O projeto atual foi financiado pela Universidade de Kansas prova de conceito de fundo. Gostaríamos de agradecer a equipe de enfermagem no KUMC (Kansas City, KS) para nos auxiliar na obtenção de humanos cordões umbilicais e David Jorgensen para ajudar com culturas cochleae.

Todos os procedimentos, incluindo a eutanásia de animais, foram conduzidos de acordo com o cuidado de Animal institucional e uso Comité (IACUC) protocolo aprovado (cálice #2014-2234) da Universidade de Kansas Medical Center (KUMC).
Nota: HWJCs foram isoladas de humanos cordões umbilicais que foram doados pelos pacientes que forneceram consentimento informado e as amostras foram utilizadas em conformidade com os protocolos aprovados pelo Comité de assuntos humanos da Universidade de Kansas (KU-IRB #15402).
1. osso Temporal colheita e isolamento da cóclea
<ol>
	<li>Decapitar um rato apropriadamente sacrificado, 15 semanas cortando entre o crânio e a primeira vértebra cervical com uma afiada tesoura cirúrgica e, em seguida, pulverizar a cabeça com etanol a 70% para desinfetar (figura 1A).</li>
	<li>Bissetriz do crânio em um médio sagital usando o mesmo par afiado de uma tesoura cirúrgica (figura 1B-C, linhas tracejadas).</li>
	<li>Remova o tecido cerebral do crânio utilizando um par de pinças (Figura 1, ponta de seta).</li>
	<li>Identifica o osso temporal através da presença do auditivo e o nervo vestibular raízes (Figura 1E-F, pontas de seta) no interior do crânio e do canal auditivo do exterior (Figura 1F, seta). Corte com cuidado através do crânio para isolar o osso temporal do resto do crânio (Figura 1). Nota: Em alguns casos, pode ser possível intrometer-se delicadamente os circundantes ossos do crânio longe do osso temporal sem a necessidade de corte.</li>
	<li>Insira a pinça bem a abertura do canal auditivo aproximadamente 5 mm (Figura 1 H), então as dicas não arrisque puncionando a cóclea. Suavemente quebrar o osso do bulla (Figura 1 H, top vermelha área sombreada) então ele fraturas (linhaFigura 1I, vermelha pontilhada). Nota: Se necessário, um microscópio de dissecação pode ajudar neste processo para garantir a colocação exata e manipulação de instrumentos.</li>
	<li>Usando a pinça fina, embora bisbilhotar o restante o bulla do osso temporal, expondo o labirinto ósseo da cóclea (Figura 1J, suportes de vermelhos).</li>
	<li>Coloque um prato de Petri grande o suficiente para segurar o osso temporal (por exemplo, 30 mm ou maior) por baixo da dissecação campo de visão do escopo e preenchê-lo com suficiente fosfato tamponado salino (PBS) para cobrir o osso temporal (por exemplo, 0,5 a 1 mL).</li>
	<li>Mergulhe no osso temporal com o bulla removido na PBS com as janelas ovais e redondas da cóclea virada para cima. Nota: A remoção do sistema vestibular da cóclea não é necessária, como o sistema vestibular serve bem como um identificador para a manipulação e orientando recursos da cóclea (Figura 1I e Figura 2B).</li>
	<li>Usando um par de microfuros de fórceps, remova a artéria estapédica, que atravessa o estribo.</li>
	<li>Inserir uma ponta da pinça ultra fina através do arco do estribo onde passou a artéria e delicadamente Levante o estribo para cima. O estribo desarticulado deve levantar fora a janela oval.</li>
	<li>Usando uma ponta da pinça ultra fina, fure o oval e redonda windows.</li>
	<li>Usar um 1X PBS preenchido 28,5 calibre seringa com tubo (diâmetro interno de 0,28 mm, diâmetro externo 0,61 mm) anexada, posicione o tubo sobre a janela oval (Figura 2A -B). Nota: A abertura do tubo pode ser manipulada usando fórceps ultra-fina, ajudando a garantir a colocação exata.<ol>
		<li>Use uma seringa fresca e tubulação para cada cóclea.</li>
	</ol>
	</li>
	<li>Perfundir 2ml de 10%-antibiótico antimicótico (anti-anti) e 10% penicilina-estreptomicina (pen-estreptococos) em PBS através da cóclea mais 5 min para remover a perilinfa. Perfusing muito rápido com muita pressão danificará as belas estruturas dentro da cóclea. Nota: Manualmente, dirigindo a seringa com a mão para perfundir o fluido através da cóclea é eficaz, embora uma bomba de perfusão pode ser usada como uma alternativa.<ol>
		<li>Se perfusing manualmente, use um par de bem, fecho automático fórceps para estabilizar a cóclea (Figura 2A) durante a perfusão, agarrando-se a porção vestibular do osso temporal. Nota: Embora não seja obrigatório, isto deixa ambas as mãos livres para lidar com instrumentos; uma mão pode conduzir a seringa (Figura 2A), enquanto o outro pode posicionar o tubo adequadamente (Figura 2B).</li>
		<li>Desta etapa da frente, tome cuidado para evitar expor a cóclea desnecessariamente ao ar. Introdução de bolhas de ar nos espaços fluidos irá bloquear a circulação do fluido, e mais a secar irá danificar as belas estruturas dentro da cóclea.</li>
	</ol>
	</li>
	<li>Remover e descartar qualquer restantes fragmentos de tecidos e ossos de músculo usando pinça fina.</li>
	<li>Se necessário, armazene cochleae isolado a 4 ° C em anti-anti 10% e 10% solução de caneta-estreptococos em PBS durante sete dias com mudanças regulares de solução antibiótica cada 48 h.</li>
</ol>
2. coclear processamento
<ol>
	<li>Decellularization
	<ol>
		<li>Para cada cóclea, encha um frasco de cintilação de vidro de 20 mL com um 1% de sódio Dodecil sulfato solução (SDS) em água deionizada (DI), que é usada para decellularize a cóclea.</li>
		<li>Corte a ponta de uma pipeta de transferência de 7 mL plástico usando uma lâmina de barbear, de modo que a abertura é grande o suficiente para uma cóclea a passagem.</li>
		<li>Com a pipeta de transferência suavemente elaborar a cóclea na pipeta então só passa por alguns milímetros da borda de corte.</li>
		<li>Expulse da cóclea para o frasco de solução cintilação SDS 1%.</li>
		<li>Circule de 2 mL de SDS 1% em água desionizada de (DI) através da cóclea no frasco de cintilação, com uma pipeta de transferência.</li>
		<li>Coloque o frasco de cintilação em rotacao e permitir que o frasco de cintilação girar a 10 rpm para 72 h à temperatura ambiente.<ol>
			<li>Altere a solução de SDS 1% cada 24 h ao longo de um período de 72 h. Tenha cuidado quando alterar SDS para Nunca exponha a cóclea ao ar.</li>
		</ol>
		</li>
		<li>Lave a cóclea três vezes por 30 min cada com água.</li></ol></li></ol>Executar as lavagens no frasco de cintilação mesma usado para as acusações de SDS; nesta fase, a cóclea é decellularized.
	
