Esta pesquisa é apoiada pelo Programa CounterACT, National Institutes of Health (NIH), Gabinete do Diretor, e do Instituto Nacional de Ciências de Saúde Ambiental (NIEHS) Número Grant U54 ES015678 (CWW). SA também é suportado pelo Hospital Infantil Escola Colorado / Colorado de Minas Collaboration Award Pilot # G0100394 e Hospital Infantil de Colorado Research Institue Award Pilot # G0100471.

1. Culturas de cardiomiócitos de rato <ol><li> Todos os experimentos foram realizados sob protocolos aprovados pelo cuidado com os animais e uso comitê institucional, IACUC. </li><li> Obter cardiomiócitos de ratos a partir de corações (ventrículos) de ratos machos (240-260 g), utilizando métodos descritos anteriormente 4. Resumidamente, anestesiar os animais com uma injecção intraperitoneal de pentobarbital (100 mg / kg; confirmar anestesia pelo método de aperto do dedo do pé) e, em seguida, remover o coração em 10.0 ml, 1 mM de Ca 2 + contendo tampão Krebs-Ringer, pH 7,4. </li><li> Lavar os corações 5-6x para remover o sangue e, em seguida, mudar para um buffer de Ca 2 + livre Krebs Ringer contendo 0,02% de protease e 0,06% de colagenase A (5,0 ml / coração). Incubar os corações nesta solução durante 10-15 min a 37 ° C com agitação ocasional. </li><li> Após 10-15 minutos, lave a solução enzimática com Ca 2 + tampão Krebs Ringer livre para um adicional de 5 min. Solte as células do tecido flácido nosção de uma pipeta 25 ml pipetando a suspensão para cima e para baixo várias vezes. </li><li> Separam-se as células a partir de tecido por filtração através de uma malha de nylon de 70 ^ m e permitir-lhes que se contentar em tampão Krebs-Ringer contendo 0,1 mM de Ca 2 +. Suspende-se o sedimento de células em tampão 1,0 ml de Krebs Ringer contendo 0,2 mM Ca 2 +. </li><li> Camada cuidadosamente ao longo de 5,0 ml de 60 ug / ml de albumina de soro bovino, BSA, em 15 ml de tubos de centrífuga, para separar os cardiomiócitos de nonmyocytes e permitir-lhes que se contentar com 30 min. Os cardiomiócitos são mais pesados ​​e se mover para a parte inferior do tubo. Retire cuidadosamente as células sobrenadante e mídia. Repetir esta etapa para purificar mais uma vez as células. </li><li> Gradualmente transição por lavagem (em etapas usando 0,25 mM, 0,5 mM, 0,75 mM, e 1,0 mM Ca 2 + contendo tampão) os miócitos / cardiomiócitos ventriculares para mM Ca 2 + contendo tampão 1,0 e novamente suspensas em meio ACCT consistindo de Dulbecco Modified Eagle Médium , DMEM containing de 2 mg / ml de BSA, 2 mM de L-carnitina, creatina 5 mM, 5 mM de taurina, 100 UI / ml de penicilina, e 100 ug / ml de estreptomicina. </li><li> Placa cardiomiócitos em meio ACCT, a uma densidade de 100 a 150 células / mm 2 a 100 mm, ou 35 milímetros laminina pratos de cultura de plástico ou 40 x 22 mm de lamelas de vidro pré-revestidas com laminina (1 mg / cm 2) revestido. Após 1 h, lavar os pratos com 2,0 ml ACCT para remover as células que não estão ligados. Adicionar 10,0 ml de meio fresco e incuba-se as células. </li></ol> 2. Diferenciada interface ar-líquido (ALI) Cultura da Human Airway células basais epiteliais <ol><li> Procure tecidos humanos traqueobrônquicas de Doenças National Research Interchange sob Institutional Review Board protocolos