Este trabalho foi apoiado pela Universidade Musculoskeletal Research Center Washington (NIH P30 AR057235), Molecular Imaging Center (NIH P50 CA094056), mechanobiology Formação Grant (NIH 5T32EB018266), NIH R21AR069804, e NIH K01AR069116. Os autores gostariam de agradecer a Patrick Wong por suas contribuições na coleta de dados.

Todos os experimentos com animais foram realizados de acordo com a Universidade de Washington em Comissão Estudos Animais St. Louis. 1. Os animais <ol><li> Obter duas estirpes de ratinhos: 10 semanas de idade BALB / c (n = 6, Balb-H, BALB / cAnNTac) e 10 semanas os animais velhos do factor nuclear kappa-B-luciferase repórter (NF-κβ-luc) criado numa ratinhos BALB / c de fundo (n = 6, ratinhos BALB / c-Tg (Rela-luc) 31Xen). </li><li> Antes da dissecção, sacrificar os animais com CO 2 sobredosagem a uma taxa de fluxo de 2,5-3 L / min durante 5 min, seguido por uma 2 min adicional de tempo de permanência. </li><li> Desinfectar a região exterior do animal por banhar-lo em um banho de etanol a 70% durante 2 min antes da dissecação. </li></ol> 2. Dissecção <ol><li> Fazer um corte vertical e longitudinal na superfície dorsal do rato usando pequenas tesouras de dissecação para expor a cavidade do corpo. </li><li> Fazer dois cortes verticais longitudinais de cada lado da espinha dorsal do animal a partir dea primeira vértebra lombar (L1) até às vértebras caudais (C8). </li><li> Usando um bisturi, uma pinça fina, e tesouras de dissecação finas, retire cuidadosamente a coluna vertebral de L1-C8 da cavidade do corpo do animal. </li><li> Cuidadosamente remover o excesso de tecidos moles em torno da coluna vertebral, enquanto assegurando a não raspar ou ferir o IVD no lado ventral. </li><li> Além disso dissecar a coluna vertebral em vértebras-disco-vértebras da coluna vertebral unidades funcionais (FSU) no L1 / L2, L3 / L4, L5 / L6, C3 / C4, C5 / C6, e discos C7 / C8. </li><li> Lavar o FSU em solução salina equilibrada de Hank suplementada com 1% de penicilina-estreptomicina durante 2 min. </li><li> Aleatoriamente atribuir os FSUs em três grupos: sem cultura e não tratada FSU (fresca), cultivadas mas não tratada FSU (Controlo), e cultivadas e tratadas facada FSU (Stab). </li><li> Encaixe congelar as amostras frescas FSU com azoto líquido imediatamente após a dissecação, e armazenar em congelador a -20 ° C. </li></ol> 3. Condições cultura de órgão<ol><li> Preparar 500 mL de meio de cultura estéril de 1: 1 de meio Dulbecco modificado por Eagle: Mistura de Nutrientes F12 (DMEM: F12) suplementado com 20% de soro fetal bovino (FBS) e 1% de penicilina-estreptomicina. </li><li> Usando 10 mL pipetas serológicas, pipeta de 2 ml de meio de cultura em cada cavidade de uma placa de cultura de 24 poços. </li><li> A seguir a dissecção e enxaguar, colocar cada FSU em uma cavidade individual na placa de 24 poços cheios com meios de comunicação. </li><li> Incubar as amostras numa incubadora esterilizada que mantém a 37 ° C, 5% de CO2, 20% de O2 e&gt; 90% de humidade. </li><li> Após um período de cultura inicial de 24 h, mecanicamente ferir as amostras Stab grupo por punção de agulha do anel fibroso usando uma agulha estéril de calibre 27, como mostrado na Figura 1A. </li><li> Mudar meios a cada 48 h através da preparação de uma nova placa de cultura de 24 poços cheios com meios de comunicação e de transferência de amostras da placa de idade para os novos usando pinças estéreis. Aspirar a velha mídia e dispose da placa de cultura de idade em um caixote do lixo de risco biológico. </li><li> No último dia da cultura, todas as amostras incubar em meios contendo 0,75 mg / mL de cloreto de nitro azul de tetrazólio durante 24 h. </li><li> Em seguida, congelar todo o FSU com azoto líquido e armazenar em congelador de -20 ° C até estar pronto para o teste mecânico ou histologia (Figura 1B). </li></ol> 4. As medições longitudinais NF-kB <ol><li> Imagem a FSU a partir de animais-NF-κβ luciferase a 1, 5, 13, e 19 dias para avaliar a expressão de NF-kB. </li><li> Adicionar 10 uL de solução a 1 mg luciferina / mL a cada poço e incubar numa incubadora estéril a 37 ° C durante 10 min. </li><li> Imagem as amostras para a bioluminescência, utilizando o sistema de imagem com um tempo de exposição de 1 min e configuração do compartimento de dois. </li><li> Ao mesmo tempo, usar a máquina para tirar uma fotografia e cobri-la com a imagem de bioluminescência para determinar a localização anatômica de luminescência no IVD. </li></ol><pclass = &quot;jove_title&quot;&gt; 5. Avaliação mecânica <ol><li> Determinar o comportamento mecânico das IVDs usando compressão dinâmica controlada pelo deslocamento 