Os autores gostariam de agradecer ao Sr. Tam Po Leung, o Sr. Wong Chi Kin e do pessoal técnico da Faculdade Science Workshop, Hong Kong Baptist University, por suas habilidades técnicas e conselhos para tornar o protótipo desta wounder.

1. Introdução às Partes do wounder (Figura 1) <ol><li> Prepare o suporte do pino, fixando os pinos ferindo pelo distâncias iguais. Segure as pontas de pipeta e ajustar a altura da ponta, movendo os pinos ferindo ajustáveis ​​para cima e para baixo. </li><li> Montar o guiamento-barra ajustável em diferentes tipos de placas de cultura de 96 poços e garantir que a área da ferida está numa posição fixa. </li><li> Fixe o pino de ferimento ajustável apertando o parafuso de hex. Fixar a barra-guia na posição apertando as tampas sextavados em ambas as extremidades. </li></ol> 2. Definir a largura da caixa Pin (Figura 2) <ol><li> Solte as tampas sextavados com a chave sextavada M5. </li><li> Inserir um número adequado de ajuste anéis de ambos os lados do suporte de pino de modo a que as barras de orientação encaixar perfeitamente com a largura da placa de cultura de 96 poços. NOTA: Corning e placas de Iwaki requer um par de anéis grandes e 1 par de anéis pequenos. Falcon e Nunc plates precisa de 1 par de anéis grandes. </li><li> Aperte o de cabeça hexagonal tampões para fixar a barra de guia. </li></ol> 3. Ajustar as Guias de (Figura 3) <ol><li> Encaixe os pinos ferindo com estéreis de 10 ul pontas de pipeta de plástico. </li><li> Solte as tampas sextavados com a chave sextavada M5. </li><li> Segurar o wounder e mergulhar os pinos ferindo em uma coluna de uma placa de cultura de 96 poços. </li><li> Ajustar a altura das barras de orientação até que todas as pontas quase tocar no fundo dos poços. </li><li> Aperte as tampas sextavados. Certifique-se de que o wounder agora se encaixa perfeitamente sobre a placa de cultura e que os pinos estão bem posicionados no meio dos poços. </li></ol> 4. A calibração dos pinos <ol><li> Segure o wounder perpendicular a uma superfície plana estéril (ou seja, uma placa de Petri) e solte todos os parafusos sextavados com a chave sextavada M3. </li><li> Toque no wounder até que todas as dicas uniformemente toque na superfície estéril plana. &lt;/ Li&gt; <li> Apertar os parafusos sextavados novamente para bloquear os pinos na posição. </li><li> Verifique a regularidade de cada ponta tocando no wounder suavemente na superfície estéril plana. </li></ol> 5. Coçar celular monocamada (Figura 4) <ol><li> Semear as células endoteliais vasculares umbilicais humanas em uma placa de 96 poços de cultura de células revestidas com gelatina a 0,1%. células de cultura em meio 199 suplementado com 20% de soro inactivado por calor fetal de bovino, 1% de penicilina / estreptomicina e 0,09 g / L de heparina. Manter as células numa incubadora humidificada com dióxido de carbono a 5% a 37 ºC durante a noite antes de ser utilizada. As células devem chegar a 100% de confluência antes ferindo. </li><li> Coloque as pontas ferir-se no mais à esquerda (ou o mais à direita) do lado de cada cavidade dentro de uma mesma coluna da placa de cultura. Deslize a wounder para o outro lado do poço; garantir que as pontas são ferindo tocar no fundo do poço. </li><li> Repita a coçar (Passo 5.2) para todas as colunas. </li><li> Depois de ferir, descarte tele meio em cada poço e substituir com compostos de teste de meio fresco contendo. </li></ol> 6. Aquisição de Dados e Análise de Imagem <ol><li> Imagem inteira, o poço com a ferida mecânica sobre a monocamada de células utilizando um microscópio de baixa potência com a câmara digital (por exemplo objectiva 10X) imediatamente após o ferimento (t = 0 h). </li><li> Adicionar os compostos de teste, se desejado e incubar durante o período de tempo desejado. Toda a imagem bem com a área da ferida novamente após o tempo de incubação desejado (t = ÔH). </li><li> Usando imagem software de análise tais como ImageJ ( <a href="https://imagej.nih.gov/ij/">https://imagej.nih.gov/ij/</a> ), medir a área ferido na imagem usando a ferramenta à mão livre. </li><li> Calcula-se a percentagem de fecho de feridas: <img alt="Equação" src="/files/ftp_upload/55411/55411eq1.jpg" /></li></ol>