aprovado. Colheita celular e realizar a cultura utilizando um procedimento publicado 5,6. Este procedimento utiliza culturas de células como eles são um modelo preciso para exposições de inalação humana para TICs, no entanto, se necessário, pode-se isolar e crescer a ALI moutilizar e / ou células epiteliais das vias respiratórias do rato 7,8. </li><li> Resumidamente, fazer pequenos pedaços (~ ¼ de polegada) de tecido após a remoção de todos os nódulos linfáticos e dos tecidos conjuntivos. Lave o tecido várias vezes em solução de Lactato de Ringer. Adicionar os tecidos para um tubo de 50 ml e adicionar solução de protease (1% Protease/0.01% de DNase em meio mínimo essencial, MEM, a proporção de tecido fluido (1:10)). </li><li> Balance o tecido a 4 ° C durante a noite. Terminar a dissociação do tecido pela adição de 10% de soro fetal bovino. Raspar a superfície epitelial com um bisturi cirúrgico e recolher as células por centrifugação (2000 rpm durante 10 min). </li><li> As células em placas a uma passagem em poços com tampa revestida com colagénio em meio especial ALI preparados como descrito em detalhe por Fulcher et al 9. Cultura durante 5 dias antes de mudar para a interface ar-líquido (ALI), removendo meios de apicais. </li><li> Alimentar as células usando meios de LPA em dias alternados, e permitir que as células se diferenciam em 2-3 semanas adicionais (observar numerosos cílios batendo e secreção de muco) antes de realizar exposições. Lavar a superfície apical com PBS quente juntamente com as mudanças de mídia. </li></ol> 3. Cloro exposição <ol><li> Contêm o sistema de exposição de cloro (CES) dentro de uma capa química qualificado com uma velocidade nominal de 100 fpm operacional que proporciona o confinamento secundário necessário para evitar a exposição do pessoal no caso de fugas acidentais do cloro. </li><li> Operar o sistema sob uma ligeira pressão positiva (0,5 polegadas de água). O ar seco é alimentado a 15 L / min, e um nível adequado de cloro é alimentado para atingir a concentração final desejada de cloro. As câmaras têm uma tampa de bloqueio com 4 mini fechaduras BCU e uma junta de silicone de baixa dureza para fornecer o selo de pressão. </li><li> Entregar a mistura Cl 2 através de um controlador de fluxo de massa. O CES usa um cilindro de gás comprimido que contém 1,0% de Cl 2, em azoto seco. </li><li> Regular o fluxo de ar de diluição com oprojetado painel de controle e regulação semelhante a concentração Cl 2 entregues para as câmaras de exposição. A bomba de amostragem baixo volume puxa a exaustão das câmaras em um analisador de cloro para monitorar as concentrações que são então gravados em um registrador de dados conectado ao analisador. </li><li> Medir as taxas de fluxo dentro das câmaras antes da exposição utilizando um medidor de fluxo de garantir taxas de fornecimento e exaustão iguais. </li><li> Prepare as culturas de células para a exposição, removendo os meios de sobrenadante e adicionando meio fresco (mídia basolateral em culturas ALI). Qualquer pré-tratamentos com agentes pode ser realizada nesta altura. </li><li> Expor as culturas de células com LPA epiteliais das vias respiratórias a Cl 2 de gás (50, 100, ou 300 ppm, durante 30 min) nas duas câmaras de polissulfona