9. </li><li> Usando um microscópio dissecção, escalpelo e pinças, remover os corpos vertebrais ósseas do FSU a partir de cada amostra, mantendo as placas terminais cartilaginosas intacto e ligado ao IVD. </li><li> Anexar as IVDs isolados para uma placa de alumínio de 1 cm x 1 cm x 0,2 centímetros utilizando cola de cianoacrilato. </li><li> Medir a altura do disco e largura, tendo uma média de três medições no eixo longitudinal de cada disco, utilizando um micrómetro de laser. Calcular a proporção da altura do disco dividindo a altura média do disco pela largura máxima do disco. </li><li> Utilizar a altura do disco medido para calcular os valores de tensão de entrada utilizados em testes mecânicos. Note-se que a altura média do disco foi de aproximadamente 690 ± 39? M? M. </li><li> Coloque as amostras em um banho de solução salina tamponada com fosfato sob a compressina máquina e pré-carregar o disco a 0,02 N. </li><li> Ciclicamente comprimir o disco, utilizando uma forma de onda sinusoidal a 1 Hz durante 20 ciclos ao nível de tensão de 1% e nível de tensão de 5% para cada 3 ensaios e gravar os valores de carga e de deslocamento da IVD. Permitir 10 minutos de descanso tempo entre ensaios para o disco para relaxar e para evitar ferimentos. </li><li> Calcular a rigidez média a partir da fase de carga do último ciclo, e calcular a perda tangente do ângulo de fase entre os dados de carga e deslocação. </li></ol> 6. proteoglicanos e colágeno Quantificação <ol><li> Seguindo o teste mecânico, medir o peso húmido do disco colocando o IVD isolado em um tubo de centrífuga previamente tarado e medindo o peso, utilizando uma balança analítica. </li><li> Digerir os IVDs isolado em 250 ul solução de digestão de papaína (0,1 M acetato de sódio, 0,01 M de EDTA, 0,005 M de cisteína HCl, pH 6,4) durante a noite a 65 ° C utilizando um aquecedor de bloco. </li><li> Centrifugar as amostras durante 10 mina 2000 xg e recolher o fluido sobrenadante. </li><li> Medir o teor de proteoglicano da IVD utilizando o ensaio de azul de dimetilmetileno (DMMB) com padrões de sulfato de condroitina. </li><li> Preparar a soluo de DMMB (21 mg DMMB, 5 mL de etanol absoluto, 2 g de formiato de sódio em 1 L de ddH 2 O). </li><li> Pipetar 30 ul dos padrões e amostras, em triplicado, numa placa de 96 pos, juntamente com 250 mL de solução de DMMB em cada poço. </li><li> Leia a absorvância da placa a 525 nm utilizando um espectrofotómetro. </li><li> Medir o teor de colagénio usando um kit de ensaio de hidroxiprolina de acordo com as instruções do fabricante. </li></ol> 7. Histologia <ol><li> Fixar um subconjunto das amostras em 4% de paraformaldeído durante 24 h, de-calcificar em ácido fórmico a 5% durante 48 h, desidratar com etanol, e incorporar-se em parafina. </li><li> Utilizando um micrótomo, as amostras de secção sagital em 10 de espessura e aplicar secções de lâminas de vidro. Manchar as seções usando safranina-O / Fast Green e DAPI. </li><li> Usando um microscópio de luz, imagem desliza em aumento de 10x. </li></ol> 8. Contraste-enhanced microComputed Tomografia (microCT) <ol><li> Digitalizar um subconjunto de amostras nos pontos de tempo 0, 2, 5, e 7 dias usando longitudinal Ioversol realce 10, e de ácido fosfomolíbdico (PMA) realce terminal no ponto de tempo de 7 dias. </li><li> 4 horas antes da microCT tempo de varredura, adicionam-se 300 uL de 350 mg / mL de solução de iodo Ioversol para o meio de cultura para uma concentração final de aproximadamente 50 mg de solução Ioversol / mL contendo iodo e incubar numa incubadora estéril a 37 ° C durante quatro h. </li><li> Após a incubação, a amostra embrulhar em gaze estéril e colocar num tubo de microcentrífuga de 1 mL. </li><li> Colocar as amostras no sistema microCT e digitalizar em 45 keVp, 177 mA, 10,5 tamanho mm voxel, e 300 ms integração. </li><li> Exportar dados do microCT como arquivos DICOM e visualizar usando software ( <em&gt; por exemplo, OsiriX). </li><li> No ponto de tempo de 7 dias, corrigir amostras usando uma solução de paraformaldeído a 4% durante 24 h, seguido por solução a 5% de PMA durante 3 dias, e digitalizar utilizando as mesmas definições como as utilizadas na etapa 8.4. </li></ol> 9. Tridimensional Modelação Elementos Finitos <ol><li> Segmento os arquivos DICOM dos IVDs usando software com limiarização manual (por exemplo, OsiriX). </li><li> Converter os volumes NP e AF do IVD em malhas de elementos finitos baseada em voxel usando MeshLab. </li><li> Combine as microestruturas da NP e AF para formar um IVD completa e aplicar condições de contorno determinadas experimentalmente no software de elementos finitos. </li></ol>

