Name: a 3d system for culturing human articular chondrocytes in synovial fluid
Uploaded: 2012-01-31T13:00:00
Description: Watch this Scientific Journal Video about A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid at JoVE.com

Gostaríamos de agradecer a Robin Nye (Tufts Medical Center), Tomoya Uchimura e Dana Cairns (Tufts University) para a prestação de ajuda com o armazenamento de líquido sinovial e centrifugação. Este trabalho foi financiado pelo NIH (1R01AR059106-01A1) para LZ

Um sistema 3D para cultivo de condrócitos articular humana em líquido sinovial Neste trabalho, nós encapsulado condrócitos articular humana em esferas de alginato modificado usando um protocolo de produção, sugeriu o encapsulamento (Lonza, e 31). Usando essas construções em 3D, nós desenvolvemos um sistema para a cultura de células em meio de cultura contendo percentuais variaram de líquido sinovial humana e avaliaram estas construções em 3D para a cartilagem da expre...

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Neste relatório, nós desenvolvemos um método que permite a cultura de condrócitos articular humana em um ambiente 3D em meio que contém altas concentrações de líquido sinovial humana. Líquido sinovial é um dos principais componentes que constituem o ambiente natural na cavidade articular, onde condrócitos articular reside. No entanto, a viscosidade do líquido sinovial tem sido um grande desafio para tridimensional de cultura de longo prazo dos condrócitos. Para superar o desafio de manter uma distribuição...

Tabela de reagentes e equipamentos específicos: <table border="1"><tr><th> Nome do reagente </th><th> Companhia </th><th> Número Catálogo </th><th> Comentários </th></tr><tr><td> Alginato (sal de sódio de ácido algínico) </td><td> Sigma </td><td> A2158-250G </td><td> Solução 2,4% armazenadas a 40 ° C </td></tr><tr><td> Cloreto de cálcio dihidratado, Granular </td><td> JT Baker </td><td> A19339 </td><td></td></tr><tr><td width="239" rowspan="2" > Condrogênicos media Crescimento </td><td width="254" rowspan="2" > Lonza </td><td width="182" > CC-3156 (base de mídia) </td><td width="141" rowspan="2" ></td></tr><tr><td width="182" > CC-4409 (suplemento) </td></tr><tr><td width="239" rowspan="2" > Condrogênicos Mídia Diferenciação </td><td width="254" rowspan="2" > LOnza </td><td width="182" > CC-3226 (base de mídia) </td><td width="141" rowspan="2" ></td></tr><tr><td width="182" > CC-4408 (suplemento) </td></tr><tr><td> Humana condrócitos articular </td><td> Lonza </td><td > CC-2550 </td><td></td></tr><tr><td> DAPI (4 &#39;,6-diamidino-2-phenylindole dicloridrato) </td><td> Sigma-Aldrich </td><td > D9542 </td><td></td></tr><tr><td> RNeasy mini-kit (para extração de RNA) </td><td> Qiagen </td><td > 74104 </td><td></td></tr><tr><td> PCR reagentes: SYBR-verde </td><td> Quanta </td><td > 95053-500 </td><td></td></tr><tr><td> 12 ml da seringa </td><td> Tyco-Kendall-Monoject </td><td > 512852 </td><td></td></tr><tr><td> 22 Gague-Agulha Hipodérmica </td><td> Tyco-Kendall-Monoject </td><td > 8881 </td><td></td></tr><tr><td> Microscópio </td><td> Olimpo </td><td > IX71 </td><td></td></tr><tr><td> Rocker plataforma </td><td> Thermolyne Thermoscientific </td><td > Vari-mix </td><td></td></tr><tr><td></td><td></td><td ></td><td></td></tr><tr><td> Seqüências de primers </td><td></td><td ></td><td></td></tr><tr><td> Colágeno IIa-forward </td><td> 5&#39;-CCA CCC TTC ATC TCT CAC AGT-3 &#39; </td><td ></td><td></td></tr><tr><td> Colágeno IIa-reverse </td><td> 5&#39;-CCTCTGCCTTGACCCGAA-3 &#39; </td><td ></td><td></td></tr><tr><td> MMP13-forward </td><td> 5&#39;-TGT GCC CTT CTT CAC ACA GAC ACT-3 &#39; </td><td ></td><td></td></tr><tr><td> MMP13-reverse </td><td> 5&#39;-GAG-AGC GTC TGA AGA CTT ATT CCG-3 &#39; </td><td ></td><td></td></tr><tr><td> Caspase 3-forward </td><td> 5&#39;-TCA TTC TTA AGG CCT GCC GTG GTA-3 &#39; </td><td ></td><td></td></tr><tr><td> Caspase 3-reverse </td><td> 5&#39;-TGG ATG AAC GAG CCA CAG TCC -3 TTT &quot; </td><td ></td><td></td></tr></table>