	
	<li>Descalcificação
	<ol>
		<li>Remova a água restante do DI o frasco de cintilação, deixando apenas o suficiente para manter a cóclea submergida.</li>
		<li>Para começar a descalcificação, adicione 5 mL de ácido etilenodiaminotetracético (EDTA) (0,02 M) a 10% em água para o frasco de cintilação que contém a cóclea.</li>
		<li>Circule de 2 mL de EDTA 10% em água DI através da cóclea no frasco de cintilação, com uma pipeta de transferência.</li>
		<li>Coloque o frasco de cintilação no rotador e permitir que o frasco de cintilação girar a 10 rpm para 72 h à temperatura ambiente. Altere a solução de EDTA 10% cada 24 h ao longo de um período de 72 h. Tenha cuidado quando alterar soluções para que a cóclea não é exposta ao ar.</li>
		<li>Enxágue a cóclea 3 vezes durante 2 h com PBS 1x; nesta fase, a cóclea é descalcificada.</li>
	</ol>
	</li>
	<li>Armazenamento
	<ol>
		<li>Armazenar os cochleae por até 72 h em uma solução 10% anti-anti, 10% caneta-estreptococos em PBS a 4 ° C. Nota: Os períodos de armazenamento podem ser possíveis com mudanças regulares de solução antibiótica; no entanto, armazenagem prolongada de cochleae transformados não foi validada neste protocolo.</li>
	</ol>
	</li>