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Os autores deste manuscrito declaram que não têm interesses financeiros concorrentes.

Este protocolo descreve uma cultura de órgão do FSU murino com ênfase sobre o controlo das alterações biológicas no IVD. A manutenção bem-sucedida dessas culturas requer técnicas estéreis cuidadosos. Em particular, a dissecção passos 2,1-2,6 e a cultura passos 3,1-3,6 requerem um cuidado especial para garantir condições estéreis são mantidas, e estes passos deverá ser realizada preferencialmente numa sala de procedimentos isolado com um fluxo de ar HEPA para minimizar co...

Dor lombar (LBP) é o principal fator para a deficiência global e perda de produtividade no local de trabalho, e os americanos sozinho gastar em excesso de 50 bilhões de dólares em tratamento LBP 1. Embora prevalente, a etiologia da LBP permanece complexa e multifatorial. No entanto, disco intervertebral (IVD) degeneração está entre os factores de risco mais importantes para a LBP 2. O IVD é feito de três microestruturas: a fibrose exterior do anel (FA), o interior do núcleo pulposo (NP), e duas placas extremas cartilaginosos que sanduíche toda a estrutura proximal e distalmente 3. Com o envelhecimento ea degeneração, os componentes IVD mudar estruturalmente, de composição, e mecanicamente 4. Estas alterações incluem a perda de proteoglicanos e hidratação da NP, diminuição da altura do disco, e deteriorou competência mecânico 5. Estas alterações sãomuitas vezes acompanhada de citoquinas que promovem uma resposta inflamatória, bem como de neutrófilos e gânglio da raiz dorsal de intrusão no espaço da articulação culminando numa cascata de eventos que levam à sintomas LBP 6. O estudo dos mecanismos de degeneração IVD é um desafio em seres humanos, porque muitas vezes não é possível isolar a causa da degeneração antes da ocorrência de dor lombar. Assim, uma abordagem redutora de simplificar o sistema experimental para baixo para o órgão IVD permite o ajuste fino mecanicista de variáveis causais e examinando os seus efeitos a jusante 5. O sistema é reduzida para apenas a população celular nativo e matriz extracelular circundante, permitindo assim que a interpretação directa dos efeitos de estulos externos sobre a degeneração IVD. Além disso, o custo mais baixo e escalabilidade de modelos de murino, bem como o grande número de animais geneticamente modificados 7, para permitir que tele triagem direcionada rápida de mecanismos degenerativas IVD e terapias potenciais. Aqui, descrevemos um sistema de cultura de órgãos de murino em que IVD estabilidade celular e do tecido é mantida ao longo de 21 dias, com foco específico dado de padrões homeostáticos, mecânicas, estruturais e inflamatórias dos IVDs. Usando esse método, podemos monitorar as mudanças funcionais dos IVDs em um modelo de lesão induzida por facada 8 de compreender os mecanismos por trás degeneração do disco. Além disso, nós descrevemos várias aplicações deste processo de cultura de órgão para conduzir mais estudos de imagiologia incluindo microCT com contraste e modelação tridimensional de alta resolução da IVD.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96 well plate</td>
			<td>Midwest Scientific</td>
			<td>TP92096</td>
