The authors would like to acknowledge Drs. Jonathan Reichner and Angle Byrd for cell experiment insight. The National Science Foundation Graduate Research Fellowship Program (GRFP) supported this work.

Mold Design 1. 3D e Peças <ol><li> Mofo <ol><li> Antes de trabalhar, obter um kit de elastômero de silicone, uma câmara de imagens de células vivas, uma lamela de vidro 22 mm, e um cubo de metal de alumínio usinado com dimensões 10,07 milímetros x 3,95 milímetros x 5,99 milímetros. Preparar a câmara de imagem de células vivas para moldagem, colocando a lamela inferior no suporte de montagem e o resto da câmara, conforme indicado pelo fornecedor. </li><li> Em seguida, usando uma pinça, coloque o cubo de alumínio usinado de metal no meio da caixa de uma câmara ao vivo imagens de células e em cima da lamela, e, em seguida, reserve. </li><li> Misturar as soluções de elastómero de silicone de acordo com o protocolo do fabricante para fazer 5 ml de elastómero. </li><li> Usando uma espátula de laboratório descartável, despeje solução de elastômero de silicone em configuração ao vivo câmara de imagens de células e garantir para não mover colocado usinado cubo de metal de alumínio. Coloque o sistema na bancada do laboratório, em um local seguro O / N para a cura. </li><li> Na manhã seguinte, desconstruira câmara de imagens de células vivas, como recomendado pelo fabricante e retirar o molde usando uma pinça. Utilizando uma pinça, extraia cuidadosamente o cubo de metal de alumínio usinado a partir do molde. Vá até a pia e lavar o molde com água deionizada. Coloque o molde em uma toalha de papel para secar. </li><li> Depois de seco, usando uma faca passatempo, cortar fendas através do molde, espaçadas 2,34 milímetros para além de cada extremidade longitudinal, no interior do molde de silicone. Verifique se as estadias do molde em um lugar seguro e seco até que você esteja pronto para construir o sistema para uma experiência. </li></ol></li><li> Hidrofílicos e hidrofóbicos As lamelas de vidro Preparação <ol><li> Para permitir que a matriz de colagénio para aderir a uma superfície, criar lamelas hidrófilos: <ol><li> Usando uma pipeta descartável, medir 150 mL de 3-aminopropil-trimetoxissilano e despeje solução num tubo de 50 ml. Adicionar 30 ml de etanol a 100% para o tubo de 50 ml com uma pipeta descartável segundo e fechar a tampa. Vortex a solução durante 2 min, assegurando uma mistura completa. Pnossa solução em uma placa de Petri de vidro e reserve. </li><li> Despeje 15 ml de álcool etílico 100% em uma segunda placa de Petri e reserve (certifique-se de rotular pratos). </li><li> Usando uma nova pipeta descartável, medir 30 ml de água desionizada e uma solução para um tubo de 50 ml, deitar. Em seguida, usando uma pipeta descartável nova medida a 1875 ul de glutaraldeído e despeje solução para o mesmo tubo de 50 ml e fechar a tampa. Vortex a solução durante 2 min, assegurando uma mistura completa. Despeje a mistura na terceira placa de Petri, e reserve. </li><li> Utilizando uma pinça, retire uma lamela de vidro 22 milímetros rodada e lave ambos os lados com 100% de mistura de etanol com uma pipeta descartável. </li><li> Coloque lamela de vidro lavadas em 3-aminopropil-trimetoxi-silano com 30 ml de etanol a 100% e solução prato permitir a sentar-se na solução durante 5 minutos. </li><li> Com uma pinça, retire lamela de vidro e lavar novamente com uma solução de etanol a 100% com pipeta descartável. </li><li> Largar lamela de vidro lavado em 1875ul de glutaraldeído e 30 ml de uma mistura de