biocontainment seladas. Os cardiomiócitos (submersas ou culturas confluentes nas membranas) são expostas a 50 ou 100 ppm Cl 2 durante 15 min. </li><li> Após a exposição lavar as câmaras com ar até o Cl 2 nível cai abaixo de 1 ppm e pode ser aberta com segurança para remover as células (no prazo de 5 min). </li></ol> 4. Resistência elétrica transepitelial (TER) Medição <ol><li> Meça o TER de ar-líquido-interface, ALI, culturas usando um voltohmmeter epitelial com um par de eletrodos de cloreto de &quot;pauzinho&quot; de prata. </li><li> Equilibrar o eletrodo pauzinho na ALI media 15 min antes do uso. </li><li> Adicionar mídia quente para a apical (1,0 ml) e basolateral (2,0 ml) de superfície e medir o TER de usar os eletrodos pauzinho do voltohmmeter. </li><li> Mergulhe o braço mais curto do eletrodo na mídia apicais e quanto mais tempo o braço na mídia basolateral. Clique no botão &quot;medida&quot; no voltohmmeter para avaliar a resistência elétrica. </li><li> O voltohmmeter tem a opção de medir ohms ou k ohms. Subtraia a resistência através de um livre de células de apoio a cultura da resistência medida em cada camada de células para produzir o transresistência epitelial (TER). </li></ol> 5. Medição Caspase <ol><li> Adicionar mídia ALI fresco (2,0 ml) para a exposição de pós superfície basolateral da célula e incubar à temperatura ambiente. Colete mídia sobrenadante a 4 e 24 horas. </li><li> Medir a caspase 3/7 actividade nas sobrenadantes de meios utilizando uma caspase comercial 3/7 Kit de ensaio. </li></ol> 6. Western Blot e Imunocitoquímica <ol><li> Realizar as transferências Western usando lisados ​​de células, como anteriormente descrito 10. Suspender o lisado de proteína (20 mg) em 5x tampão de amostra reduzida e deixe ferver por 5 min. Sujeitou-se o lisado de proteína a SDS-PAGE (4-15%) e transferir as proteínas separadas para uma membrana de nitrocelulose por transferência electroforética. </li><li> Bloquear a ligação não-específica por incubação da membrana com leite a 5% em tampão de lavagem (PBS + 0,1% de detergente) e sondar as membranas com anticorpos primários contra SERCA2 ou actina sarcomérica na diluição 1:1.000, durante a noite a 4 ° C. Em seguida, lavar e incubar as membranas com os respectivos anticorpos secundários conjugados com peroxidase e desenvolvimento para a detecção, tal como descrito antes de 10 usando o kit comercial de detecção com peroxidase. </li><li> Para imunocitoquímica, o tratamento de células vivas cultivadas em insertos ou lamelas de vidro em placas de 6 poços com 0,4% de Triton-X-100 em tampão de citrato de sódio 10 mM durante 20 min, após a lavagem com PBS. </li><li> Bloco de ligação não específica por tratamento das células com 5% de soro de burro, durante 20 min, e em seguida, incuba-se as células com IgG não específicas ou os anticorpos primários individuais específicos para actina sarcomérica ou Ki-67. </li><li> Lavar as células com PBS e incuba-se com anticorpos secundários conjugados fluorescentes para detectar o anticorpo primário e um corante nuclear (1 ug / ml de DAPI). </li><li> Lavar com PBS e montar lamelas usando a mídia de montagem comerciais. </li><li> Visualizar a coloração utilizando um microscópio de fluorescência. </li></ol>