			<td>Used for biochemical assays</td>
		</tr>
		<tr>
			<td>24 well plate</td>
			<td>Midwest Scientific</td>
			<td>TP92024</td>
			<td>Used for organ culture</td>
		</tr>
		<tr>
			<td>25 ml pipettes</td>
			<td>Midwest Scientific</td>
			<td>TP94024</td>
			<td>Used for organ culture</td>
		</tr>
		<tr>
			<td>10 ml pipettes</td>
			<td>Midwest Scientific</td>
			<td>TP94010</td>
			<td>Used for organ culture</td>
		</tr>
		<tr>
			<td>5 ml pipettes</td>
			<td>Midwest Scientific</td>
			<td>TP94005</td>
			<td>Used for organ culture</td>
		</tr>
		<tr>
			<td>500 ml bottle top filters, 22um</td>
			<td>Midwest Scientific</td>
			<td>TP99505</td>
			<td>Used for filter media</td>
		</tr>
		<tr>
			<td>10 ul pipette tips</td>
			<td>Midwest Scientific</td>
			<td>NP89140098</td>
			<td>Used for biochemical assays</td>
		</tr>
		<tr>
			<td>200 ul pipette tips</td>
			<td>Midwest Scientific</td>
			<td>NP89140900</td>
			<td>Used for biochemical assays</td>
		</tr>
		<tr>
			<td>1000 ul pipette tips</td>
			<td>Midwest Scientific</td>
			<td>NP89140920</td>
			<td>Used for biochemical assays</td>
		</tr>
		<tr>
			<td>DMEM /F-12</td>
			<td>Invitrogen</td>
			<td>11330032</td>
			<td>Used for culture media</td>
		</tr>
		<tr>
			<td>Optiray 350</td>
			<td>Guebert</td>
			<td>19133341</td>
			<td>Used for contrast enhanced microCT</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma</td>
			<td>F2442</td>
			<td>Used for culture media</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin&#160;</td>
			<td>Sigma</td>
			<td>P4333</td>
			<td>Used for culture media</td>
		</tr>
		<tr>
			<td>Tetrazolium Blue Chloride</td>
			<td>Sigma</td>
			<td>T4375</td>
			<td>Used for biochemical assays</td>
		</tr>
		<tr>
			<td>D-Luciferin</td>
			<td>Sigma</td>
			<td>L6152</td>
			<td>Used for bioluminescence imaging</td>
		</tr>
		<tr>
			<td>Chondroitin Sulfate</td>
			<td>Sigma</td>
			<td>C9819</td>
			<td>Used for biochemical assays</td>
		</tr>
		<tr>
			<td>10% Phosphomolybdic Acid Solution</td>
			<td>Sigma</td>
			<td>HT152</td>
			<td>Used for contrast enhanced microCT</td>
		</tr>
		<tr>
			<td>Safranin O</td>
			<td>Sigma</td>
			<td>S8884</td>
			<td>diluted to .1% concentration (in water)</td>
		</tr>
		<tr>
			<td>Fast Green FCF</td>
			<td>Sigma</td>
			<td>F7258</td>
			<td>.001% concentration</td>
		</tr>
		<tr>
			<td>Papain from papaya latex</td>
			<td>Sigma&#160;</td>
			<td>P3125</td>
			<td>Used for biochemical assays</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td>Nucleic acid staining</td>
		</tr>
		<tr>
			<td>Cyanoacrylate Glue</td>
			<td>Loctite</td>
			<td>234790</td>
			<td>Adhesive&#160;</td>
		</tr>
		<tr>
			<td>1.5 ml Microcentrifuge Tubes&#160;</td>
			<td>Fischer Scientific</td>
			<td>S348903</td>
			<td>Used for biochemical assays</td>
		</tr>
		<tr>
			<td>Big Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BioDent</td>
			<td>ActiveLife</td>
			<td></td>
			<td>For mechanical testing</td>
		</tr>
		<tr>
			<td>Cytation 5</td>
			<td>Biotek</td>
			<td></td>
			<td>Spectrophotometer</td>
		</tr>
		<tr>
			<td>AxioCam503</td>
			<td>Carl Zeiss AG</td>
			<td></td>
			<td>Microscope</td>
		</tr>
		<tr>
			<td>VivaCT40</td>