3. a colheita e a expansão de hWJCs
<ol>
	<li>Isole o hWJCs de acordo com protocolos previamente publicados22. Brevemente, segmento cordões umbilicais em seções de 3 cm.</li>
	<li>Com cuidado faça uma incisão superficial com um bisturi longitudinalmente em cada segmento de cordão umbilical para desenrolar e expor a geleia de Wharton, do cordão umbilical. Use a pinça para remover os vasos sanguíneos.</li>
	<li>Lave o cordão umbilical segmentos duas vezes com suficiente PBS (por exemplo, 40 mL em uma placa de Petri de 60 mm) para submergir o tecido. Picar finamente segmentos de tecido com um bisturi. Digerir o tecido em uma placa de Petri com 50 mL de meio de digestão de 100mm (colagenase tipo 2 de 0,2%, 1% penicilina-estreptomicina, em baixo-glicose médio (DMEM modificado águia de Dulbecco)).</li>
	<li>Coloque o tecido em digerir médio em um agitador orbital a 50 rpm durante a noite em um 5% CO2, incubadora 37 ° C. No dia seguinte, diluir meio digestão na proporção de 01:16 em 2% antibiótico antimicótico em PBS, pelota e células por centrifugação a 500 x g à temperatura ambiente (~ 27 ° C) por 10 min. Discard sobrenadante.</li>
	<li>Resuspenda pelotas em Mesenchymal Stem Cell crescimento médio (MSCGM) e recombinar as células. Células de placa em uma densidade de 7 x 103 células/cm2 em cultura de tecidos trataram frascos de T-75. Cultura HWJCs em MSCGM e expandir a passagem 5 para experimentos. Nota: HWJCs pode ser sub cultivadas em T-frascos maiores (p. ex., T-150, T-300) quando a passagem para aumentar o rendimento de hWJCs para experimentos.</li>
</ol>
4. infusão de hWJCs em Decellularized Cochleae
<ol>
	<li>Preparar cochleae decellularized
	<ol>
		<li>Utilizando técnica asséptica, lave cochleae armazenado em 1X PBS três vezes para 30 min cada.</li>
		<li>Transferir cochleae em separado poços de uma placa de 24 e adicionar 1 mL de 37 ° C pré aquecido MSCGM.</li>
		<li>Incube cochleae em um 5% CO2, incubadora de cultura de célula de 37 ° C por um período mínimo de 1 h antes da infusão de células.</li>
	</ol>
	</li>
	<li>Dissociar a hWJCs e ressuspender
	<ol>
		<li>HWJCs de lavagem com 37 ° C pré aquecido 1X PBS duas vezes.</li>
		<li>Adicionar suficiente 37 ° C pré aquecido tripsina com 0,5% de EDTA para cobrir a superfície do vaso de célula.</li>
		<li>Incube a hWJCs para até 5 min em um 5% CO2, incubadora de cultura de células de 37 ° C.<ol>
			<li>Verifique-se que 90% das células ter retirado a superfície de cultura de células exibindo células sob um microscópio invertido. Nota: As células que se movem quando o navio de cultura suavemente é aproveitado, tem destacado. As células que permanecem estacionárias, não ter destacado.</li>
			<li>Bata levemente os lados do balão para desalojar mais células anexadas.</li>
		</ol>
		</li>
		<li>HWJCs transferência do navio cultura para um tubo cônico de 50 mL, contendo uma quantidade equivalente de MSCGM para o volume de tripsina utilizando técnica asséptica, usado para dissociar as células.</li>
		<li>O hWJCs de pelotas por centrifugação a 500 x g, à temperatura ambiente (~ 27 ° C) por 5 min.</li>
		<li>Aspire o sobrenadante e ressuspender o hWJCs em MSCGM em uma concentração de 500.000 células/mL.</li>
	</ol>
	</li>
	<li>Perfundir cochleae
	<ol>
		<li>Transferência cochleae para uma nova placa de 24 usando uma pipeta de transferência.</li>
		<li>Adicione uma gota de MSCGM para cada cóclea para impedir qualquer cóclea desseque.</li>
		<li>Delicadamente, Oriente a cóclea usando fórceps ultra fino, de modo que as janelas ovais e redondas estão virada para cima. Nota: Tenha muito cuidado ao manusear diretamente cada cóclea cóclea é facilmente marcado.</li>
		<li>Utilizando uma seringa de insulina estéril de 28,5 mm com tubos conectados, elaborar 0,2 mL resuspended hWJCs (100.000 células).</li>
		<li>Usando a pinça bem esterilizada, posicione tubulação sobre a janela oval.</li>
		<li>Delicada e lentamente perfundir a cóclea com 0,2 mL de ressuspensão hWJCs utilizando uma seringa de insulina 28,5 mm conectada à tubulação de polietileno (diâmetro exterior: 0,61 mm, diâmetro interno: 0,28 mm) ao longo de cerca de 5 min.</li>
		<li>Após a perfusão, adicionar 0,8 mL de 37 ° C pré aquecido MSCGM para o bem que contém a cóclea para trazer o volume total no poço de 1 mL.</li>
		<li>Repita as etapas 4.3.3-4.3.7 para cada cóclea, utilizando uma seringa de fresca e tubos cada vez.</li>
		<li>Lugar perfundidos cochleae em um 5% CO2, incubadora de cultura de células de 37 ° C e mudança mídia três vezes por semana.</li>
	</ol>
	</li>
</ol>
5. preservação e colheita de cóclea
Nota: Cochleae podem ser cultivadas e colhidas em qualquer ponto do tempo de post-perfusão de até 30 dias.
<ol>
	<li>Para preservar os cochleae, corrigir em paraformaldeído 4% em 1X PBS durante a noite a 4 ° C, em uma plataforma de balanço.</li>
	<li>Após a fixação, lave cochleae com PBS 1x, três vezes por 5 min cada.</li>
	<li>Gradualmente, desidratar a cóclea com etanol e limpar com um solvente Hidrocarboneto alifático antes de incorporação em parafina.</li>
	<li>Secção de amostras a uma espessura de 10 µm usando um micrótomo e montar em vidro corrediças do microscópio. Nota: As amostras agora estão prontas para histológica ou imuno-histoquímica de processamento usando protocolos padrão.</li>
</ol>