<ol>
	<li>Friedl, P., Wolf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour-cell+invasion+and+migration:+diversity+and+escape+mechanisms.">Tumour-cell invasion and migration: diversity and escape mechanisms.</a> Nat Rev Cancer. 3 (5), 362-374 (2003).</li><li>Friedl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prespecification+and+plasticity:+shifting+mechanisms+of+cell+migration.">Prespecification and plasticity: shifting mechanisms of cell migration.</a> Curr Opin Cell Biol. 16 (1), 14-23 (2004).</li><li>Lampugnani, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration+into+a+wounded+area+in+vitro.">Cell migration into a wounded area in vitro.</a> Methods Mol Biol. 96, 177-182 (1999).</li><li>Sholley, M. M., Gimbrone, M. A., Cotran, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+migration+and+replication+in+endothelial+regeneration:+a+study+using+irradiated+endothelial+cultures.">Cellular migration and replication in endothelial regeneration: a study using irradiated endothelial cultures.</a> Lab Invest. 36 (1), 18-25 (1977).</li><li>Gotlieb, A. I., Spector, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+into+an+in+vitro+experimental+wound:+a+comparison+of+porcine+aortic+endothelial+and+smooth+muscle+cells+and+the+effect+of+culture+irradiation.">Migration into an in vitro experimental wound: a comparison of porcine aortic endothelial and smooth muscle cells and the effect of culture irradiation.</a> Am J Pathol. 103 (2), 271-282 (1981).</li><li>Chen, Y. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+migration+chip+for+chemotaxis-based+microfluidic+selection+of+heterogeneous+cell+populations.">Single-cell migration chip for chemotaxis-based microfluidic selection of heterogeneous cell populations.</a> Sci Rep. 18 (5), 9980 (2015).</li><li>Yarrow, J. C., Periman, Z. E., Westwood, N. J., Mitchison, T. 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+high-throughput+cell+migration+assay+using+scratch+wound+healing,+a+comparsion+of+image-based+readout+methods.">A high-throughput cell migration assay using scratch wound healing, a comparsion of image-based readout methods.</a> BMC Biotechnol. 4, 21 (2004).</li><li>Lauder, H., Frost, E. E., Hiley, C. R., Fan, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+the+repair+process+involved+in+the+repair+of+a+cell+monolayer+using+an+in+vitro+model+of+mechanical+injury.">Quantification of the repair process involved in the repair of a cell monolayer using an in vitro model of mechanical injury.</a> Angiogenesis. 2 (1), 67-80 (1998).</li><li>Yue, P. Y. K., Leung, E. P. Y., Mak, N. K., Wong, R. N. 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+simplified+method+for+quantifying+cell+migration/+wound+healing+in+96-well+plates.">A simplified method for quantifying cell migration/ wound healing in 96-well plates.</a> J. Biomol Screen. 15 (4), 427-433 (2010).</li><li>Yue, P. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elucidation+of+the+mechanisms+underlying+the+angiogenic+effects+of+ginsenoside+Rg(1)+in+vivo+and+in+vitro.">Elucidation of the mechanisms underlying the angiogenic effects of ginsenoside Rg(1) in vivo and in vitro.</a> Angiogenesis. 8 (3), 205-216 (2005).</li><li>Kwok, H. H., Chan, L. S., Poon, P. Y., Yue, P. Y., Wong, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ginsenoside-Rg1+induces+angiogenesis+by+the+inverse+regulation+of+MET+tyrosine+kinase+receptor+expression+through+miR-23a.">Ginsenoside-Rg1 induces angiogenesis by the inverse regulation of MET tyrosine kinase receptor expression through miR-23a.</a> Toxicol Appl Pharmacol. 287 (3), 276-283 (2015).</li></ol>

Os autores declaram que não têm interesses financeiros concorrentes.