			<td>Scanco</td>
			<td></td>
			<td>MicroCT</td>
		</tr>
		<tr>
			<td>Analytical balance</td>
			<td>Denver Instrument Company</td>
			<td>A-200DS</td>
			<td>Analytical balance</td>
		</tr>
		<tr>
			<td>Incubator HERAcell 150i</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>Organ Culture</td>
		</tr>
		<tr>
			<td>Dissection Scope</td>
			<td>VistaVision</td>
			<td></td>
			<td>Used during dissection</td>
		</tr>
		<tr>
			<td>Laser Micrometer</td>
			<td>Keyence</td>
			<td>LK-081</td>
			<td>Measuring disc height</td>
		</tr>
		<tr>
			<td>Microcentrifuge 5810 R</td>
			<td>Eppendorf</td>
			<td></td>
			<td>Used for biochemical assays</td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica&#160;</td>
			<td>RM2255</td>
			<td>Used for histology</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Prism 7</td>
			<td>GraphPad</td>
			<td></td>
			<td>For statistics</td>
		</tr>
		<tr>
			<td>MATLAB R2014a</td>
			<td>Mathworks</td>
			<td></td>
			<td>For modeling</td>
		</tr>
		<tr>
			<td>Osiri-LXIV</td>
			<td>Pixmeo</td>
			<td></td>
			<td>Open Source</td>
		</tr>
		<tr>
			<td>MeshLab v1.3.3</td>
			<td>Visual Computing Lab - ISTI - CNR</td>
			<td></td>
			<td>Open Source</td>
		</tr>
		<tr>
			<td>PreView/FEBio 2.3</td>
			<td>Utah MRL &#38; Columbia MBL</td>
			<td></td>
			<td>Open Source</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Windows</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection Tools</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cohan-Vannas Spring Scissors&#160;</td>
			<td>Fine Science Tools&#160;&#160;</td>
			<td>15000-02</td>
			<td>Or any nice pair of spring scissors</td>
		</tr>
		<tr>
			<td>Fine Scissors - Sharp&#160; (small)</td>
			<td>Fine Science Tools&#160;&#160;</td>
			<td>14060-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors - Sharp&#160; (larger)</td>
			<td>Fine Science Tools&#160;&#160;</td>
			<td>14060-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine Science Tools&#160;&#160;</td>
			<td>11252-40</td>
			<td>At least 2; can also use #3&#160;</td>
		</tr>
		<tr>
			<td>Extra Fine Graefe Forceps, serrated</td>
			<td>Fine Science Tools&#160;&#160;</td>
			<td>11150-10</td>
			<td>At least 2</td>
		</tr>
		<tr>
			<td>Micro-Adson Forceps, serrated</td>
			<td>World Precision Instruments</td>
			<td>503719-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Adson Forceps, teeth</td>
			<td>World Precision Instruments</td>
			<td>501244</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel Handle - #3</td>
			<td>Fine Science Tools&#160;&#160;</td>
			<td>10003-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel Handle - #4</td>
			<td>Fine Science Tools&#160;&#160;</td>
			<td>10004-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel Blades - #23</td>
			<td>Fine Science Tools&#160;&#160;</td>
			<td>10023-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Insect Pins , size 000</td>
			<td>Fine Science Tools&#160;&#160;</td>
			<td>26000-25</td>
			<td></td>
		</tr>
		<tr>
			<td>27G Needle</td>
			<td>BD PrecisionGlide Needles</td>
			<td>BD305109</td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vitro organ culture model of the murine intervertebral disc