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<li>Nonoyama, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Investigation+of+the+ototoxicity+of+gadoteridol+%28ProHance%29+and+gadodiamide+%28Omniscan%29+in+mice%5BTitle%5D)+AND+%22Acta+Otolaryngol%22%5BJournal%5D)">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/pubmed?term=((Rhesus+Cochlear+and+Vestibular+Functions+Are+Preserved+After+Inner+Ear+Injection+of+Saline+Volume+Sufficient+for+Gene+Therapy+Delivery%5BTitle%5D)+AND+%22J+Assoc+Res+Otolaryngol%22%5BJournal%5D)">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/pubmed?term=((The+bioactivity+of+cartilage+extracellular+matrix+in+articular+cartilage+regeneration%5BTitle%5D)+AND+%22Adv+Healthc+Mater%22%5BJournal%5D)">The bioactivity of cartilage extracellular matrix in articular cartilage regeneration.</a> Adv Healthc Mater. 4 (1), 29-39 (2015).</li>
</ol>

Os autores não têm nada para divulgar.

Temos com sucesso demonstrado que células cocleares nativas podem ser removidas da cóclea através de um processo de decellularization, que permite a utilização da cóclea como um andaime de tecido tridimensional, intrincado. Santi et al. 15 desenvolveu o método inicial para decellularizing cochleae e estimaram com precisão os volumes de muitas estruturas cocleares através com o auxílio de microscopia de luz folha23. Tal trabalho inicial serviu como uma bas...

A cóclea é uma estrutura espiral intrincado encontrada no osso temporal. Ele é composto de um labirinto ósseo exterior e interior, concêntricos labirinto membranoso1. O labirinto membranoso é composto por três espaços fluidos: Scala vestibuli, Scala Scala tympani1e mídia. A mídia scala abriga o epitélio sensorial, que é composto de uma infinidade de tipos de células, mas as células sensoriais de cabelo (HC), que transduce energia mecânica em ondas sonoras para impulsos nervosos2, são de particular interesse. Exposição ao trauma acústico3,4,5, medicação6, doença7,8e envelhecimento9 tudo o que pode resultar em insuficiência auditiva através da morte HC. Perda de cabelo celular em mamíferos é permanente, ao contrário de HCs aviária, que podem se regenerar após lesão10.
Uma variedade de esforços de pesquisa contemporânea procuraram restaurar perdidos HCs, embora as abordagens experimentais específicas variam. Manipulação da expressão do gene no epitélio sensorial e implante de células-tronco diferenciadas fora do corpo são abordagens dominantes no campo, apesar de tem sido métodos de indução que buscam diferenciar as células-tronco em organoids coclear tentativa de11,12,13. Cada uma dessas abordagens é também dependente diretamente as células-tronco, ou as pistas do desenvolvimento, usadas por células-tronco; no entanto, um segundo compartilhados, e potencialmente crítico, o elemento é o ECM da cóclea em si14,15.
O ECM não só fornece suporte físico para as células e tecidos, que inclui uma superfície de adesão celular, sobrevivência, proliferação e migração, mas também tem um papel crítico no desenvolvimento de HCs e a espiral do gânglio15,16 ,17. Naturalmente ocorrendo ECM fornece sinais indutivos que podem orientar a determinação do fenótipo celular e/ou18, de adesão, proliferação e sobrevivência celular. Consequentemente, o uso da cóclea decellularized em combinação com hWJCs cultivadas oferecem uma oportunidade única de explorar o papel da regeneração ECM e HC. HWJCs são um tipo de célula prontamente disponível, sem controvérsias, isolado de humanos cordões umbilicais que se comportar como células-tronco mesenquimais19. HWJCs tem demonstrado a capacidade de diferenciar-se abaixo de20,de linhagens de célula marcante21. Assim, o atual protocolo detalha o isolamento, decellularization e perfusão de cochleae de carcaças de rato C57BL com hWJCs para engenharia de tecidos do ouvido 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