Nossa wounder célula tem várias características únicas em resolver os problemas dos ensaios de migração celular tradicionais. A célula wounder mecânica 8-canal é feita de aço inoxidável de alto grau (Aço 304) com uma longa vida útil que pode ser esterilizado em autoclave. Quase todas as marcas comerciais de placas de 96 poços de cultura disponíveis no mercado podem ser usadas com esta wounder célula mecânica por causa da barra de orientação ajustável. O desenho da barra de orientação ajustável também assegura que a área de coçar é no centro de cada poço. A ferida em posição central do bem pode facilitar a captura de imagem utilizando um microscópio. As imagens possuem algumas variações, enquanto tomadas em diferentes pontos de tempo, mas eles não afetam a análise de dados, porque quase 90% da área do poço pode ser visto pelo microscópio com uma objectiva de baixa ampliação (ou seja, 10X). Assim, uma análise semi-elevado rendimento pode ser realizado em placas de 96 poços de cultura de 7, &lt;sup class = &quot;xref&quot;&gt; 8. No entanto, este método requer ainda um grande número de imagens, que necessitam de uma análise de dados longa. Isso pode ser resolvido se um sistema de aquisição de imagem automática está disponível. O design dos pinos ajustáveis ​​individuais do wounder permite que mesmo contacte com a superfície da placa de 96 poços para atingir feridas idênticas, lisas e afiadas na monocamada de células. Os pinos coçar são comerciais dicas plástico pipetas, que podem ser esterilizados e eliminados depois de cada arranhão, e podem fornecer grande elasticidade entrar em contato com a superfície do bem, sem danos mecânicos. Estas vantagens podem minimizar ainda mais a contaminação cruzada de amostras durante a experiência ea influência sobre o movimento de células em toda a área desnudo. No futuro, vamos desenvolver um pino controlada por mola que pode eliminar o passo pino de ajuste de altura. O passo mais crítico para usar este dispositivo é a força aplicada sobre a monocamada de células durante o coçar. O wounder deveser realizada perpendicularmente contra a placa de cultura, de modo que oito pontas de pipeta são tocar no fundo do poço. Com efeito, o wounder proporciona um método para efectuar o ensaio de migração de células sem o uso de equipamento caro e complicado. O protocolo para este wounder é simples e direta. Em conclusão, nosso wounder mecânica de 8 canais é um dispositivo fácil de usar para os ensaios de migração celular que não requer habilidades especiais. Os utilizadores podem efectuar o ensaio de migração de células em placas de 96 poços com um número relativamente grande de amostras de um modo rápido e simples, 9, 10, 11.

A migração celular desempenha um papel importante nos processos celulares, tais como a embriogénese, neurogénese, a angiogénese, a cura de feridas, a reparação da mucosa,-epiteliais mesenquimais-transições em desenvolvimento normal e uma situação de doença. É um processo complicado que envolve a coordenação de numerosos eventos inter e intra-celulares, incluindo as interacções molécula de sinalização, célula de polarização, a reorganização do citoesqueleto, remodelação da matriz, protrusão membrana e célula-célula dinâmica de modulação da adesão 2. Ao estudar a migração celular, pode ser determinada a descoberta e validação de produtos químicos ou biomoléculas que conduzem a movimentação das células e dos seus percursos bioquímicos relacionados. Este processo fundamental pode ser usado beneficamente como um proxy para diversas aplicações, tais como o tratamento de doença e drogas segmentação. O ensaio ferindo é um dos ensaios de migração de células 3. Aqui, umamostra individual de células irá ser parcialmente removido por meios mecânicos para produzir uma área desnudada onde as células vão migrar para cobrir a área. A percentagem de recuperação em um ponto de tempo em particular irá ser monitorizada. Ao manusear grande número de amostras, questões como o custo experimental e contaminação cruzada dentro de amostras pode ser problemático. Apesar de muitas ferramentas comerciais e ensaios estão disponíveis para estudar a migração de células 4, 5, 6, muitos deles necessários equipamentos caros e sofisticados e melhorias ainda são necessários. Por estas razões, uma célula wounder mecânica de 8 canais (Figura 1) é desenvolvido. O nosso wounder célula mecânica de 8 canais incorporou várias características únicas em resolver os problemas acima mencionados. Ele proporciona a flexibilidade para efectuar a migração de células / ferindo ensaios em formato de placa de cultura de 96 poços. A gu ajustávelbar design Iding assegura que a área de zero é colocada na posição central de cada poço. Além disso, a altura das barras de orientação pode ser ajustada de modo que o wounder é aplicável para vários tipos de placas de cultura. Finalmente, o desenho ferimento pin ajustável permite que um mesmo contato das pontas de pipeta com a superfície da placa de cultura para alcançar simultânea e reprodutível ferimento 7, 8.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-well cell culture plate</td>
			<td>Nunc</td>
			<td>167008</td>
			<td>Other brands of 96-well cell culture plate can also be used</td>
		</tr>
		<tr>
			<td>P10 pipette tips</td>
			<td>Axygen</td>
			<td>301-03-051</td>
			<td>Short P10 pipette tip is more easy to create a clear wound</td>
		</tr>
		<tr>
			<td>Wounder</td>
			<td>R&#38;P Technology Limited</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Medium 199</td>
			<td>Sigma</td>
			<td>M2520-1L</td>
			<td>For cell culture of human umbilical vein endothelial cells. 
			Use appropirate culture medium and condition for other type of cells.</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>26140079</td>
			<td>For cell culture of human umbilical vein endothelial cells. 
			Use appropirate culture medium and condition for other type of cells.</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>For cell culture of human umbilical vein endothelial cells. 
			Use appropirate culture medium and condition for other type of cells.</td>
		</tr>
		<tr>
			<td>Heparin sodium salt from porcine intestinal mucosa</td>
			<td>Sigma</td>
			<td>H3393</td>
			<td>For cell culture of human umbilical vein endothelial cells. 
			Use appropirate culture medium and condition for other type of cells.</td>
		</tr>
		<tr>
			<td>Gelatin from bovine skin</td>
			<td>Sigma</td>
			<td>G9391</td>
			<td>For cell culture of human umbilical vein endothelial cells. 
			Use appropirate culture medium and condition for other type of cells.</td>
		</tr>
	</tbody>
</table>