degeneração do disco intervertebral (IVD) é um contribuinte significativo para a dor lombar. O IVD é uma joint fibrocartilaginous que serve para transmitir e amortecer cargas na coluna vertebral. O IVD consiste de um pulposo rica em proteoglicanos núcleo (NP) e um anel fibrose rico em colagénio (AF) ensanduichado por placas terminais cartilaginosas. Juntamente com as vértebras adjacentes, a estrutura vértebras-IVD forma uma unidade funcional coluna (FSU). Estas microestruturas contêm tipos de células únicas, bem como matrizes extracelulares únicas. cultura de órgão inteiro do FSU preserva a matriz extracelular nativa, fenótipos de diferenciação celular, e interacções de matriz celular. Assim, as técnicas de cultura de órgãos são particularmente úteis para a investigação dos mecanismos biológicos complexos da IVD. Aqui, descrevemos uma abordagem de alto rendimento para a cultura FSUs rato lombar inteiro que fornece uma plataforma ideal para estudar os mecanismos da doença e terapias para o IVD. Além disso, descrevemos several aplicações que utilizam este processo de cultura de órgão para conduzir mais estudos de imagiologia incluindo microCT com contraste e tridimensional de alta resolução de modelagem de elemento finito da IVD.

As Figuras 2-3 mostram resultados representativos de distribuição de proteoglicano, a expressão de NF-kB, a rigidez, a viscosidade, a altura do disco, e peso molhado para IVDs cultivadas de ratinho. Se cultivadas adequadamente, os parâmetros IVD do grupo de controlo não deve ser significativamente diferente do grupo de fresco. Se a cultura está infectado ou de outro modo comprometida, o grupo de controlo irá ser diferente do grupo de fresco, especialmente na expressão de NF-kB e de distribuiçã...

Watch this Scientific Journal Video about An In Vitro Organ Culture Model of the Murine Intervertebral Disc at JoVE.com

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

cultura de órgão inteiro do disco intervertebral (IVD) preserva a matriz extracelular nativa, fenótipos celulares, e interacções de matriz celular. Aqui nós descrevemos um sistema de cultura IVD utilizando lombar do mouse e IVDs caudal em suas unidades da coluna vertebral funcionais e várias aplicações que utilizam este sistema.

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 ...

an-in-vitro-organ-culture-model-of-the-murine-intervertebral-disc

Research

JoVE Journal

Bioengineering

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

9.4K visualizações.  Washington University in St. Louis.  cultura de órgão inteiro do disco intervertebral (IVD) preserva a matriz extracelular nativa, fenótipos celulares, e interacções de matriz celular. Aqui nós descrevemos um sistema de cultura IVD utilizando lombar do mouse e IVDs caudal em suas unidades da coluna vertebral funcionais e várias aplicações que utilizam este sistema. 