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The authors declare that they have no competing financial interests.

O tipo mais comum de exposição tóxica aguda ocorre quando se respira um produto químico venenoso para os pulmões. Estes produtos químicos também podem ser tomadas rapidamente na corrente sanguínea e pode afetar outros órgãos, como o cérebro eo coração. Inalação de toxicidade de vários agentes que utilizam modelos animais são amplamente estudado e relatado, no entanto, os mecanismos são menos bem compreendidas. Este é um grande obstáculo no desenvolvimento de terapias eficazes. Ausência de em</em...

A exposição a produtos químicos tóxicos inalados (tiques) / gases, tais como o cloro (Cl 2) continua a ser um problema de saúde em curso em exposição acidental, assim como no seu uso potencial como um agente de ameaça química. Embora os pulmões são o principal alvo, órgãos como o coração eo cérebro também são afetados 1-3. Modelos in vivo são geralmente utilizados para testes de toxicidade de TICs, mas em ensaios in vitro para avaliação de toxicidade são mais simples, mais rápido e mais rentável. In vitro modelos também permitem extensa investigação de interações célula-agente que podem ser difíceis de avaliar in vivo. Tais sistemas de exposição in vitro são raros e, além disso, em alguns dos modelos convencionais, onde os agentes tóxicos são adicionadas ao meio de cultura no qual as células são submergidas, as propriedades dos agentes pode mudar devido a interacções e ligação de componentes do meio. Em tais cenários sistemas de cultura de células, como a interface ar-líquido (ALI) culturas de células primárias das vias respiratórias humanas epiteliais, aqui propostas, que podem ser diretamente expostos a agentes gasosos pode ser promissor. As células epiteliais que revestem as vias aéreas são as primeiras linhas de defesa contra substâncias químicas tóxicas inaladas. O epitélio das vias respiratórias humanas forma uma barreira física entre o lúmen e as células subjacentes no pulmão e participa na resposta do pulmão. Ela produz um número de citocinas e outros agentes pro-e anti-inflamatórias bem como segrega líquido superfície muco / vias aéreas (ASL) que cobre o epitélio. Uma das limitações em convencional submerso em sistemas de cultura in vitro é também que o ASL e muco que cobre a superfície epitelial é removido ou diluído. Isto não reflecte o estado fisiológico das células epiteliais pulmonares, que são expostas ao ar. Assim, um ideal sistema in vitro para testes de toxicidade TIC deve replicar esta arquitetura. Há um grande interesse no desenvolvimento rápido de triagem métodos que predizem em toxicidade in vivo. As células epiteliais cultivadas no ALI diferenciar e têm estruturas e funções bem diferenciadas em comparação com células cultivadas submersas e servir um modelo superior das vias respiratórias. Neste estudo, descrevemos o uso de cultura de ar-líquido interface de vias respiratórias humanas (traqueobrônquicos) células epiteliais para testar a toxicidade do gás inalado venenoso e compará-lo com uma cultura de células submersa de cardiomiócitos, portanto, estudar um outro alvo importante de toxicidade.

<table border="0" cellpadding="0" cellspacing="0" width="1128">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Name</td>
			<td style="width:329px;">Company</td>
			<td style="width:147px;">Catalog Number</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Rats</td>
			<td style="width:329px;">Harlan Laboratories</td>
			<td>Sprague-Dawley&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Pentobarbital</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">P3761</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Chlorine</td>
			<td style="width:329px;">AirGas, Inc</td>
			<td style="width:147px;">X02NI99CP163LS1</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Caspase 3/7 kit&#160;</td>
			<td style="width:329px;">Promega</td>
			<td style="width:147px;">G8091</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Epithelial voltohmmeter and chopstick electrode</td>
			<td style="width:329px;">World Precision Instruments</td>
			<td style="width:147px;">EVOM and STX2</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Snapwell inserts</td>
			<td style="width:329px;">Corning</td>
			<td style="width:147px;">07-200-708</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">70 micron nylon cell strainer</td>
			<td style="width:329px;">Corning</td>
			<td style="width:147px;">#352360</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polysulfone biocontainment chambers&#160;</td>
			<td style="width:329px;">BCU, Allentown Cage Equipment</td>
			<td style="width:147px;">BCU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td style="width:329px;">Life technologies</td>
			<td style="width:147px;">12491-015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Sarcomeric actin antibody</td>
			<td style="width:329px;">Abcam Cambridge, MA</td>
			<td style="width:147px;">ab28052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SERCA2 antibody</td>
			<td style="width:329px;">&#160;Affinity Bioreagents, Golden, CO</td>
			<td style="width:147px;">MA3-9191</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ki-67 antibody</td>
			<td style="width:329px;">&#160;Dako, Carpinteria, CA</td>
			<td style="width:147px;">M7248</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa-488-conjugated secondary antibody</td>
			<td style="width:329px;">&#160;Invitrogen, Grand Island, NY</td>
			<td style="width:147px;">A11029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">BSA</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">A9418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Carnitine</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">C0283</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Taurine</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">T8691</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Creatinine</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">C6257</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Krebs Ringer Buffer</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">K4002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Protease</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">P5147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Collagenase</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">C6885</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; DNAase</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">DN-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Lactated Ringer solution</td>
			<td style="width:329px;">Abott Laboratories</td>
			<td style="width:147px;">7953</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Donkey serum</td>
			<td style="width:329px;">Fisher Scientific</td>
			<td style="width:147px;">017-000-001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">PBS, phosphate buffered saline</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">D1408</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">4-15% SDS-PAGE gels</td>
			<td style="width:329px;">Bio-Rad</td>
			<td style="width:147px;">456-1083</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Nitrocellulose membrane</td>
			<td style="width:329px;">Bio-Rad</td>
			<td style="width:147px;">162-0115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Dergent, Tween&#160;</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">P1379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Peroxidase detection kit</td>
			<td style="width:329px;">Pierce</td>
			<td align="right" style="width:147px;">3402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">DAPI</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">D9542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Mounting media, Fluormount G</td>
			<td style="width:329px;">eBiosciences</td>
			<td style="width:147px;">00-4958-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Sodium citrate</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">71497</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Collagen</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">C7521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">MEM</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">M8028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Laminin</td>
			<td style="width:329px;">BD biosciences</td>
			<td style="width:147px;">354259</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Penicillin/Streptomycin</td>
			<td style="width:329px;">Life Technologies</td>
			<td style="width:147px;">15070063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">FBS</td>
			<td style="width:329px;">Gibco</td>
			<td style="width:147px;">200-6140AJ</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro cell culture model for toxic inhaled chemical testing