a device for performing cell migration/wound healing in a 96-well plate

A migração de células / ferindo ensaio é um método vulgarmente utilizado para estudar a migração celular e outros processos biológicos, tais como angiogénese e metástase do tumor. Neste ensaio, as células são cultivadas para formar uma monocamada confluente e um ferimento mecânico é criado por raspagem com um dispositivo. Em seguida, a taxa de migração das células em relação à área desnudada pode ser monitorizada por meio de imagens. O nosso wounder mecânica de 8 canais foi concebida para resolver a maior parte dos problemas associados com o ensaio de migração celular. Em primeiro lugar, o nosso wounder pode ser facilmente esterilizados por autoclave ou com desinfectantes comuns. Em segundo lugar, os pinos individuais ajustáveis ​​permitem mesmo contacte com a placa de cultura celular de modo a que as feridas afiadas e reprodutíveis pode ser criado. Em terceiro lugar, as barras de orientadores em ambos os lados da posição de ferimento wounder assegurar consistente em cada poço. O uso de descartáveis ​​de plástico dicas de pipeta para ferindo pode fornecer mais melhor manuseio do wounder, bem como para minimizar cross-de contaminantesíon. Em conclusão, a wounder célula pode proporcionar aos investigadores um dispositivo reprodutível e amigável do utilizador para realizar o ensaio de migração de células usando a placa de cultura de 96-poços padrão.

Este wounder mecânica 8-canal é construído para arranhar uma monocamada de células, a fim de realizar o ensaio de migração celular. É um dispositivo fácil de usar que pode realizar ensaios coçando de células em placas de 96 poços em menos de um minuto, sem treinamento especial. Este wounder pode introduzir áreas feridas em monocamadas de células com uma largura uniforme de cerca de 600 mm e com bordas afiadas (Figuras 5 e 6). A largura das feridas depende da marca de pontas de pipeta e desempenho do usuário. Esta gama de largura ferida é adequado para a imagem comum a captura sob ampliação de baixa potência (Figura 7). Após a captura da imagem, a percentagem do fecho da ferida pode ser calculada. Este wounder celular incorporou estratégias diferentes para resolver os problemas encontrados com o ensaio de migração celular. Além disso, ele proporciona flexibilidade na realização de ensaios de migração de células em vários pl de 96 poços de culturacomeu formatos. <img alt="figura 1" src="/files/ftp_upload/55411/55411fig1.jpg" /> Figura 1: wounder célula mecânica de oito canais. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55411/55411fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 2" src="/files/ftp_upload/55411/55411fig2.jpg" /> Figura 2: Ajustar a largura do suporte do pino. Inserir os anéis de ajuste, de modo a montar a placa de 96 poços. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55411/55411fig2large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 3" src="/files/ftp_upload/55411/55411fig3.jpg" /> Figura 3. Ajuste as barras orientadores para caber diferentes formatos de placas. </strong&gt; Deslize a barra de guia para cima e para baixo para ajustar a placa de 96 poços. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55411/55411fig3large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 4" src="/files/ftp_upload/55411/55411fig4.jpg" /> Figura 4: Riscando direção da ponta. Arranhar as células deslizando o wounder a partir do um lado do fundo do poço para o outro lado do fundo do poço. <img alt="Figura 5" src="/files/ftp_upload/55411/55411fig5.jpg" /> Figura 5: Visão geral da placa de 96 poços após o ferimento. A monocamada de células é ferido pela wounder produzindo arestas com largura uniforme de cerca de 600 mm. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55411/55411fig5large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> &lt;<html> / P&gt; <img alt="Figura 6" src="/files/ftp_upload/55411/55411fig6.jpg" /> Figura 6: Imagens representativas de uma fileira e uma coluna de poços imediatamente após arranhão. A análise quantitativa da largura da cada ferida. As imagens são capturadas sob uma objetiva de 10X. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55411/55411fig6large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 7" src="/files/ftp_upload/55411/55411fig7.jpg" /> Figura 7: As imagens captadas ferida (objectiva 10X) em t = 0 horas e t = ÔH. A linha ponteada indica a área da ferida e a linha a cheio indica a área da migração celular. A) As células endoteliais da veia umbilical humana. B) fibroblastos dérmicos humanos.obter = &quot;_ blank&quot;&gt; Clique aqui para ver uma versão maior desta figura.