Vídeo: An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

Hidrogéis alginato para Tridimensional Cultura Órgão de ovários e tubas uterinas

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

Bioengenharia

Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture

The intervertebral disc (IVD) is the joint of the spine connecting vertebra to vertebra. It functions to transmit loading of the spine and give flexibility to the spine. It composes of three compartments: the innermost nucleus pulposus (NP) encompassing by the annulus fibrosus (AF), and two cartilaginous endplates connecting the NP and AF to the vertebral body on both sides. Discogenic pain possibly caused by degenerative intervertebral disc disease (DDD) and disc herniations has been identified as a major problem in our modern society. To study possible mechanisms of IVD degeneration, in vitro organ culture systems with live disc cells are highly appealing. The in vitro culture of intact bovine coccygeal IVDs has advanced to a relevant model system, which allows the study of mechano-biological aspects in a well-controlled physiological and mechanical environment. Bovine tail IVDs can be obtained relatively easy in higher numbers and are very similar to the human lumbar IVDs with respect to cell density, cell population and dimensions. However, previous bovine caudal IVD harvesting techniques retaining cartilaginous endplates and bony endplates failed after 1-2 days of culture since the nutrition pathways were obviously blocked by clotted blood. IVDs are the biggest avascular organs, thus, the nutrients to the cells in the NP are solely dependent on diffusion via the capillary buds from the adjacent vertebral body. Presence of bone debris and clotted blood on the endplate surfaces can hinder nutrient diffusion into the center of the disc and compromise cell viability. Our group established a relatively quick protocol to "crack"-out the IVDs from the tail with a low risk for contamination. We are able to permeabilize the freshly-cut bony endplate surfaces by using a surgical jet lavage system, which removes the blood clots and cutting debris and very efficiently reopens the nutrition diffusion pathway to the center of the IVD. The presence of growth plates on both sides of the vertebral bone has to be avoided and to be removed prior to culture. In this video, we outline the crucial steps during preparation and demonstrate the key to a successful organ culture maintaining high cell viability for 14 days under free swelling culture. The culture time could be extended when appropriate mechanical environment can be maintained by using mechanical loading bioreactor. The technique demonstrated here can be extended to other animal species such as porcine, ovine and leporine caudal and lumbar IVD isolation.

This protocol illustrates a harvesting technique for coccygeal bovine intervertebral discs for organ culture for in vitro organ culture.

Preparação de Intact Discos cauda bovina Intervertebral de Cultura de Órgãos

The intervertebral disc (IVD) is the joint of the spine connecting  vertebra to vertebra. It functions to transmit loading of the spine and give  ...

Tissue Engineering of the Intestine in a Murine Model

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to preoperative weights within 40 days.1 In humans, massive small bowel resection can result in short bowel syndrome, a functional malabsorptive state that confers significant morbidity, mortality, and healthcare costs including parenteral nutrition dependence, liver failure and cirrhosis, and the need for multivisceral organ transplantation.2 In this paper, we describe and document our protocol for creating tissue-engineered intestine in a mouse model with a multicellular organoid units-on-scaffold approach. Organoid units are multicellular aggregates derived from the intestine that contain both mucosal and mesenchymal elements,3 the relationship between which preserves the intestinal stem cell niche.4 In ongoing and future research, the transition of our technique into the mouse will allow for investigation of the processes involved during TESI formation by utilizing the transgenic tools available in this species.5The availability of immunocompromised mouse strains will also permit us to apply the technique to human intestinal tissue and optimize the formation of human TESI as a mouse xenograft before its transition into humans. Our method employs good manufacturing practice (GMP) reagents and materials that have already been approved for use in human patients, and therefore offers a significant advantage over approaches that rely upon decellularized animal tissues. The ultimate goal of this method is its translation to humans as a regenerative medicine therapeutic strategy for short bowel syndrome.

This article and the accompanying video present our protocol for generating tissue-engineered intestine in the mouse, using an organoid units-on-scaffold approach.

Engenharia de Tecidos do Intestino num modelo murino

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

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 model well differentiated human airway epithelium and heart muscle cells. This could be an invaluable tool to study the deleterious effects of toxic inhaled chemicals, such as chlorine, that can normally interact with the cell surfaces, and form various byproducts upon reacting with water, and limiting their effects in submerged cultures. Our model using well differentiated human airway epithelial cell cultures at air-liqiuid interface circumvents this limitation as well as provides an opportunity to evaluate critical mechanisms of toxicity of potential poisonous inhaled chemicals. We describe enhanced loss of membrane integrity, caspase release and death upon toxic inhaled chemical such as chlorine exposure. In this article, we propose methods to model chlorine exposure in mammalian heart and airway epithelial cells in culture and simple tests to evaluate its effect on these cell types.

