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 expressão gênica. 1. Prepare condrócitos articular humana (HAC) por três-dimensional encapsulamento (3D) <ol><li> Descongelar um frasco (1 ml) de condrócitos articular humana (HAC) (Lonza) (Passage 2) em 37 ° C banho-maria por 1 min. </li><li> Misture com 1 ml de meio de crescimento de condrócitos (Lonza) e centrifugar a 3000 rpm por 3-5 min para coletar as células. Desprezar o sobrenadante. </li><li> Ressuspender celular em meio de crescimento de condrócitos (Lonza). </li><li> Células da placa em um tecido de 10 cmplaca de cultura (BD Biosciences), e cultura em meios de crescimento de condrócitos (Lonza) até as células são confluentes. HACS deve ser usado em um número passagem não superior a 3 ou 4 (P3 ou P4). </li><li> Lavar HACS na placa com 155mm NaCl em vez do PBS padrão, seguido de trypsinzation para coletar células para encapsulamento de alginato talão. Uma vez que as células são separadas, spin para baixo as células a 3000rpm por 5min. As células estão agora prontos para encapsulamento 3D. </li></ol> 2. Encapsular HACS em grânulos 3D Ressuspender o HACS em solução de alginato de 1,2% (Sigma) a uma densidade de 8x10 5 células / ml. Número de células foram determinados antes de encapsulamento, utilizando um contador de células padrão. É muito importante para misturar bem para garantir uma distribuição uniforme das células as contas. <ol><li> Pipetar os HAC / mistura solução de alginato em uma seringa 12 ml conectada a uma agulha de calibre 22 (Tyco Healthcare, Inc.). </li><li> Enquanto isso, prepare um copo de 50 mL com 102 mM CaCl 2 em um volume 5 vezes maior do que a solução HAC / aginate. Coloque uma barra de agitação no interior do copo e agitar a 102 mM CaCl 2 solução lentamente (aproximadamente a 150 rpm). </li><li> Segure a ponta da seringa cerca de 6 centímetros acima da superfície da solução de CaCl 2 e adicione a mistura HAC / alginato na solução de CaCl 2 gotas. Em geral, os grânulos de alginato resultantes são dois milímetros de diâmetro, com uma densidade de encapsulamento de cerca de 10 4 células / talão. Nota: A altura da seringa sobre a solução CaCl 2 é importante. Distância mais curta irá resultar em gota em forma esférica, em vez de contas em forma, fazendo com que a integridade mecânica irregular e encapsulamento de células. </li><li> Vamos agitar as contas na solução CaCl 2 por 20-25 min. Enquanto outros protocolos de encapsulamento alginato indicam um tempo de incubação mais curto de 10 minutos, descobrimos que o tempo de incubação com a solução de CaCl 2 maior a integridade do seranúncios. </li><li> Remover a solução CaCl 2, lavar as contas em 2-3, 2-3 vezes volumes de NaCl, e depois uma vez em meio de diferenciação dos condrócitos (Lonza). Enquanto outros alginato de protocolos de encapsulamento envolvem o uso de talão de um filtro para os procedimentos de lavagem acima, descobrimos que os grânulos de alginato, muitas vezes ficam presas no filtro para fora e seca, reduzindo assim a eficiência de encapsulamento. Encontrámo-lo mais eficiente para permitir que os grânulos se depositem no fundo do copo antes de descartar a solução. </li><li> Use uma espátula padrão para transferir as contas em placas de cultura. Nós cultura normalmente de 12 contas por três centímetros bem para dois dias em meio de crescimento de condrócitos para permitir que os condrócitos se ajustar ao processo de encapsulamento no meio em que foram cultivadas em meio antes de mudar para a diferenciação de condrócitos (Lonza) com líquido sinovial. </li></ol> 3. Condrócitos cultura em meio de cultura líquido sinovial <ol><li> Líquido sinovial fresco pode ser obtido a partir de um oclínica utpatient (Obtivemos líquido sinovial de Tufts Medical Center). O líquido sinovial pode ser transferido em tubos de 15ml falcon para o laboratório, e imediatamente centrifugadas a 3000 rpm por 15 min para remover restos celulares. Alíquota livre de células do líquido sinovial em tubos de 1,5 mL de microcentrífuga, para evitar