Watch this Scientific Journal Video about A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate at JoVE.com

A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate

Aqui, nós apresentamos um protocolo para realizar um ensaio de migração de células semi-elevado rendimento sobre uma placa de cultura celular de 96 poços. Este protocolo é um método rápido, simples e econômica para criar feridas scratch consistentes sobre a monocamada de células.

Um dispositivo para executar a migração celular / cicatrização de feridas em uma placa de 96 poços

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor ...

a-device-for-performing-cell-migrationwound-healing-in-a-96-well-plate

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

10.4K visualizações.  Hong Kong Baptist University.  Aqui, nós apresentamos um protocolo para realizar um ensaio de migração de células semi-elevado rendimento sobre uma placa de cultura celular de 96 poços. Este protocolo é um método rápido, simples e econômica para criar feridas scratch consistentes sobre a monocamada de células. 

Vídeo: A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

Compreender Organogenesis precoce Usando um Simplificado<em&gt; In Situ</em&gt; Protocolo de hibridação em<em&gt; Xenopus</em

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

Biologia do Desenvolvimento

Automated Quantification of Hematopoietic Cell &#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.

A strategy to quantitatively analyze histological data in the bone marrow is presented. Confocal microscopy of fluorescently labeled cells in tissue sections results in 2-dimensional images, which are automatically analyzed. Co-localization analyses of different cell types are compared to data from simulated images, giving quantitative information about cellular interactions.

Automated Quantificação de células hematopoiéticas - Interações de células estromais em imagens histológicas de osso não descalcificadas

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. ...

Scalable 96-well Plate Based iPSC Culture and Production Using a Robotic Liquid Handling System

Continued advancement in pluripotent stem cell culture is closing the gap between bench and bedside for using these cells in regenerative medicine, drug discovery and safety testing. In order to produce stem cell derived biopharmaceutics and cells for tissue engineering and transplantation, a cost-effective cell-manufacturing technology is essential. Maintenance of pluripotency and stable performance of cells in downstream applications (e.g., cell differentiation) over time is paramount to large scale cell production. Yet that can be difficult to achieve especially if cells are cultured manually where the operator can introduce significant variability as well as be prohibitively expensive to scale-up. To enable high-throughput, large-scale stem cell production and remove operator influence novel stem cell culture protocols using a bench-top multi-channel liquid handling robot were developed that require minimal technician involvement or experience. With these protocols human induced pluripotent stem cells (iPSCs) were cultured in feeder-free conditions directly from a frozen stock and maintained in 96-well plates. Depending on cell line and desired scale-up rate, the operator can easily determine when to passage based on a series of images showing the optimal colony densities for splitting. Then the necessary reagents are prepared to perform a colony split to new plates without a centrifugation step. After 20 passages (~3 months), two iPSC lines maintained stable karyotypes, expressed stem cell markers, and differentiated into cardiomyocytes with high efficiency. The system can perform subsequent high-throughput screening of new differentiation protocols or genetic manipulation designed for 96-well plates. This technology will reduce the labor and technical burden to produce large numbers of identical stem cells for a myriad of applications.

The protocols described allow laboratories to perform scalable, adherent stem cell culture in high throughput with minimal labor, experience and equipment investment cost using a programmable liquid handling robot and 96-well plates. iPSCs passaged more than 20 times on this system maintained pluripotency, normal karyotypes and differentiated into cardiomyocytes.