As culturas celulares são indispensáveis ​​para desenvolver e estudar a eficácia de agentes terapêuticos, antes da sua utilização em modelos animais. Nós temos a capacidade única de modelar células do epitélio das vias aéreas humana e do músculo cardíaco bem diferenciadas. Esta poderia ser uma ferramenta de valor inestimável para estudar os efeitos deletérios de produtos químicos tóxicos inalados, tais como o cloro, que normalmente podem interagir com as superfícies das células, e formam vários subprodutos quando reagem com água, e limitar os seus efeitos em culturas submersas. Nosso modelo utilizando culturas de células epiteliais das vias aéreas o bem diferenciadas em interface ar-liqiuid contorna essa limitação, bem como fornece uma oportunidade para avaliar mecanismos críticos de toxicidade de potenciais químicos inalados venenosas. Descrevemos aprimorada perda da integridade da membrana, liberação caspase e morte sobre química inalado tóxico, como a exposição de cloro. Neste artigo, propomos métodos para modelar exposição de cloro no coração de mamíferos e das vias aéreas epitelial cvaras de cultura e testes simples para avaliar seu efeito sobre esses tipos de células.

Haste primária cardiomiócitos em forma anexar em matrizes de laminina e se espalhar e se diferenciar em culturas confluentes (Figura 1A e sua inserção). Estas células foram adicionalmente caracterizados com base na expressão de actina e sarcomérica SERCA2 (Figuras 1B e 1C). Rato cardiomiócitos são altamente suscetíveis à toxicidade de cloro como 15 minutos de exposição a 100 ppm de cloro causou extensos arredondamento celular e morte em culturas submersas e...

Watch this Scientific Journal Video about In vitro Cell Culture Model for Toxic Inhaled Chemical Testing at JoVE.com

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Este protocolo destina-se a demonstrar o método de exposição de culturas de células a produtos químicos tóxicos inalados. Exposição de diferenciado interface ar-líquido (LPA) culturas de células epiteliais das vias aéreas oferece um modelo único de exposição das vias aéreas de gases tóxicos como o cloro. Nesse artigo, descrevem efeito de exposição ao cloro em culturas de interface ar-líquido de células epiteliais e de cultura submersa de cardiomiócitos. Em sistemas de exposição in vitro permitem estudos sobre os mecanismos importantes para avaliar as vias que poderia, então, ser utilizados para o desenvolvimento de novos agentes terapêuticos.