repetidos de congelamento-descongelamento. Sobrenadante pode ser armazenada a -80 ° C até o uso. </li><li> Antes de cultivo, misture o líquido sinovial com meio de diferenciação dos condrócitos (CDM, Lonza) em diferentes proporções volumétricas, com uma concentração constante de ácido ascórbico 100mM e 9 mM CaCl 2 (esta quantidade traço de CaCl 2 é garantir a integridade do alginato esferas). </li><li> Manter a placa em uma plataforma de balanço em uma incubadora de 37 ° C (freqüência de balanço: cerca de 75 vezes / min) para ajudar a distribuição de nutrientes dentro do alginato, e para reduzir a aglutinação das contas. Isto é especialmente importante à medida que a cultura destes condrócitos por um período prolongado de tempo (até 4 semanas). Nota: Embora o crescimento de condrócitos e mídia diferenciação (Lonza) contêm dois antibióticos comumente utilizados gentamicina e anfotericina, não tínhamos qualquer suplemento de antibióticos quando usamos diferentes percentuais de líquido sinovial para cultura de condrócitos. Nenhuma contaminação foi já observado. </li><li> Alterar media misturas cada 2-3 dias. A viscosidade do líquido sinovial tem sido um grande obstáculo em condrócitos cultura encapsulado em grânulos de alginato durante a longo prazo culturas. Encontrámo-lo mais eficaz para diluir meio fluido sinovial suplementado com 102mm CaCl 2 (50% V / V), que também fortalece a integridade das esferas de alginato. Desta forma, o meio pode lentamente ser removido com uma pipeta, sem prejudicar as contas. </li></ol> 4. HACS colheita em grânulos de alginato para a análise de expressão gênica Cuidados especiais devem ser tomados quando a colheita da HACS dos grânulos de alginato para a análise da expressão gênica. <ol><li> Lavar as esferas de alginato 3 times em 102 mM CaCl 2 para cerca de 5 min de cada vez. </li><li> Recuperar os condrócitos, adicionando 55 NaCitrate mM em um volume 4-5 vezes maior do que os grânulos. É importante que os grânulos de alginato são totalmente imersos na solução NaCitrate. Agitar ou rocha por 20-30 min. </li><li> Girar as células e desprezar o sobrenadante. </li><li> RT-PCR para análise, ressuspender o pellet celular em tampão de lise celular (Qiagen RNA isolamento kit), e prosseguir com purificação de RNA. </li></ol> 5. Fix HACS em grânulos de alginato para análise histológica Cuidados especiais devem ser tomados para colher o HACS dos grânulos 3D para análise histológica. <ol><li> Lavar as esferas de alginato de 3 vezes em 102 mM CaCl 2 para cerca de 5 min de cada vez. Completamente lavar o fluido viscoso sinovial, achamos mais eficaz de lavagem com meio de diferenciação dos condrócitos (Lonza) durante a noite, balançando em um 37 ° C incubadora frequência de balanço (: sobre o tempo 75s / min). O meio de crescimento de condrócitos foi usado para garantir a sobrevivência de condrócitos neste lavagem prolongada e para permitir a máxima penetração do agente de fixação. </li><li> Corrigir as contas em etanol 70% durante a noite, e prosseguir com a análise histológica. Para executar a coloração DAPI, incubar alginato com DAPI solução (500ng/ml) para 1hr sob balanço delicado, e lavar por 3 vezes com 102mm CaCl 2 por 15 min a cada vez. DAPI imagens podem ser vistas sob um microscópio fluorescente. As contas também pode ser seccionado para alcian blue e coloração H &amp; E. </li></ol> 6. Resultados representante Nosso método de cultivo em 3D para condrócitos humanos em elevadas percentagens de líquido sinovial é descrita no diagrama esquemático mostrado na Fig.1. Depois de condrócitos humanos foram encapsuladas em esferas de alginato, eles foram autorizados a crescer em meio suplementado com diferentes proporções de líquido sinovial. Por causa da viscosidade do fluido sinovial, é essencialcondrócitos cultura em condições de balanço constante para evitar o acúmulo de construções cartilagem e para assegurar uma distribuição uniforme dos nutrientes. Também é essencial lavar as esferas de alginato extensivamente antes de recuperar os condrócitos, de modo que a fixação ou tampão de lise podem penetrar as esferas (Fig.1). Brilhante campo (BF) imagens de condrócitos na alginato são mostrados na Fig.2. Coloração DAPI foi realizada para confirmar a distribuição usniform das células dentro das contas (Fig. 2). No final do período de cultura de 21 dias, a análise de expressão gênica foi realizada por qRT-PCR. A referência gene GAPDH foi utilizada para a normalização de todos os PCRs, pois estava determinado a ser um dos genes de referência mais confiável para análise de qPCR condrócitos 32. Um exemplo é mostrado na figura. 