Escalável placa de 96 poços Baseado iPSC Cultura e Produção Usando um sistema de manipulação robótica Líquido

Continued advancement in pluripotent stem cell culture is closing the gap between bench and bedside for using these cells in regenerative medicine, drug ...

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to follow and manipulate the wound response ex vivo in an environment that closely mimics that of epithelial tissue injury in vivo. This issue was addressed by creating a clinically relevant epithelial ex vivo injury-repair model based on cataract surgery. In this culture model, the response of the lens epithelium to wounding can be followed live in the cells&#8217; native microenvironment, and the molecular mediators of wound repair easily manipulated during the repair process. To prepare the cultures, lenses are removed from the eye and a small incision is made in the anterior of the lens from which the inner mass of lens fiber cells is removed. This procedure creates a circular wound on the posterior lens capsule, the thick basement membrane that surrounds the lens. This wound area where the fiber cells were attached is located just adjacent to a continuous monolayer of lens epithelial cells that remains linked to the lens capsule during the surgical procedure. The wounded epithelium, the cell type from which fiber cells are derived during development, responds to the injury of fiber cell removal by moving collectively across the wound area, led by a population of vimentin-rich repair cells whose mesenchymal progenitors are endogenous to the lens1. These properties are typical of a normal epithelial wound healing response. In this model, as in vivo, wound repair is dependent on signals supplied by the endogenous environment that is uniquely maintained in this ex vivo culture system, providing an ideal opportunity for discovery of the mechanisms that regulate repair of an epithelium following wounding.

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment.

Estabelecimento de um clinicamente relevantes<em&gt; Ex Vivo</em&gt; Mock Cataract Surgery Modelo para investigar Epithelial Wound Repair em um microambiente Native

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to ...

Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and behavioral phenotypes. The remarkably diverse phenotypes that have evolved as marine populations adapt to countless freshwater environments, combined with the ability to cross marine and freshwater forms, provide a rare vertebrate system in which genetics can be used to map genomic regions controlling evolved traits. Excellent genomic resources are now available, facilitating molecular genetic dissection of evolved changes. While mapping experiments generate lists of interesting candidate genes, functional genetic manipulations are required to test the roles of these genes. Gene regulation can be studied with transgenic reporter plasmids and BACs integrated into the genome using the Tol2 transposase system. Functions of specific candidate genes and cis-regulatory elements can be assessed by inducing targeted mutations with TALEN and CRISPR/Cas9 genome editing reagents. All methods require introducing nucleic acids into fertilized one-cell stickleback embryos, a task made challenging by the thick chorion of stickleback embryos and the relatively small and thin blastomere. Here, a detailed protocol for microinjection of nucleic acids into stickleback embryos is described for transgenic and genome editing applications to study gene expression and function, as well as techniques to assess the success of transgenesis and recover stable lines.

Transgenic manipulations and genome editing are critical for functionally testing the roles of genes and cis-regulatory elements. Here a detailed microinjection protocol for the generation of genomic modifications (including Tol2-mediated fluorescent reporter transgene constructs, TALENs, and CRISPRs) is presented for the emergent model fish, the threespine stickleback.

Microinjeção para Transgénese e Genoma Edição na Threespine Sticklebacks

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and ...

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

An example of adenoviral gene therapy in the human diabetic organ-cultured corneas is presented towards the normalization of delayed wound healing and markedly reduced epithelial stem cell marker expression in these corneas. It also describes the optimization of this process in stem cell-enriched limbal epithelial cultures.

Adenoviral terapia genética para Diabetic Ceratopatia: Efeitos sobre a cicatrização de feridas e Stem Cell marcador expressão em humanos Corneas Organ-cultivadas e células epiteliais Limbal

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene ...

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own body. The ability to grow bone de novo using a patient's own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient's own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.

Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

Isolamento rápido de BMPR-IB + adiposo-derivados de células estromais para uso em um Defeito Modelo Cura calota craniana

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

Uma nova técnica de transplante de almofada de gordura mamária para visualizar a geração de vasos de células vasculares endoteliais vasculares

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

Geração de insulina tridimensional, livre de andaime expressando Pancreatoids do Mouse progenitoras pancreáticas em Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

Visualizando o nó e placa Notochordal em Gastrulating embriões de rato usando microscopia eletrônica e imunofluorescência de montagem inteira

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...