts of the cochlea are the facial nerve, semicircular canals, and the vestibule. In the middle, the inferior borders of the cochlea are identified by the round window and the basal turn of the cochlea. In the anterior border, one encounters the carotid artery; the landmark for the superior border is the genicular ganglion (GG) of the facial nerve. The medial structures are determined by the locations of the internal auditory canal, the superior semicircular canal, and the canal of the internal carotid artery.
In this article, we present a method for extracting the cochlea reliably out of the temporal bone by drill-out while following several anatomical landmarks.

<meta charset="utf8"></head><body>Extracción de la cóclea de un hueso temporal humano: un protocolo cadavérico

This article presents a reliable method for extracting the human cochlea from the cadaver temporal bone by drill-out while following distinct anatomical landmarks.

Extracción de la cóclea de un hueso temporal humano: un protocolo cadavérico

The extraction of the cochlea from a cadaver human temporal bone may be required for different studies of the inner ear. For histological evaluations, the ...