Este projecto foi apoiado pela National Science Foundation suíço (SNF # 310030-127586/1).

1. A colheita do disco intervertebral <ol><li> Cauda bovina toda comprimento é obtido a partir de um matadouro local, se possível sem pele, pois a presença de pele aumenta a chance de contaminação (Fig. 2). </li><li> Prepare tábua de cortar e preparar grandes estação de trabalho estéril e instrumentos em cima de uma tábua de corte (Fig. 2). </li><li> Prepare sob a gaze estéril capa de fluxo laminar estéril umedecido com cloreto de sódio 0,9% contendo citrato de sódio 55mm e colocado em cada poço da placa de 6 poços. </li><li> Prepare uma bacia e diluir 1:100 de solução Betadine com água da torneira. </li><li> Mergulhe a cauda inteira bovina em uma bacia contendo solução de Betadine 1% por 5 min. </li><li> Brevemente cauda seca com gaze estéril e colocá-lo na estação de trabalho estéril. </li><li> Remova cuidadosamente muscular ao redor da cauda com lâmina de bisturi # 22. </li><li> Corta fora as partes da cauda, ​​que não são necessários, normalmente ambas as extremidades da cauda, ​​que contêm relativamente grandes e muito pequenosIVDs (Fig. 3). </li><li> Guarnição ainda mais o músculo e tendões ao redor do IVD utilizando lâmina de bisturi n º 10. Cuidado para não cortar o anel externo dos discos. </li><li> Marca anterior da IVD com um marcador de pele cirúrgica (este passo é opcional, ele ajuda a determinar o centro de rotação axial em nosso biorreator). </li><li> Localize o IVD e vértebra ponto de conexão, movendo a cauda suavemente. Sinta-se a fronteira entre IVD e osso com o lado chato de uma lâmina de bisturi. O site clivagem deve ser de 1-2 mm de distância do IVD para a vértebra. Personalizado lugar feito lâmina industrial no site clivagem (Fig. 4). </li><li> Cleave e IVD vértebra martelando em cima do suporte de lâmina custom-made industrial (Fig. 3). </li><li> Repita do outro lado da conexão IVD-vértebra. </li><li> Enrole isolado IVD com gaze esterilizada umedecida com cloreto de sódio 0,9% contendo citrato de sódio 55mm. </li><li> Continue o isolamento de IVDs para o número da amostra desejada (geralmente em torno de 6 podeser colhida com diâmetro entre 10-20 mm). </li><li> Conectar o sistema de lavagem a jato (Zimmer, inc.) Com solução de Ringer Lactato estéril é, 5L sacos são um tamanho útil (PBS estéril ou, alternativamente solução salina 0,9%). </li><li> Segure o IVD com uma pinça e jato de lavagem ambos os lados da superfície de placa terminal usando Zimmer Pulsavac sistema de jato de lavagem (Fig. 1C). A arma do jato deveria ser fuzilada na superfície da placa terminal em um ângulo entre 30 - 60 ° por cerca de 30s de cada lado. (Figs. 1 e 7) </li><li> Coloque o IVDs volta para a placa 6-bem e enrole com uma gaze umedecida, enquanto a jato de lavar outra amostra do disco. </li><li> IVDs estão prontos para a cultura de órgãos ea jusante aplicações (por exemplo, a viabilidade celular, carga mecânica, a cultura de órgãos) (Figura 1D). </li></ol> 2. Resultados representante Avaliação dos resultados de julgar uma preparação do disco intervertebral de sucesso pode ser uma experiência de difusão onde um corante fluorescente de baixo peso molecular (por exemplo, vermelho Procion)é preparado como uma solução a 1% em PBS 2. O IVD é mantida com pelo menos 100 mL de solução corante e do corante é permitido então difusa passivamente no IVD por 24h sob inchaço livre. O IVD é, então, flash-congelados em nitrogênio líquido e trazido de volta à temperatura ambiente e desidratados por uma série de transferências de acetona (primeiro pré-resfriada em -80 ° C, em seguida, -20 ° C, então 4 ° C e, finalmente, à temperatura ambiente ). O disco pode então ser incorporados em poli-metil-acrilato (PMMA) e cortar com uma lâmina afiada para produzir 100 mm de espessura. Essas seções podem ser visualizados por qualquer microscópio de campo claro padrão, mas preferencialmente pelo uso de microscopia de fluorescência desde Procion vermelha emite uma fluorescência vermelha (Figs. 5-6). O trabéculas da superfície óssea aparecem muito limpo após a etapa lavaging jet (Fig. 7). Parâmetros para uma cultura de órgão bem-sucedidos são, antes de tudo a ausência de contaminação viabilidade celular, mantido (Figs. 8-9), altura do disco e&quot;integridade do disco&quot; em geral, medida com cortes histológicos e safranina O / verde rápido mancha ou Hematoxilina de Meyer 3. Nós desenvolvemos protocolos e macros publicados em outros lugares 4,5 a mancha tecido do disco ao vivo e para verificar a viabilidade das células em 3D usando um microscópio de varredura confocal a laser eo kit de coloração ao vivo / morto (Molecular Probes, Invitrogen, Basel, Suíça). Nós ainda desenvolveu uma macro e de rotina para realizar a contagem de células automatizado para estimar a viabilidade das células no tecido 3D ou portadores 3D usando a plataforma ImageJ NIH 4-6. Protocolos alternativos têm sido propostos para determinar a viabilidade das células disco pela digestão inicial usando a digestão sequencial da matriz extracelular utilizando pronase colagenase e tipo 2 e em seguida, para manchar a suspensão