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

This protocol is designed to demonstrate exposure method of cell cultures to inhaled toxic chemicals. Exposure of differentiated air-liquid&#160;interface (ALI) cultures of airway epithelial cells provides a unique model of airway exposure to toxic gases such as chlorine. In this manuscript we describe effect of chlorine exposure on air-liquid&#160;interface cultures of epithelial cells and submerged culture of cardiomyocytes. In vitro exposure systems allow important mechanistic studies to evaluate pathways that could then be utilized to develop novel therapeutic agents.

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 ...

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 ...

Decellularization of Whole Human Heart Inside a Pressurized Pouch in an Inverted Orientation

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ultimately rejection can occur. Creating a functional, autologous bio-artificial heart could solve these challenges. Biofabrication of a heart comprised of scaffold and cells is one option. A natural scaffold with tissue-specific composition as well as micro- and macro-architecture can be obtained by decellularizing hearts from humans or large animals such as pigs. Decellularization involves washing out cellular debris while preserving 3D extracellular matrix and vasculature and allowing &#34;cellularization&#34; at a later timepoint. Capitalizing on our novel finding that perfusion decellularization of complex organs is possible, we developed a more &#34;physiological&#34; method to decellularize non-transplantable human hearts by placing them inside a pressurized pouch, in an inverted orientation, under controlled pressure. The purpose of using a pressurized pouch is to create pressure gradients across the aortic valve to keep it closed and improve myocardial perfusion. Simultaneous assessment of flow dynamics and cellular debris removal during decellularization allowed us to monitor both fluid inflow and debris outflow, thereby generating a scaffold that can be used either for simple cardiac repair (e.g. as a patch or valve scaffold) or as a whole-organ scaffold.

This method enables decellularization of a complex solid organ using a simple protocol based on osmotic shock and perfusion of ionic detergent with minimal organ matrix disruption. It comprises a novel decellularization technique for human hearts inside a pressurized pouch with real-time monitoring of flow dynamics and cellular debris outflow.

Decellularization do coração humana dentro de uma bolsa pressurizada em uma orientação invertida

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ...

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 ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

Modelos avançados de fígado 3D para testes de genotoxicidade in vitro após exposição nanomaterial de longo prazo

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

An Open Source Technology Platform to Manufacture Hydrogel-Based 3D Culture Models in an Automated and Standardized Fashion

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These issues represent drawbacks in workflows that can ultimately result in irreproducibility of research findings. Manual-based workflows are further limiting the advancement and widespread adoption of viscous materials, such as hydrogels used for biomedical applications. These challenges can be overcome by using automated workflows with standardized mixing processes to increase reproducibility. In this study, we present step-by-step instructions to use an open source protocol designer, to operate an open source workstation, and to identify reproducible mixtures. Specifically, the open source protocol designer guides the user through the experimental parameter selection and generates a ready-to-use protocol code to operate the workstation. This workstation is optimized for pipetting of viscous materials to enable automated and highly reliable handling by the integration of temperature docks for thermoresponsive materials, positive displacement pipettes for viscous materials, and an optional tip touch dock to remove excess material from the pipette tip. The validation and verification of mixtures are performed by a fast and inexpensive absorbance measurement of Orange G. This protocol presents results to obtain 80% (v/v) glycerol mixtures, a dilution series for gelatin methacryloyl (GelMA), and double network hydrogels of 5% (w/v) GelMA and 2% (w/v) alginate. A troubleshooting guide is included to support users with protocol adoption. The described workflow can be broadly applied to a number of viscous materials to generate user-defined concentrations in an automated fashion.

This protocol serves as a comprehensive tutorial for standardized and reproducible mixing of viscous materials with a novel open source automation technology. Detailed instructions are provided on the operation of a newly developed open source workstation, the usage of an open source protocol designer, and the validation and verification to identify reproducible mixtures.

Uma plataforma de tecnologia de código aberto para fabricar modelos de cultura 3D à base de hidrogel de forma automatizada e padronizada

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These ...

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering.

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

Transplante de um Patch Bioimpresso 3D em um modelo murino de infarto do miocárdio

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...

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