água desionizada e retiradas durante 30 min. </li><li> Após 30 min, remover lamela de vidro com uma pinça e enxaguar com água desionizada e coloque-O tecido seco / N para secar à temperatura ambiente. </li><li> Dipping lamela Repita e movendo-se para o maior número de lamelas, conforme necessário com as mesmas soluções geradas. </li></ol></li><li> Faça lamelas hidrofóbicas pelo protocolo determinado. <ol><li> Adicionar 500 ul de tridecafluoro-1,1,2,2-tetrahydrooctyl, 100 ul de ácido acético e 19,4 ml de hexano em um tubo de 50 mL e agitar com vortex durante 2 min. Despeje solução em vidro Petri prato e reserve. </li><li> Utilizando uma pinça limpo, tire uma lamela de vidro e cair na solução mista preparado por 2 min. Após 2 min passaram, tirar lamela de vidro com uma pinça e enxágüe com água deionizada usando uma pipeta e lugar lamela descartáveis ​​para O tecido seco / N para secar em temperatura ambiente. Lamela loja em pratos de Petri de plástico até ser utilizado. </li><li> Repita dipp lamelaing e movendo-se para o maior número de lamelas, conforme necessário com as mesmas soluções geradas. </li></ol></li></ol></li></ol> Assembléia 2. Mold <ol><li> Antes de usar moldes, usando uma pipeta descartável lave o molde com álcool etílico a 90% ao longo de um recipiente para resíduos químicos. Em seguida, colocar o molde numa placa de Petri cheia de água desionizada e permitir a sentar O / N. </li><li> Tome molde da solução usando uma pinça e coloque em uma toalha para o ar seco antes de iniciar a montagem de molde. </li><li> Enquanto o molde é o ar de secagem, corte as lamelas hidrofílicos quadrados em dois retângulos um pouco maiores do que 3,95 milímetros x 5,99 milímetros usando uma ferramenta de traçagem de diamante de alta precisão. Deslize cada lamela corte retângulo nas ranhuras cortadas no molde de silicone. </li><li> Vire o molde de cabeça para baixo e aplique graxa de vácuo ao longo da parte inferior do molde de silicone com uma pipeta descartável. Em seguida, vire o molde de volta com o lado direito para cima e pressione a parte inferior do molde para as circulares tampas de vidro hidrofílicoslábio para criar uma vedação. </li><li> Com luvas descartáveis, pegar o molde montado e coloque em um prato descartável Petri e, em seguida, em um bio-hood. Ligue lâmpada UV Bio-capa para 1 hora para esterilizar o molde antes de ocorrer a experimentação. </li></ol> 3. O colagénio mistura e Matrix 3D <ol><li> Antes de preparar a mistura de colagénio, mancha e preparar as células para a imagiologia de acordo com protocolos padrão 28. </li><li> Utilizando técnicas laboratoriais padrão e protocolos 29,30, num bio-capa, e meios da mistura de células em quimioatractor um tubo de 15 ml com a concentração desejada quimioatractor. Pipeta 5 ml de solução a concentração dos media-chemoattractant célula específica em um tubo de 15 ml e mover a solução em um banho de água aquecida. </li><li> Usando uma pipeta, 5 ml de extracto de células de apenas meios de comunicação para um segundo tubo de 15 ml e colocar num banho de água aquecida para aquecer a solução para as experiências de microscopia. </li><li> Em um bio-hood, pipeta 30 ul de 10x tampão fosfatoed solução (PBS) para dentro do tubo de micro centrífuga. Adicionar 6 ul de 1 N hidróxido de sódio (NaOH) em mesma micro tubo de centrifugação e de vórtice durante 15 seg. </li><li> Obter solução de cauda de rato colágeno I da geladeira e passar para a bio-capa utilizando técnicas laboratoriais padrão e protocolos 29. Verifique se o colágeno ainda