de células 7. Uma versão anterior do protocolo envolveu um passo de incubação no PBS contendo dez vezes mais concentrado concentração de penicilina / estreptomicina por 10 min após a colheita dos discos anteriores órgão cuLTURE. Com o passo a lavagem a jato esta etapa torna-se obsoleta, pois esta etapa parece benéfica para fins de cultura. <img alt="Figura 1" src="/files/ftp_upload/3490/3490fig1.jpg" /> Figura 1. Visão geral das etapas metodológicas para preparar bovina coccígea discos intervertebrais com placas terminais intactas e uma fina camada (1-2mm) do osso. A) Visão esquemática do disco intervertebral (IVD) e seus caminhos nutricionais. B) Procedimento para cortar o IVD com um personalizado porta-lâmina e uma lâmina afiada industrial. C) lavagem Jet passo para garantir a viabilidade das células de alta e para permitir a troca de nutrientes mantendo a placas e 1-2 mm de osso anexado em ambos os lados. D) Finalmente, o IVD podem ser cultivadas na câmara de espécime de um biorreator ou ser usado para qualquer aplicação a jusante. <img alt="Figura 2" src="/files/ftp_upload/3490/3490fig2.jpg" /> Figura 2 TOP:. Todo o comprimento da cauda de bovino fresca (de preferência obtaINED dentro de 2-3 horas post-mortem para garantir a viabilidade das células de altura. Baixo: imagem de raios-X de uma cauda de bovinos com idade entre cerca de 6 meses, ilustrando a existência de placas de crescimento (GP), centro secundário de ossificação, entre as placas ósseas (EP) e os corpos vertebrais (VB). <img alt="Figura 3" src="/files/ftp_upload/3490/3490fig3.jpg" /> Figura 3. A Tools) para dissecar a cauda bovina em condições estéreis. B) Customized porta-lâmina para cortar-out segmentos dinâmicas da cauda de bovinos de corte em ação, enquanto o disco intervertebral. C) Vista lateral do porta-lâmina personalizado com uma lâmina de padrão industrial (Lutz, Alemanha). <img alt="Figura 4" src="/files/ftp_upload/3490/3490fig4.jpg" /> Figura 4. Desenho e imagem da cauda bovina após a remoção do ligamento muscular circundante e ilustrando os sites cleaving para cortar os segmentos de dinâmicas. Vértebra parcial deve ser &quot;rachado&quot; wiª o porta-lâmina custom-made idealmente 1-2 mm de distância da placas terminais cartilaginosas para garantir superfície óssea completa. <img alt="Figura 5" src="/files/ftp_upload/3490/3490fig5.jpg" /> Figura 5. Demonstração da limpeza eficaz da etapa de lavagem a jato, antes (A) e após (B). Observe as superfícies limpas óssea dos segmentos do disco intervertebral após o procedimento de pulverização. <img alt="Figura 6" src="/files/ftp_upload/3490/3490fig6.jpg" /> Figura 6. Free-inchaço experiência difusão de recém-preparados excisadas discos cauda bovina intervertebral (IVDs) saiu por 24 horas em solução Procion 1% vermelho, demonstrando efeito bloqueio pela placa de crescimento. A vista digitais de raios-X sagital de um IVD ainda contendo a placa de crescimento e uma segunda camada de imagem de microscopia fluorescente de osso corte sagital através do explante disco intervertebral coccígea bovina com placa terminal cartilaginosa e óssea após 24h livre difusão. GP: placa de crescimento, 2ndry CO: centro secundário de ossificação. <img alt="Figura 7" src="/files/ftp_upload/3490/3490fig7.jpg" /> Figura 7. Free-inchaço experiência de recém-preparados excisadas discos cauda bovina intervertebral (IVDs) saiu por 24 horas em solução Procion 1% vermelho, demonstrando efeito do tratamento de limpeza a jato lavagem. Sagital de espessura (~ 100 mm) de excisadas IVDs bovina com ~ 1,5 mmthick placas final ósseo que sofreu lavagem a jato de tratamento (Esquerda) lado controle sem tratamento (Direito) jet-lavaged tratamento. Entrada mostra o padrão de pulverização, que foi usado a partir do sistema Zimmer desbridamento da ferida 24h experimentos livre difusão de bovinos explantes disco intervertebral em vermelho Procion 1%. <img alt="Figura 8" src="/files/ftp_upload/3490/3490fig8.jpg" /> Figura 8. Images Live / Dead of projeções de pilhas confocal Imaging (250μm), proveniente de bovinsegmentos do disco intervertebral e preparado com este método a jato de lavagem mantidos sob livre inchaço para 0 e 14 dias respectivamente para o núcleo pulposo (NP), o interior anel fibroso (IA) eo anel fibroso exterior (OA). Células verde = células vivas manchada pelo calceína AM (acetil metílico), glóbulos vermelhos = núcleo células mortas coradas pelo homodímero de etídio-1. <img alt="Figura 9" src="/files/ftp_upload/3490/3490fig9.jpg" /> Figura 9. Viabilidade celular de disco intervertebral usando microscopia confocal a laser de varredura em tecido vivo e digitalização 3D de pilha. A viabilidade das células de núcleo pulposo (NP) e fibrose do anel (AF) ao longo dos 21 dias de cultura em free-inchaço condição. (Média ± EPM, N = 6) As diferenças estatísticas foram testados usando não-paramétrico Kruskal-Wallis assinado Rank Sum Test entre os grupos. Foram encontradas diferenças significativas entre o dia 21 e todos os pontos de outra época da NP. Enquanto na AF diferenças significativas foram encontradas entre os dias 7 adia 0. (* P &lt;0,05, ** p &lt;0,01).