A cultura in vitro modelo celular para Toxic Chemical Testing Inhaled

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to ...

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Research

JoVE Journal

Bioengineering

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

10.6K visualizações.  University of Colorado.  Este protocolo destina-se a demonstrar o método de exposição de culturas de células a produtos químicos tóxicos inalados. Exposição de diferenciado interface ar-líquido (LPA) culturas de células epiteliais das vias aéreas oferece um modelo único de exposição das vias aéreas de gases tóxicos como o cloro. Nesse artigo, descrevem efeito de exposição ao cloro em culturas de interface ar-líquido de células epiteliais e de cultura submersa de cardiomiócit...

Vídeo: In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

Dispositivo microfluídicos para Recriando um microambiente do tumor<em&gt; In Vitro</em

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

Bioengenharia

Shape Memory Polymers for Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape.
Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture.
Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions.

A method for developing cell culture substrates with the ability to change topography during culture is described. The method makes use of smart materials known as shape memory polymers that have the ability to memorize a permanent shape. This concept is adaptable to a wide range of materials and applications.

Forma Polímeros Memória da Cultura célula ativa

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk of thromboemboli formation1,2,3. We have developed a method to line the inside surface of Ti tubes with autologous blood-derived human or porcine endothelial progenitor cells (EPCs)4. By implanting Ti tubes containing a confluent layer of porcine EPCs in the inferior vena cava (IVC) of pigs, we tested the improved biocompatibility of the cell-seeded surface in the prothrombotic environment of a large animal model and compared it to unmodified bare metal surfaces5,6,7 (Figure 1). This method can be used to endothelialize devices within minutes of implantation and test their antithrombotic function in vivo. 
Peripheral blood was obtained from 50 kg Yorkshire swine and its mononuclear cell fraction cultured to isolate EPCs4,8. Ti tubes (9.4 mm ID) were pre-cut into three 4.5 cm longitudinal sections and reassembled with heat-shrink tubing. A seeding device was built, which allows for slow rotation of the Ti tubes. 
We performed a laparotomy on the pigs and externalized the intestine and urinary bladder. Sharp and blunt dissection was used to skeletonize the IVC from its bifurcation distal to the right renal artery proximal. The Ti tubes were then filled with fluorescently-labeled autologous EPC suspension and rotated at 10 RPH x 30 min to achieve uniform cell-coating9. After administration of 100 USP/ kg heparin, both ends of the IVC and a lumbar vein were clamped. A 4 cm veinotomy was performed and the device inserted and filled with phosphate-buffered saline. As the veinotomy was closed with a 4-0 Prolene running suture, one clamp was removed to de-air the IVC. At the end of the procedure, the fascia was approximated with 0-PDS (polydioxanone suture), the subcutaneous space closed with 2-0 Vicryl and the skin stapled closed. 
After 3 - 21 days, pigs were euthanized, the device explanted en-block and fixed. The Ti tubes were disassembled and the inner surfaces imaged with a fluorescent microscope.
We found that the bare metal Ti tubes fully occluded whereas the EPC-seeded tubes remained patent. Further, we were able to demonstrate a confluent layer of EPCs on the inside blood-contacting surface.
Concluding, our technology can be used to endothelialize Ti tubes within minutes of implantation with autologous EPCs to prevent thrombosis of the device. Our surgical method allows for testing the improved biocompatibility of such modified devices with minimal blood loss and EPC-seeded surface disruption.

A method for seeding titanium blood-contacting biomaterials with autologous cells and testing biocompatibility is described. This method uses endothelial progenitor cells and titanium tubes, seeded within minutes of surgical implantation into porcine venae cavae. This technique is adaptable to many other implantable biomedical devices.

Autólogas progenitoras endoteliais Cell Technology Semear e Teste de biocompatibilidade para dispositivos Cardiovascular em Modelo Animal Grande

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk ...