está frio. Pipetar 168,3 ul de colagénio no mesmo tubo de micro centrífuga. </li><li> Em seguida, adicionar 18 ul de amarelo-verde microesferas modificadas com carboxilato fluorescentes, utilizando uma pipeta para o mesmo tubo de micro centrífuga. Vortex do tubo de microcentrífuga com todas as soluções durante 30 seg. </li><li> Adicionar 77,7 uL de mistura de células meio celular pretendida (cerca de 2 X 10 6 células / ml de concentração) para dentro do tubo de micro centrífuga e misture utilizando a ponta da pipeta durante 20 seg. Conforme necessário, alterar a densidade da matriz seguindo-se a mistura de colagénio costume adaptado para a adição de contas fluorescentes e mesa de viabilidade celular (Tabela 1). </li><li> Pipetar 300 &amp; #181; l de mistura de células-colagénio para o centro do poço (bem como 2) do conjunto de molde preparado. Coloque o molde sobre um prato de Petri descartáveis ​​e movê-lo para uma incubadora padrão durante 20 minutos a 37 ° C com 5% de CO 2. </li><li> Retirar sistema da incubadora e coloque de volta para bio-hood. Utilizando uma pinça, retire ambas as lamelas hidrofóbicos por aperto na parte superior de cada lamela e puxando para cima e longe do molde. </li><li> Mover todo o sistema para a microscopia e imagem desejada para garantir os lados do molde de colagénio são ainda recta para permitir um gradiente de difusão planar. </li><li> Com o sistema ainda sob o microscópio, utilizando uma pipeta de canal único 100 ul, adicionar 100 uL de meio celular para o poço # 1. Em seguida, usando uma pipeta de canal único 100 ul adicionar 100 ul de meios de células com a concentração desejada em quimioatractivo bem # 3. </li><li> Imediatamente após a adição de soluções em ambos os poços, início de imagem. </li></ol> 4. Criação de Imagens e DifusãoModelagem <ol><li> Se se quer encontrar o coeficiente de difusão para a densidade específica de colagénio para calcular a concentração específica, seguir o protocolo abaixo: <ol><li> Siga o protocolo dado acima intitulado &quot;Mistura de colágeno e Matrix 3D&quot;, em vez de gerar meios de células com a concentração chemoattractant desejar, faça rhodamine 5 M em um tubo de 15 ml usando cálculos e protocolos 30 padrão. </li><li> Imagem as matrizes de colágeno 3D com um sistema confocal montado em um microscópio invertido usando um laser de argônio (488 nm) para capturar a fluorescência. Imagem a cada 2 ou 3 segundos para até 7 hr. </li></ol></li><li> Para Modelagem Difusão: Usando o modelo matemático apresentado abaixo, escrever um código de software computacional para processamento e análise com estas etapas gerais. <img alt="Equação 1" src="/files/ftp_upload/52948/52948eq1.jpg" /><ol><li> Importar imagens para software computacional para redimensionamento e normalizando pelo inten máximosidade. Converta imagens para um mapa de cor cinza da imagem para selecionar a borda. Escolha de um eixo ao longo do centro da imagem, para a direita do centro e para a esquerda do centro. </li><li> Usando o código de software computacional, traçar a intensidade normalizada, juntamente com o verdadeiro intensidade e a diferença entre os dois. </li><li> A seguir, introduza os parâmetros necessários para a montagem (concentração, tempo e duração). </li><li> Traça-se a concentração normalizada em função do tempo e o perfil de concentração ao longo do eixo-x com um ajuste polinomial para os dados. </li><li> Calcular o coeficiente de difusão utilizando o código de software computacional. </li></ol></li></ol> 5. As medições experimentais <ol><li> Referindo de novo a equação de difusão, reescrever a equação para encontrar a concentração específica em qualquer local dentro do sistema como mostrado abaixo. <img alt="Equação 2" src="/files/ftp_upload/52948/52948eq2.jpg" /> Onde: L = Comprimento do thmatriz de colágeno e x = localização sob investigação D = coeficiente de difusão T = tempo decorrido </li><li> Durante as experiências de quimiotaxia, garantir para registrar o tempo específico que a quimiocina momento foi adicionado ao sistema. Uma vez que o movimento de migração celular é encontrado, garantir a gravar confocal gravação de lapso de tempo, e observe o tempo específico e localização da borda onde imaging ocorreu. </li><li> Durante a análise de dados, conecte o observado o tempo decorrido, a localização e o coeficiente de difusão para a célula específica na equação para encontrar a concentração normalizada específica em qualquer ponto dentro do colagénio, tanto para difusão e experiências com células. </li></ol> 6. Acompanhamento de migração celular Utilizando TFM <ol><li> Para controlar a migração de células usando técnicas de TFM / DVC seguir o protocolo abaixo: <ol><li> Após a adição da concentração desejada em quimioatractor bem # 3, iniciar o Imaging as matrizes de colagénio em 3D com um sistema confocal montado num micr invertidooscope para encontrar uma célula para investigar. </li><li> Uma vez que uma célula se encontra em movimento, colocar a célula no meio do campo de vista e utilizar um motor piezoeléctrico (para a imagem no eixo z), cada imagem 1 ou 2 minutos. </li><li> Para calcular a geração de força e deformação celular, gerar um código computacional para encontrar os deslocamentos dos grânulos de fluorescência seguindo os protocolos TFM / DVC 31. </li></ol></li></ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Calculations for Molecular Biology and Biotechnology: A Guide to Mathematics in the Laboratory 2e. , (2010).</li><li>Bar-Kochba, E., Toyjanova, J., Andrews, E., Kim, K., Franck, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Fast+Iterative+Digital+Volume+Correlation+Algorithm+for+Large+Deformations.">A Fast Iterative Digital Volume Correlation Algorithm for Large Deformations.</a> Exp. Mech. In press, (2014).</li><li>Shenoy, V., Rosenblatt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+of+Macromolecules+in+Collagen+and+Hyaluronic+Acid,+Rigid-Rod-Flexible+Polymer+Composite+Matrixes.">Diffusion of Macromolecules in Collagen and Hyaluronic Acid, Rigid-Rod-Flexible Polymer Composite Matrixes.</a> Macromolecules. 28 (26), 8751-8758 (1995).</li><li>Leddy, H., Guilak, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-Specific+Molecular+Diffusion+in+Articular+Cartilage+Measured+using+Fluorescence+Recovery+after+Photobleaching.">Site-Specific Molecular Diffusion in Articular Cartilage Measured using Fluorescence Recovery after Photobleaching.</a> Ann. Biomed. Eng. 31 (7), 753-760 (2003).</li></ol>

The authors declare that they have no competing financial interests.

Os passos mais críticos para as experiências bem-sucedidas de difusão com ou sem células são: configurar corretamente o conjunto de molde; desenvolver a destreza manual necessário para evitar danos durante a extracção de lamelas hidrofóbicos; garantindo a encontrar uma linha muito boa de partida linear para calcular correctamente o coeficiente de difusão; corrigir cálculos experimentais de colagénio e quimioatractiva; usar corretamente do sistema de imagens de células vivas para garantir matriz não secar; ...