<ol>
	<li>Lee, C. R., Iatridis, J. C., Poveda, L., Alini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+organ+culture+of+the+bovine+intervertebral+disc:+effects+of+vertebral+endplate+and+potential+for+mechanobiology+studies.">In vitro organ culture of the bovine intervertebral disc: effects of vertebral endplate and potential for mechanobiology studies.</a> Spine (Phila Pa 1976). 31, 515-522 (1976).</li><li>Chan, S. C. W., Gantenbein-Ritter, B., Leung, V. Y., Chan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreserved+intervertebral+disc+with+injected+bone+marrow-derived+stromal+cells:+a+feasibility+study+using+organ+culture.">Cryopreserved intervertebral disc with injected bone marrow-derived stromal cells: a feasibility study using organ culture.</a> Spine. J. 10 (6), 486-496 (2010).</li><li>Chan, S. C., Ferguson, S. J., Wuertz, K., Gantenbein-Ritter, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+Response+of+the+Intervertebral+Disc+to+Repetitive+Short+Term+Cyclic+Torsion.">Biological Response of the Intervertebral Disc to Repetitive Short Term Cyclic Torsion.</a> Spine (Phila Pa 1976). , (2011).</li><li>Gantenbein-Ritter, B., Sprecher, C. M., Chan, S., Illien-J&#252;nger, S., Grad, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+imaging+protocols+for+live/dead+staining+in+three-dimensional+carriers.">Confocal imaging protocols for live/dead staining in three-dimensional carriers.</a> Methods Mol. Biol. 740, 127-140 (2011).</li><li>Gantenbein-Ritter, B., Potier, E., Zeiter, S., van der Werf, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+of+three+techniques+to+determine+cell+viability+in+3D+tissues+or+scaffolds.">Accuracy of three techniques to determine cell viability in 3D tissues or scaffolds.</a> Tissue Engineering Part C Methods. 14, 353-358 (2008).</li><li>Rasband, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ImageJ. , (1997).</li><li>Haschtmann, D., Stoyanov, J. V., Ettinger, L., Nolte, L. P., Ferguson, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+novel+intervertebral+disc/endplate+culture+model:+analysis+of+an+ex+vivo+in+vitro+whole-organ+rabbit+culture+system.">Establishment of a novel intervertebral disc/endplate culture model: analysis of an ex vivo in vitro whole-organ rabbit culture system.</a> Spine. 31, 2918-2925 (2006).</li><li>Gantenbein, B., Gr&#252;nhagen, T., Lee, C. R., van Donkelaar, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+organ+culturing+system+for+intervertebral+disc+explants+with+vertebral+endplates:+a+feasibility+study+with+ovine+caudal+discs.">An in vitro organ culturing system for intervertebral disc explants with vertebral endplates: a feasibility study with ovine caudal discs.</a> Spine. 31, 2665-2673 (2006).</li><li>Korecki, C. L., Kuo, C. K., Tuan, R. S., Iatridis, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intervertebral+disc+cell+response+to+dynamic+compression+is+age+and+frequency+dependent.">Intervertebral disc cell response to dynamic compression is age and frequency dependent.</a> J. Orthop. Res. 27, 800-806 (2009).</li><li>Korecki, C. L., MacLean, J. J., Iatridis, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+compression+effects+on+intervertebral+disc+mechanics+and+biology.">Dynamic compression effects on intervertebral disc mechanics and biology.</a> Spine (Phila Pa 1976). 33, 1403-1409 (1976).</li><li>Jim, B., Steffen, T., Moir, J., Roughley, P., Haglund, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+intact+intervertebral+disc+organ+culture+system+in+which+degeneration+can+be+induced+as+a+prelude+to+studying+repair+potential.">Development of an intact intervertebral disc organ culture system in which degeneration can be induced as a prelude to studying repair potential.</a> European Spine Journal. , 1-11 (2011).</li></ol>