Nos mamíferos, mechanosensory células ciliadas que facilitam a falta de audiência a capacidade de regenerar, que limitou a tratamentos para perda auditiva. Estratégias atuais de medicina regenerativa concentraram-se no transplante de células-tronco ou manipulação genética de em torno de células de suporte na orelha interna para incentivar a substituição de células-tronco danificadas para corrigir a perda de audição. Ainda, a matriz extracelular (ECM) pode desempenhar um papel fundamental na indução e manutenção da função das células de cabelo e não tem sido suficientemente estudada. Usando o coclear ECM como um andaime para cultivar células-tronco adultas pode fornecer insights exclusivos sobre como a composição e arquitetura do ambiente extracelular aids células em sustentar a função auditiva. Aqui nós apresentamos um método para isolar e decellularizing cochleae de ratos para usar como andaimes aceitando perfundidas células-tronco adultas. O protocolo atual, cochleae são isolados da eutanásia de ratos, decellularized e descalcificadas. Depois, células de geleia humana da Wharton (hWJCs) que foram isoladas a partir do cordão umbilical foram cuidadosamente pintadas em cada cóclea. Os cochleae foram usados como biorreatores, e as células foram cultivadas por 30 dias antes de sofrer a transformação para análise. Decellularized cochleae mantidos identificáveis estruturas extracelulares, mas não revelou a presença de células ou fragmentos visíveis de DNA. Células perfundidas na cóclea invadiram a maior parte do interior e exterior da cóclea e cresceram sem incidentes em uma duração de 30 dias. Assim, o método atual pode ser usado para estudar como coclear ECM afeta células desenvolvimento e comportamento.

Usando os métodos apresentados aqui, sucesso decellularization de cochleae foi avaliado examinando-se a presença ou ausência de DNA através de 4', coloração 6-diamidino-2-phenylindole (DAPI). Cochleae foram considerados totalmente decellularized, se o DNA não foi identificado dentro da cóclea decellularized. Uma cóclea nativa de uma experiência anterior que não passam por decellularization ou descalcificação foi usada como controle positivo para ilustrar as estruturas e célu...

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

Um protocolo para Decellularizing Cochleae Mouse para engenharia de tecidos do ouvido interno

O objetivo do presente protocolo é demonstrar um método eficaz para decellularize e descalcificar cochleae rato para utilização como andaimes para aplicações de engenharia de tecidos.

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

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Research

JoVE Journal

Bioengineering

10.8K visualizações.  University of Kansas Medical Center. O objetivo do presente protocolo é demonstrar um método eficaz para decellularize e descalcificar cochleae rato para utilização como andaimes para aplicações de engenharia de tecidos.

Vídeo: 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 da embrionárias mouse cocleares explantes e Gene Transferência de Eletroporação

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

Biologia do Desenvolvimento

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.

Longo prazo Time Lapse Imagem do mouse 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 ...

Neurociência

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.

A abordagem do rato Round-janela para ototóxicos Delivery Agent: A rápida e técnica confiável para induzir a degeneração das células da cóclea

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.

Avaliação da Planar-Cell-Polaridade fenótipos em Ciliopathy rato 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.

Crioseccionamento e imunocoloração do tecido do ouvido interno de camundongos: dos estágios embrionário ao adulto

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.

Etiquetagem Dextran e Captação em Células Capilares Murina Coclear

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

Bioengenharia

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.

Preparação da superfície coclear no mouse 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 Neonatais Totais 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.

Um Estudo Comparativo de drogas métodos de entrega direcionados à orelha de rato Inner: Bullostomy<em&gt; Versus</em&gt; Injeção transtimpânica

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. ...

Biologia

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>Extração da cóclea de um osso temporal humano: um 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.

Extração da cóclea de um osso temporal humano: um 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 ...