3, onde foram analisados ​​os resultados de condrócitos articular humana cultivadas no líquido sinovial obtidos a partir de seis pacientes com osteoartrite. Consistente com o fato de que os condrócitos a partirLonza foram expandidos em culturas 2D, o que conduziria inevitavelmente a de diferenciação-33, descobrimos que o Dia 0 condrócitos expressa níveis mínimos de genes matriz da cartilagem (Fig. 3). 3D cultura de condrócitos com meio de diferenciação dos condrócitos (Lonza) ou meio suplementado com líquido sinovial aumentado significativamente cartilagem expressão gênica de colágeno, aggrecan e MMP13, o que indica condrócitos re-diferenciação por Dia 21 (Fig.3) 34. Aumento da percentagem de líquido sinovial na mídia resultou em níveis comparáveis ​​da matriz da cartilagem marcadores de colágeno II e da expressão de mRNA aggrecan (Fig.3 e 3B). Além disso, condrócitos cultivados em 100% fluido sinovial mesmo exibiu uma diminuição na cartilagem degradante expressão do mRNA da enzima MMP13, em comparação com aquelas cultivadas em meio sozinho (Fig.3C). Curiosamente, o nível de expressão de morte celular indicador de caspase 3 diminuiu gradualmente com relações cada vez maior de líquido sinovial, sugerindo que scultura líquido ynovial levou a níveis de apoptose diminuiu (Fig. 3D). Portanto, nossos resultados mostram que os condrócitos articular humana cultivo em altos níveis de líquido sinovial em um ambiente 3D é uma tecnologia viável. Tabelas e Figuras <img alt="Figura 1." src="/files/ftp_upload/3587/3587fig1.jpg" /> Figura 1. Diagrama esquemático do método para a cultura humana condrócitos articular em elevadas percentagens de líquido sinovial em grânulos de alginato 3D. Primeiro, condrócitos e solução de alginato são misturados. Quando aplicado gota a gota, à solução CaCl 2, condrócitos são imobilizadas dentro do reticulado grânulos de alginato-Ca hidrogel. Essas construções 3D são então cultivados em meio de diferenciação dos condrócitos (Lonza) com diferentes proporções de líquido sinovial humana. Após 21 dias de cultivo sob uma condição de balanço, contas de alginato contendo células são lavadas exaustivamente após a qual as células são obtidas para o geneanálise de expressão. <img alt="Figura 2." src="/files/ftp_upload/3587/3587fig2.jpg" /> Figura 2. Campo Bright (BF) e imagens DAPI de condrócitos articular humana culturas encapsulado com 0%, 30%, 50%, 70% e 100% líquido sinovial. Condrócitos foram esférico na forma em todas as condições de cultura. Imagens de campo claro (BF) e DAPI foram sobrepostos para confirmar a localização e distribuição dos condrócitos. Inserções, as imagens ampliadas. Pontas de flechas, co-localização de condrócitos em BF e imagens coloração DAPI. <img alt="Figura 3." src="/files/ftp_upload/3587/3587fig3.jpg" /> Figura 3. QRT-PCR análise de condrócitos articular humana encapsulado no dia 0 (D0) uma após 21 dias de cultivo (D21), em meio suplementado com proporções variáveis ​​de líquido sinovial (SF) (0%, 30%, 50%, 70 % e 100%). Resultados de quatro amostras independentes são mostrados aqui. GAPDH foi utilizado como referência interna para todos os PCRs. <strong&gt; A. Expressão de mRNA de colágeno II. B. Agrecan expressão mRNA. C. MMP13 expressão mRNA. D. Caspase expressão de RNAm 3. Significância estatística foi avaliada para o Dia 0 Dia amostras e 21 amostras de 0% e 100% líquido sinovial (SF) cultura, utilizando o software Instat. * Indica P &lt;0,05.