Não temos nada a revelar.

O primeiro passo para a cultura de órgão bem-sucedido é ter certeza de que o explante não deve ser contaminada. A cauda deve ser descascado antes de começar com o procedimento. Qualquer pêlos levados a um laboratório estéril pode ser problemático em termos de contaminação. A cauda bovina deveriam idealmente ser o mais fresco possível (isso afeta a viabilidade celular inicial). Além disso, a etapa de lavagem betadine é recomendada para reduzir o risco de novas contaminações. Em vez de usar uma lâmina cus...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comments (opcional) </td></tr><tr><td colspan="4"> Tecido do disco intervertebral de bovino fresca da cauda de bovinos, de matadouro local (de preferência dentro de horas post-mortem e sem pele). </td></tr><tr><td> Além disso Pulsavac AC Sistema </td><td> Zimmer inc., Suíça </td><td> 00-5150-486-01 </td><td> Melhor desempenho com a cabeça hip-spray e com fonte de alimentação AC (aquele com os 8 AA bateria que também trabalham, mas é menos conveniente) </td></tr><tr><td> Alta Capacidade Fan spray w / Splash Shield, 12,7 centímetros de comprimento </td><td> Zimmer inc., Suíça </td><td> 00-5150-175-00 </td><td> Existem vários pulverizadores disponíveis, nós testamos esta um sucesso </td></tr><tr><td> Lâminas de bisturi # 22 e # 10 </td><td> Swann-Mortem, Inglaterra </td><td> # 10: 0201 # 22: 0208 </td><td></td></tr><tr><td> Titular lâmina de bisturi # 3 e # 4 </td><td> Hausmann, Alemanha </td><td> # 3: 06.103.00 # 4: 06.104.00 </td><td></td></tr><tr><td> Lutz industrial blade </td><td> Lutz, Alemanha </td><td> 1022.0884 </td><td></td></tr><tr><td> Tampão fosfato salino (PBS) </td><td> Invitrogen, Suíça </td><td> 10010-023 </td><td></td></tr><tr><td> Modificado Dulbecco Eagle Medium (DMEM) </td><td> Gibco, Suíça </td><td> 11960-044 </td><td></td></tr><tr><td> Solução de Ringer com lactato (sem glicose) </td><td> Bichsel, Suíça </td><td> 133 0002 </td><td></td></tr><tr><td> 6 bem-placa multi-bem </td><td> TPP, Suíça </td><td> 92006 </td><td></td></tr><tr><td> Solução de Betadine </td><td> Mundipharma, Suíça </td&gt; <td> 10055025 </td><td></td></tr><tr><td> Marcador cirúrgico da pele </td><td> Porex Cirúrgica, Suíça </td><td> 9560 </td><td></td></tr><tr><td> Tábua de corte grande </td><td></td><td></td><td> Qualquer marca é possível </td></tr></tbody></table>

preparation of intact bovine tail intervertebral discs for organ culture

O disco intervertebral (IVD) é a articulação da coluna vertebral conectando vértebra em vértebra. Ele funciona para transmitir carga de coluna vertebral e dar flexibilidade à coluna vertebral. Compõe de três compartimentos: o núcleo pulposo interior (NP) que abrange pelo anel fibroso (AF) e duas placas terminais cartilaginosas que liga o NP e AF do corpo vertebral em ambos os lados. Dor discogênica possivelmente causada por uma doença degenerativa do disco intervertebral (DDD) e hérnia discal tem sido identificada como um problema grave em nossa sociedade moderna. Para estudar possíveis mecanismos de degeneração IVD, em sistemas de órgãos de cultura in vitro com células de discos ao vivo são altamente atraente. O cultivo in vitro de bovinos intactos IVDs coccígea tem avançado a um sistema-modelo relevante, que permite o estudo de mecano-biológico aspectos em um ambiente bem controlado fisiológicas e mecânicas. IVDs cauda de bovinos podem ser obtidos relativamente fácil em maior número umd são muito semelhantes ao humano IVDs lombar em relação à densidade celular, a população de células e dimensões. No entanto, anterior caudal bovina técnicas de colheita IVD retenção placas terminais cartilaginosas e ósseas placas terminais falhou após 1-2 dias de cultura desde as vias de nutrição foram, obviamente, bloqueada por sangue coagulado. IVDs são os maiores órgãos avascular, assim, os nutrientes para as células do NP são exclusivamente dependentes de difusão através da brotos capilares do corpo vertebral adjacente. Presença de resíduos de osso e sangue coagulado nas superfícies placa motora pode dificultar a difusão de nutrientes para o centro do disco e viabilidade celular compromisso. O nosso grupo estabeleceu um protocolo relativamente rápida de &quot;crack&quot;-out do IVDs da cauda com um baixo risco de contaminação. Somos capazes de permeabilizar o recém-cortadas superfícies ósseas placa terminal usando um sistema de lavagem a jato cirúrgica, que remove os coágulos de sangue e restos de corte e de forma muito eficiente reabre o caminho de difusão de nutriçãopara o centro do IVD. A presença de placas de crescimento em ambos os lados do osso vertebral tem que ser evitado e ser removidos antes da cultura. Neste vídeo, que descrevem as etapas cruciais durante a preparação e demonstrar a chave para uma cultura de órgão bem-sucedido manter a viabilidade celular de alta por 14 dias, sob inchaço cultura livre. O tempo de cultura poderia ser estendido quando o ambiente mecânico adequado pode ser mantida usando biorreator carga mecânica. A técnica demonstrada aqui pode ser estendido a outras espécies animais, como suínos, ovinos e isolamento caudal e lombar leporinos IVD.

Watch this Scientific Journal Video about Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture at JoVE.com

Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture

Este protocolo ilustra uma técnica de colheita, para coccígea bovina discos intervertebrais para a cultura de órgãos para<em&gt; In vitro</em&gt; Cultura do órgão.

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  ...

preparation-intact-bovine-tail-intervertebral-discs-for-organ

Research

JoVE Journal

Bioengineering

14.4K visualizações.  University of Bern. Este protocolo ilustra uma técnica de colheita, para coccígea bovina discos intervertebrais para a cultura de órgãos para In vitro Cultura do órgão.

Vídeo: Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture

Preparation of Complaint Matrices for Quantifying Cellular Contraction

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling. Focal adhesions are macromolecular assemblies that couple the contractile F-actin cytoskeleton to the ECM. This connection allows for the transmission of intracellular mechanical forces across the cell membrane to the underlying substrate. Recent work has shown the mechanical properties of the ECM regulate focal adhesion and F-actin morphology as well as numerous physiological processes, including cell differentiation, division, proliferation and migration. Thus, the use of cell culture substrates has become an increasingly prevalent method to precisely control and modulate ECM mechanical properties.
To quantify traction forces at focal adhesions in an adherent cell, compliant substrates are used in conjunction with high-resolution imaging and computational techniques in a method termed traction force microscopy (TFM). This technique relies on measurements of the local magnitude and direction of substrate deformations induced by cellular contraction. In combination with high-resolution fluorescence microscopy of fluorescently tagged proteins, it is possible to correlate cytoskeletal organization and remodeling with traction forces.
Here we present a detailed experimental protocol for the preparation of two-dimensional, compliant matrices for the purpose of creating a cell culture substrate with a well-characterized, tunable mechanical stiffness, which is suitable for measuring cellular contraction. These protocols include the fabrication of polyacrylamide hydrogels, coating of ECM proteins on such gels, plating cells on gels, and high-resolution confocal microscopy using a perfusion chamber. Additionally, we provide a representative sample of data demonstrating location and magnitude of cellular forces using cited TFM protocols.

In this video, we demonstrate the experimental techniques used to fabricate compliant, extracellular matrix (ECM) coated substrates suitable for cell culture, and which are amenable to traction force microscopy and observing effects of ECM stiffness on cell behavior.

Preparação de Matrizes Reclamação para Quantificar contração celular

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling.  Focal adhesions are ...

Bioengenharia

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 ...

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

Preparação de fibrina Andaimes 3D para aplicações de cultura de células-tronco

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

Amostra Estratégias Preparação para Mass Spectrometry imagem da cultura celular 3D Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments,&#160;as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier,&#160;more quantifiable extracellular relay of signals to the cells. 
For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

O Chip Multi-organ - Uma plataforma microfluídica para longo prazo Coculture Multi-tecidos

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

Estudando a estequiometria do Receptor do Factor de Crescimento Epidérmico em células intactas utilizando Microscopia Correlativo

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

Preparação de Thermoresponsive nanoestruturados Surfaces para Engenharia de Tecidos

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of visible light with thermally induced acoustic waves or phonons propagating at a speed of a few km/sec. Information on the elasticity and structure of the material is obtained in a nondestructive contactless manner, hence opening the way to in vivo applications and potential diagnosis of pathology. This work describes the application of Brillouin spectroscopy to the study of biomechanics in elastin and trypsin-digested type I collagen fibers of the extracellular matrix. Fibrous proteins of the extracellular matrix are the building blocks of biological tissues and investigating their mechanical and physical behavior is key to establishing structure-function relationships in normal tissues and the changes which occur in disease. The procedures of sample preparation followed by measurement of Brillouin spectra using a reflective substrate are presented together with details of the optical system and methods of spectral data analysis.

We present a protocol for the application of Brillouin light scattering spectroscopy to elastin and trypsin-purified type I collagen fibers of the extracellular matrix to extract their full elastic properties.

Preparação de Matriz Extracelular fibras de proteína de Brillouin Spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

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

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

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

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

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

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

Fabricação e validação de um sistema de órgãos-em-microplaqueta com eléctrodos integrados para quantificar diretamente a resistência elétrica de Transendothelial

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...