A deslocação preferida de células para um gradiente de concentração, conhecido como quimiotaxia, desempenha um papel importante em processos fisiológicos e patológicos no organismo. Tais exemplos são a pele e mucosa 1 a cicatrização de feridas, a morfogénese 2, 3 inflamação, e 4,5 o crescimento do tumor. Também tem sido mostrado que as células cancerosas podem migrar através de duas estratégias individuais e colectivos-migração celular 6. Além disso, os mecanismos de difusão de instabilidade pode induzir a separação de células individuais ou agrupados a partir de um corpo / objeto tumoral e, em seguida, podem emigrar para uma fonte de nutrientes e, portanto, invadir áreas mais largas e 7 tecidos. Além disso, foi demonstrado que diversos mecanismos de migração podem ser activa em 2D e em 3D, devido a diferentes funções de moléculas de adesão 8. Portanto, um movimento para fisiologicamente relevante em ensaios in vitro para investigar a motilidade celular em um measureable de modo simples e é de importância na compreensão de fenômenos movimento de 9 células. Infelizmente, a dificuldade em analisar a migração de células, um ensaio de quimiotaxia quantificável abrangente geralmente requer um método trabalhoso longo, fundada na medição da motilidade celular e fenômenos de transporte modelos imparciais. Abordagens experimentais passadas para investigar quimiotaxia de células incluem a câmara de Boyden 10 eo ensaio sob agarose 11. No entanto, dentro destes primeiros ensaios, as experiências de migração de células não acompanhar o movimento em relação ao tempo. Mais, o mais importante, os gradientes de concentração utilizados para as experiências não foram bem definidas ou completamente compreendido, enquanto apenas para sustentar a sinalização para não mais do que algumas horas. Além disso, as tentativas de câmara de quimiotaxia precoce restrito a migração de células para duas dimensões e não permitem controlar a cinética da migração 12. Olhando para a câmara de Boyden, um ensaio endpointnão permitiria ao pesquisador observar visualmente a migração e não poderia diferenciar diretamente quimiotaxia (movimento direcional) de quimiocinese (movimento aleatório). Além disso, várias variáveis ​​diferenças no tamanho de poro e espessura de membranas feitas a câmara muito difícil de reproduzir de forma fácil e oculta a reacção migrante de células para quimiocinas 13,14. Com o novo entendimento de microfluídica, novas câmaras e micro-dispositivos têm sido investigados como um instrumento para investigar a locomoção de células sob condições de fluxo intersticiais ou quimiotaxia 15,16. De acordo com estes novos dispositivos, novas métricas de células foram introduzidos e investigados, como o efeito da tensão de cisalhamento em uma célula 17,18. Infelizmente, câmaras de quimiotaxia microfluídicos passadas e atuais limitam estudos de migração celular para substratos um 2D revés importante, pois muitos processos biológicos, incluindo a invasão de células de tumores e metástases, e immigração celular Mune, envolvem a migração 3D. Câmaras-onde a observação direta de uma solução chemoattractant está em contacto com um gel 3D contendo células também têm sido também relatados 19,20. Estas câmaras têm dois compartimentos, um contendo um quimioatractor e uma contendo células, são unidas ao lado uma da outra na horizontal 21 ou como anéis concêntricos 22. Estes sistemas são apontados na direção certa, mas não manter um sistema de quimiotaxia por um período prolongado de tempo. Além disso, os investigadores examinaram também difusividade através de membranas de colagénio em células de diálise, bem como a difusão de moléculas marcadoras através de amostras de colagénio submetido a pressão hidrostática 23-25. Algumas experiências de difusão em géis de colagénio dependem de alterações físicas e químicas do gel usando campos magnéticos e incorporação química 26. Um método popular para a modelagem diffusivity em collatecidos endógenos baseia-se na imagem de fluorescência de ponto fotodegradação contínua. Este método revelou anisotropia nos coeficientes de difusão de macromoléculas em tecidos de colagénio orientadas. No entanto, a fotodegradação foi usado na cartilagem articular e não matrizes