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R., Syftestad, G., Benton, H., Silliman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+human+knee+synovial+fluid+on+chondrogenesis+in+vitro.">Effects of human knee synovial fluid on chondrogenesis in vitro.</a> Am. J. Knee. Surg. 8, 124-129 (1995).</li><li>van den Hoogen, B. M., van de Lest, C. H., van Weeren, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loading-induced+changes+in+synovial+fluid+affect+cartilage+metabolism.">Loading-induced changes in synovial fluid affect cartilage metabolism.</a> Br. J. Rheumatol. 37, 671-676 (1998).</li><li>Webb, G. R., Westacott, C. I., Elson, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoarthritic+synovial+fluid+and+synovium+supernatants+up-regulate+tumor+necrosis+factor+receptors+on+human+articular+chondrocytes.">Osteoarthritic synovial fluid and synovium supernatants up-regulate tumor necrosis factor receptors on human articular chondrocytes.</a> Osteoarthritis Cartilage. 6, 167-176 (1998).</li><li>Kook, S. H., Son, Y. O., Lee, K. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+affects+positively+the+proliferation+of+bovine+satellite+cells+and+their+myogenic+differentiation+through+up-regulation+of+MyoD.">Hypoxia affects positively the proliferation of bovine satellite cells and their myogenic differentiation through up-regulation of MyoD.</a> Cell. Biol. Int. 32, 871-878 (2008).</li><li>Knobloch, T. J., Madhavan, S., Nam, J., Agarwal, S., Agarwal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+chondrocytic+gene+expression+by+biomechanical+signals.">Regulation of chondrocytic gene expression by biomechanical signals.</a> Crit. Rev. Eukaryot. Gene. Expr. 18, 139-150 (2008).</li><li>Guo, J. F., Jourdian, G. W., MacCallum, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+and+growth+characteristics+of+chondrocytes+encapsulated+in+alginate+beads.">Culture and growth characteristics of chondrocytes encapsulated in alginate beads.</a> Connect. Tissue. Res. 19, 277-297 (1989).</li><li>Toegel, S., Huang, W., Piana, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+reliable+reference+genes+for+qPCR+studies+on+chondroprotective+action.">Selection of reliable reference genes for qPCR studies on chondroprotective action.</a> BMC. Mol. Biol. 8, 13-13 (2007).</li><li>Lin, Z., Fitzgerald, J. B., Xu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiles+of+human+chondrocytes+during+passaged+monolayer+cultivation.">Gene expression profiles of human chondrocytes during passaged monolayer cultivation.</a> J. Orthop. Res. 26, 1230-1237 (2008).</li><li>Goessler, U. R., Bieback, K., Bugert, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+chondrocytes+differentially+express+matrix+modulators+during+in+vitro+expansion+for+tissue+engineering.">Human chondrocytes differentially express matrix modulators during in vitro expansion for tissue engineering.</a> Int. J. Mol. Med. 16, 509-515 (2005).</li><li>Anat, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 121, 107-118 (1976).</li></ol>