a 3d system for culturing human articular chondrocytes in synovial fluid

Destruição da cartilagem é uma característica central patológicas da osteoartrose, uma das principais causas de incapacidade em os EUA. Cartilagem no adulto não se regenera de forma muito eficiente in vivo, e como resultado, osteoartrite causa a perda irreversível da cartilagem e é acompanhada por dor crônica e 1,2 imobilidade. Cartilagem engenharia de tecidos oferece grande potencial para regenerar e restaurar a função do tecido. Esta tecnologia envolve tipicamente condrócitos semeadura em andaimes natural ou sintético e cultivar o 3D, resultando em um meio de construir equilibrada ao longo de um período de tempo com um gol de um tecido de engenharia bioquímica e biomecanicamente madura que pode ser transplantado para um local do defeito in vivo 3-6 . Atingir uma condição ideal para o crescimento de condrócitos e deposição de matriz é essencial para o sucesso da cartilagem engenharia de tecidos. Cartilagem na articulação cavidade nativa, na articular superfície do osso é banhada no líquido sinovial. Este líquido claro e viscoso fornece nutrientes para a cartilagem articular avascular e contém fatores de crescimento, citocinas e enzimas que são importantes para o metabolismo dos condrócitos 7,8. Além disso, o líquido sinovial facilita o movimento de baixa fricção entre superfícies cartilaginosas principalmente através de secretar dois componentes-chave, ácido hialurônico e lubricin 9 10. Em contraste, a cartilagem da engenharia de tecidos é mais freqüentemente cultivados em meio artificial. Enquanto esses meios são susceptíveis capaz de proporcionar condições mais definido para o estudo do metabolismo dos condrócitos, líquido sinovial reflete com mais precisão o ambiente natural do qual condrócitos articular residem dentro De fato, o líquido sinovial tem a vantagem de ser fácil de obter e armazenar, e muitas vezes podem ser reabastecidos regularmente pelo corpo. Vários grupos completaram o meio de cultura com líquido sinovial em crescimento humano, coelho, bovinos e cães chondrocytes, mas principalmente utilizados apenas os baixos níveis de líquido sinovial (abaixo de 20%) 11-25. Enquanto condrócitos cavalo, galinha e humanos foram cultivadas em meio com maior percentual de líquido sinovial, estes sistemas de cultura eram bidimensionais 26-28. Aqui apresentamos o nosso método de cultura de condrócitos articular humana em um sistema 3D, com uma alta porcentagem de líquido sinovial (até 100%) durante um período de 21 dias. Ao fazer isso, superamos um grande obstáculo apresentado pela alta viscosidade do fluido sinovial. Este sistema oferece a possibilidade de estudar os condrócitos humanos em líquido sinovial em um ambiente 3D, que pode ainda ser combinado com outros dois fatores importantes (tensão de oxigênio e carga mecânica) 29,30 que constituem o ambiente natural para a cartilagem para imitar o ambiente natural para crescimento da cartilagem. Além disso, este sistema também pode ser utilizada para a dosagem de atividade líquido sinovial em condrócitos e fornecer uma plataforma para o desenvolvimentoregeneração da cartilagem tecnologias e opções terapêuticas para a artrite.

Watch this Scientific Journal Video about A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid at JoVE.com

A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

Um sistema 3D de condrócitos articular humana cultivo em altos níveis de líquido sinovial é descrita. Líquido sinovial reflete o microambiente mais natural para a cartilagem articular, e pode ser facilmente obtido e armazenado. Esse sistema, portanto, pode ser usado para estudar a regeneração da cartilagem e para terapêutica de triagem para tratamento da artrite.

Um Sistema de 3D para condrócitos Cultivar Humanos Articular no líquido sinovial

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Biologia

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

Derivação eficiente dos recursos humanos Precursores cardíaca e Cardiomyocytes pluripotentes a partir de células estaminais embrionárias humanas com a indução de moléculas pequenas

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

A System for ex vivo Culturing of Embryonic Pancreas

A System for ex vivo Culturing of Embryonic Pancreas

Um Sistema de<em&gt; Ex vivo</em&gt; A cultura de Pâncreas embrionárias

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer's Vesicle

Análise da função do gene e Visualização de Cilia-Generated fluxo de fluidos em vesículas de Kupffer

Internal organs such as the  heart, brain, and gut develop left-right (LR) asymmetries that are critical for  their normal functions1. Motile cilia are ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

A cultura de células epiteliais nasais Humano na interface ar líquido

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

A Method for Culturing Embryonic C. elegans Cells

A Method for Culturing Embryonic C. elegans Cells

Um Método para o cultivo embrionário<em&gt; C. elegans</em&gt; Células

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

Gap juncional intercelular comunicação: um biomarcador funcional para avaliar os efeitos adversos de tóxicos e toxinas, e Benefícios para a saúde de produtos naturais

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

A Assembleia e Aplicação de &#39;tesoura Anéis&#39;: A endotelial novo modelo de Orbital, unidirecional e periódica fluxo de fluido e tensão de cisalhamento

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Magnético-ativado da pilha classificação estratégias para isolar e purificar sinovial fluidos derivados mesenquimais células-tronco de um modelo de coelho

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...