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

Sistemas Célula de rotação de cultura para cultura de células humanas: células trofoblásticas Humanos como um modelo

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

Irritação dos olhos Test (EIT) para identificação de perigos de olho irritantes químicos usando reconstruída Humano Córnea-like epitelial (RHCE) Modelo Tissue

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Formação Light-mediada e padronização de hidrogéis para aplicações de cultura celular

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

A<em&gt; In Vitro</em&gt; Modelo Cultura Órgão da Murino Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are an emerging treatment for photoreceptor degenerative diseases that seek to restore vision by artificially stimulating the surviving retinal neurons in the hope of eliciting comprehensible visual perception in patients. Current retinal prostheses have demonstrated success in restoring limited vision to patients using an array of electrodes to electrically stimulate the retina but face substantial physical barriers in restoring high acuity, natural vision to patients. Chemical neurostimulation using native neurotransmitters is a biomimetic alternative to electrical stimulation and could bypass the fundamental limitations associated with retinal prostheses using electrical neurostimulation. Specifically, chemical neurostimulation has the potential to restore more natural vision with comparable or better visual acuities to patients by injecting very small quantities of neurotransmitters, the same natural agents of communication used by retinal chemical synapses, at much finer resolution than current electrical prostheses. However, as a relatively unexplored stimulation paradigm, there is no established protocol for achieving chemical stimulation of the retina in vitro. The purpose of this work is to provide a detailed framework for accomplishing chemical stimulation of the retina for investigators who wish to study the potential of chemical neuromodulation of the retina or similar neural tissues in vitro. In this work, we describe the experimental setup and methodology for eliciting retinal ganglion cell (RGC) spike responses similar to visual light responses in wild-type and photoreceptor-degenerated wholemount rat retinas by injecting controlled volumes of the neurotransmitter glutamate into the subretinal space using glass micropipettes and a custom multiport microfluidic device. This methodology and protocol are general enough to be adapted for neuromodulation using other neurotransmitters or even other neural tissues.

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

This protocol describes a novel method for investigating a form of chemical neurostimulation of wholemount rat retinas in vitro with the neurotransmitter glutamate. Chemical neurostimulation is a promising alternative to the conventional electrical neurostimulation of retinal neurons for treating irreversible blindness caused by photoreceptor degenerative diseases.

Metodologia para neuromodulação Biomimetic químico de Retinas de ratos com o neurotransmissor glutamato em Vitro

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

Um modelo de cultura de matriz-adipócito extracelular humano para estudar crosstalk metabólico de matriz e células

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

In Vitro Model of Human Cutaneous Hypertrophic Scarring using Macromolecular Crowding

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol applies a macromolecular crowding (MMC) technique to mimic this physiological crowding through the addition of neutral polymers (crowders) to cell cultures in vitro. Previous studies using Ficoll or dextran as crowders demonstrate that the expression of collagen I and fibronectin in WI38 and WS-1 cell lines are significantly enhanced using the MMC technique. However, this technique has not been validated in primary hypertrophic scar-derived human skin fibroblasts (hHSFs). As hypertrophic scarring arises from the excessive deposition of collagen, this protocol aims to construct a collagen-rich in vitro hypertrophic scar model by applying the MMC technique with hHSFs. This optimized MMC model has been shown to possess more similarities with in vivo scar tissue compared to traditional 2-dimensional (2-D) cell culture systems. In addition, it is cost-effective, time-efficient, and ethically desirable compared to animal models. Therefore, the optimized model reported here offers an advanced &#8220;in vivo-like&#8221; model for hypertrophic scar-related studies.

This protocol describes the use of macromolecular crowding to create an in vitro human hypertrophic scar tissue model that resembles in vivo conditions. When cultivated in a crowded macromolecular environment, human skin fibroblasts exhibit phenotypes, biochemistry, physiology, and functional characteristics resembling scar tissue.

Modelo in vitro de cicatrizes hipertróficas cutâneas humanas usando aglomeração macromolecular

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol ...