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

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

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

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

Hidrogéis alginato para Tridimensional Cultura Órgão de ovários e tubas uterinas

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

Bioengenharia

Preparation of 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 ...

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 ...

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 ...

Fragility Assessment of Bovine Cortical Bone Using Scratch Tests

Bone is a complex hierarchical material with five distinct levels of organization. Factors like aging and diseases like osteoporosis increase the fragility of bone, making it fracture-prone. Owing to the large socio-economic impact of bone fracture in our society, there is a need for novel ways to assess the mechanical performance of each hierarchical level of bone. Although stiffness and strength can be probed at all scales &#8211; nano-, micro-, meso-, and macroscopic &#8211; fracture assessment has so far been confined to macroscopic testing. This limitation restricts our understanding of bone fracture and constrains the scope of laboratory and clinical studies. In this research, we investigate the fracture resistance of bone from the microscopic to the mesoscopic length scales using micro scratch tests combined with nonlinear fracture mechanics. The tests are performed in the short longitudinal orientation on bovine cortical bone specimens. A meticulous experimental protocol is developed and a large number (102) of tests are conducted to assess the fracture toughness of cortical bone specimens while accounting for the heterogeneity associated with bone microstructure.

This study assesses the fracture toughness of bovine cortical bone at the sub-meso levels using microscopic scratch tests. This is an original, objective, rigorous, and reproducible method proposed to probe fracture toughness below the macroscopic scale. Potential applications are studying changes in bone fragility due to diseases like osteoporosis.

Avaliação da fragilidade do osso Cortical bovino, usando testes de zero

Bone is a complex hierarchical material with five distinct levels of organization. Factors like aging and diseases like osteoporosis increase the ...

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human patients. Human Organ-on-a-Chip (Organ Chip) microfluidic cell culture devices, which provide an experimental in vitro platform to assess efficacy, toxicity, and pharmacokinetic (PK) profiles in humans, may be better predictors of therapeutic efficacy and safety in the clinic compared to animal studies. These devices may be used to model the function of virtually any organ type and can be fluidically linked through common endothelium-lined microchannels to perform in vitro studies on human organ-level and whole body-level physiology without having to conduct experiments on people. These Organ Chips consist of two perfused microfluidic channels separated by a permeable elastomeric membrane with organ-specific parenchymal cells on one side and microvascular endothelium on the other, which can be cyclically stretched to provide organ-specific mechanical cues (e.g., breathing motions in lung). This protocol details the fabrication of flexible, dual channel, Organ Chips through casting of parts using 3D printed molds, enabling combination of multiple casting and post-processing steps. Porous poly (dimethyl siloxane) (PDMS) membranes are cast with micrometer sized through-holes using silicon pillar arrays under compression. Fabrication and assembly of Organ Chips involves equipment and steps that can be implemented outside of a traditional cleanroom. This protocol provides researchers with access to Organ Chip technology for in vitro organ- and body-level studies in drug discovery, safety and efficacy testing, as well as mechanistic studies of fundamental biological processes.

Here, we present a protocol that describes the fabrication of stretchable, dual channel, organ chip microfluidic cell culture devices for recapitulating organ-level functionality in vitro.

Fabricação escalável de Stretchable, Dual Channel, órgão Microfluidic Chips

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human ...

Anterior Segment Organ Culture Platform for Tracking Open Globe Injuries and Therapeutic Performance

Open globe injuries have poor visual outcomes, often resulting in permanent loss of vision. This is partly due to an extended delay between injury and medical intervention in rural environments and military medicine applications where ophthalmic care is not readily available. Untreated injuries are susceptible to infection after the eye has lost its watertight seal, as well as loss of tissue viability due to intraocular hypotension. Therapeutics to temporarily seal open globe injuries, if properly developed, may be able to restore intraocular pressure and prevent infection until proper ophthalmic care is possible. To facilitate product development, detailed here is the use of an anterior segment organ culture open globe injury platform for tracking therapeutic performance for at least 72 h post-injury. Porcine anterior segment tissue can be maintained in custom-designed organ culture dishes and held at physiological intraocular pressure. Puncture injuries can be created with a pneumatic-powered system capable of generating injury sizes up to 4.5 mm in diameter, similar to military-relevant injury sizes. Loss of intraocular pressure can be observed for 72 h post-injury confirming proper injury induction and loss of the eye&#39;s watertight seal. Therapeutic performance can be tracked by application to the eye after injury induction and then tracking intraocular pressure for multiple days. Further, the anterior segment injury model is applicable to widely used methods for functionally and biologically tracking anterior segment physiology, such as assessing transparency, ocular mechanics, corneal epithelium health, and tissue viability. Overall, the method described here is a necessary next step toward developing biomaterial therapeutics for temporarily sealing open globe injuries when ophthalmic care is not readily available.

Open globe eye injuries may go untreated for multiple days in rural or military-relevant scenarios, resulting in blindness. Therapeutics are needed to minimize loss of vision. Here, we detail an organ culture open globe injury model. With this model, potential therapeutics for stabilizing these injuries can be properly evaluated.

Plataforma de Cultura de Órgãos do Segmento Anterior para Rastreamento de Lesões no Globo Aberto e Desempenho Terapêutico

Open globe injuries have poor visual outcomes, often resulting in permanent loss of vision. This is partly due to an extended delay between injury and ...

Reconstituting Cytoarchitecture and Function of Human Epithelial Tissues on an Open-Top Organ-Chip

Nearly all human organs are lined with epithelial tissues, comprising one or multiple layers of tightly connected cells organized into three-dimensional (3D) structures. One of the main functions of epithelia is the formation of barriers that protect the underlining tissues against physical and chemical insults and infectious agents. In addition, epithelia mediate the transport of nutrients, hormones, and other signaling molecules, often creating biochemical gradients that guide cell positioning and compartmentalization within the organ. Owing to their central role in determining organ-structure and function, epithelia are important therapeutic targets for many human diseases that are not always captured by animal models. Besides the obvious species-to-species differences, conducting research studies on barrier function and transport properties of epithelia in animals is further compounded by the difficulty of accessing these tissues in a living system. While two-dimensional (2D) human cell cultures are useful for answering basic scientific questions, they often yield poor in vivo predictions. To overcome these limitations, in the last decade, a plethora of micro-engineered biomimetic platforms, known as organs-on-a-chip, have emerged as a promising alternative to traditional in vitro and animal testing. Here, we describe an Open-Top Organ-Chip (or Open-Top Chip), a platform designed for modeling organ-specific epithelial tissues, including skin, lungs, and the intestines. This chip offers new opportunities for reconstituting the multicellular architecture and function of epithelial tissues, including the capability to recreate a 3D stromal component by incorporating tissue-specific fibroblasts and endothelial cells within a mechanically active system. This Open-Top Chip provides an unprecedented tool for studying epithelial/mesenchymal and vascular interactions at multiple scales of resolution, from single cells to multi-layer tissue constructs, thus allowing molecular dissection of the intercellular crosstalk of epithelialized organs in health and disease.

The present protocol describes the capabilities and the essential culture modalities of the Open-Top Organ-Chip for the successful establishment and maturation of full-thickness organ-on-chip cultures of primary tissues (skin, alveolus, airway, and intestine), providing the opportunity to investigate different functional aspects of the human epithelial/mesenchymal and vascular niche interface in vitro.

Reconstituindo a citoarquitetura e a função de tecidos epiteliais humanos em um chip de órgão de topo aberto

Nearly all human organs are lined with epithelial tissues, comprising one or multiple layers of tightly connected cells organized into three-dimensional ...