Não temos nada a revelar.

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

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not ...

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Research

JoVE Journal

Biology

20.7K visualizações.  Tufts University School of Medicine. 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.

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

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

Um sistema para a cultura de células epiteliais pigmentadas da íris para estudo da regeneração Lens em Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

Biologia

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

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 transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

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

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

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

Internal organs such as the heart, brain, and gut develop left-right (LR) asymmetries that are critical for their normal functions1. Motile cilia are involved in establishing LR asymmetry in vertebrate embryos, including mouse, frog, and zebrafish2-6. These 'LR cilia' generate asymmetric fluid flow that is necessary to trigger a conserved asymmetric Nodal (TGF-&beta; superfamily) signaling cascade in the left lateral plate mesoderm, which is thought to provide LR patterning information for developing organs7. Thus, to understand mechanisms underlying LR patterning, it is essential to identify genes that regulate the organization of LR ciliated cells, the motility and length of LR cilia and their ability to generate robust asymmetric flow.
In the zebrafish embryo, LR cilia are located in Kupffer's vesicle (KV)2,4,5. KV is comprised of a single layer of monociliated epithelial cells that enclose a fluid-filled lumen. Fate mapping has shown that KV is derived from a group of ~20-30 cells known as dorsal forerunner cells (DFCs) that migrate at the dorsal blastoderm margin during epiboly stages8,9. During early somite stages, DFCs cluster and differentiate into ciliated epithelial cells to form KV in the tailbud of the embryo10,11. The ability to identify and track DFCs&mdash;in combination with optical transparency and rapid development of the zebrafish embryo&mdash;make zebrafish KV an excellent model system to study LR ciliated cells.
Interestingly, progenitors of the DFC/KV cell lineage retain cytoplasmic bridges between the yolk cell up to 4 hr post-fertilization (hpf), whereas cytoplasmic bridges between the yolk cell and other embryonic cells close after 2 hpf8. Taking advantage of these cytoplasmic bridges, we developed a stage-specific injection strategy to deliver morpholino oligonucleotides (MO) exclusively to DFCs and knockdown the function of a targeted gene in these cells12. This technique creates chimeric embryos in which gene function is knocked down in the DFC/KV lineage developing in the context of a wild-type embryo. To analyze asymmetric fluid flow in KV, we inject fluorescent microbeads into the KV lumen and record bead movement using videomicroscopy2. Fluid flow is easily visualized and can be quantified by tracking bead displacement over time.
Here, using the stage-specific DFC-targeted gene knockdown technique and injection of fluorescent microbeads into KV to visualize flow, we present a protocol that provides an effective approach to characterize the role of a particular gene during KV development and function.

Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer&#039;s Vesicle

Cilia-generated fluid flow in Kupffer&#x2019;s Vesicle (KV) controls left-right patterning of the zebrafish embryo. Here, we describe a technique to modulate gene function specifically in KV cells. In addition, we show how to deliver fluorescent beads into KV to visualize fluid flow.

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

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

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 ...

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 function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

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

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

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

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

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

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

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

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

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 ...

3D Culturing of Organoids from the Intestinal Villi Epithelium Undergoing Dedifferentiation

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is compartmentalized into crypts and villi: the stem and proliferating cells are confined to the crypts, whereas the villi epithelium contains only differentiated cells. Hence, the normal intestinal crypts, but not the villi, can give rise to organoids in 3D cultures. The procedure described here is applicable only to villus epithelium undergoing dedifferentiation leading to stemness. The method described uses the Smad4-loss-of-function:&#946;-catenin gain-of-function (Smad4KO:&#946;-cateninGOF) conditional mutant mouse. The mutation causes the intestinal villi to dedifferentiate and generate stem cells in the villi. Intestinal villi undergoing dedifferentiation are scraped off the intestine using glass slides, placed in a 70 &#181;m strainer and washed several times to filter out any loose cells or crypts prior to plating in BME-R1 matrix to determine their organoid-forming potential. Two main criteria were used to ensure that the resulting organoids were developed from the dedifferentiating villus compartment and not from the crypts: 1) microscopically evaluating&#160;the isolated villi to ensure absence of any tethered crypts, both before and after plating in the 3D matrix, and 2) monitoring the time course of organoid development from the villi. Organoid initiation from the villi occurs only two to five days after plating and appears irregularly shaped, whereas the crypt-derived organoids from the same intestinal epithelium are apparent within sixteen hours of plating and appear spherical. The limitation of the method, however, is that the number of organoids formed, and the time required for organoid initiation from the villi vary depending on the degree of dedifferentiation. Hence, depending upon the specificity of the mutation or the insult causing the dedifferentiation, the optimal stage at which villi can be harvested to assay their organoid forming potential, must be determined empirically.

The procedure describes isolation of the villi from the mouse intestinal epithelium undergoing dedifferentiation to determine their organoid forming potential.

Culturação 3D de Organoides do Epitélio Intestinal Villi passando por Dediferente

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is ...