de colagénio. Embora semelhantes, os experimentos de modelagem necessárias devem ser realizadas através da compreensão especificamente o coeficiente de difusão de géis de colágeno. Mais importante ainda, os sistemas não utilizam um método para medir a força de geração de células. Infelizmente, a maioria dos sistemas parecem estar faltando um ou dois elementos-chave para um sistema ideal: o que permite rastreamento de celular, uma compreensão gradiente de difusão com um fator quimiotático através da matriz, um conjunto relativamente simples com uma facilidade de reprodutibilidade, a minimização de célula-célula interações, e a capacidade de medir unidades dimensionais para quantificação (isto é, velocidade, força, concentração específica). Moghe et ai. </em&gt; 27 proposto um sistema que cumpriu a maioria destas exigências em que as células foram inicialmente dispersos por todo o gel em vez de concentradas na superfície do filtro, mas foi difícil de medir as forças que a célula gera. Para este fim, apresentamos um sistema de gradiente de difusão planar para investigar quimiotaxia em uma matriz de colágeno 3D, que permite superar as limitações modernas câmara de difusão de ensaios existentes, que é baseado na microscopia de lapso de tempo, juntamente com técnicas de análise de imagem para medir celular forças em um ambiente 3D. Este protocolo proporciona uma maneira simples, contudo inovadora de criar uma câmara de difusão 3D simples que pode ser utilizado para investigar a quimiotaxia 3D em células diferentes.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Silicone elastomer kit</td>
			<td>Dow Corning Corp</td>
			<td>182 SIL ELAST KIT .5KG</td>
			<td>a two-part misture with a 10:1 mix</td>
		</tr>
		<tr>
			<td>Live cell imaging chamber</td>
			<td>Live Cell Instrument</td>
			<td>CM-B18-1</td>
			<td>CMB for 18mm round coverslips</td>
		</tr>
		<tr>
			<td>22mm Glass Coverslip</td>
			<td>Fisher Scientific</td>
			<td>NC0180281</td>
			<td>Neuvitro Corp. cover slip 22mm 1.5</td>
		</tr>
		<tr>
			<td>Machined aluminum metal cube</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hobby utility knife</td>
			<td>X-Acto</td>
			<td>X3201</td>
			<td></td>
		</tr>
		<tr>
			<td>3-(aminopropyl) trimethoxysilane</td>
			<td>Sigma-Aldrich</td>
			<td>281778-5ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Polysciences, Inc</td>
			<td>00216A-10</td>
			<td>Glutaraldehyde, EM Grade, 8%</td>
		</tr>
		<tr>
			<td>50 ml tube</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
			<td>Standard floor model and tabletop centrifuges</td>
		</tr>
		<tr>
			<td>Glass petri dish</td>
			<td>Fisher Scientific</td>
			<td>08-747A</td>
			<td>Reusable Petri Dishes: Complete (60 x 15mm)</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fisher Scientific</td>
			<td>22-327-379</td>
			<td>Fine Point Forceps</td>
		</tr>
		<tr>
			<td>Cover glasses</td>
			<td>Fisher Scientific</td>
			<td>12-518-105A</td>
			<td>Rectangle; 30 x 22mm; Thickness No. 1</td>
		</tr>
		<tr>
			<td>Tridecafluoro-1,1,2,2-tetrahydrooctyl</td>
			<td>Gelest</td>
			<td>SIT8174.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>320099</td>
			<td>Acetic acid ACS reagent, &#8805;99.7%</td>
		</tr>
		<tr>
			<td>Hexane</td>
			<td>Sigma-Aldrich</td>
			<td>296090</td>
			<td>Hexane</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>anhydrous, 95%</td>
		</tr>
		<tr>
			<td>Ethyl alcohol</td>
			<td>Sigma-Aldrich</td>
			<td>E7023</td>
			<td>200 proof, for molecular biology</td>
		</tr>
		<tr>
			<td>High-precision diamond scribing tool</td>
			<td>Lunzer</td>
			<td>PV-081-3</td>
			<td>Straight extended tip scribe, .020&#34; (.50mm) diameter by .200&#34; (5.0mm) tip length</td>
		</tr>
		<tr>
			<td>Vacuum grease</td>
			<td>Dow Corning</td>
			<td>14-635-5C</td>
			<td>High-Vacuum Grease</td>
		</tr>
		<tr>
			<td>15 ml tube</td>
			<td>Fisher Scientific</td>
			<td>14-959-49D</td>
			<td>15 ml conical centrifuge tubes with hydrophobic, biologically inert surface</td>
		</tr>
		<tr>
			<td>10X phosphate buffered solution</td>
			<td>Fisher Scientific</td>
			<td>BP399-500</td>
			<td>1.37 M Sodium Chloride, 0.027 M Potassium Chloride, and 0.119 M Phosphate Buffer</td>
		</tr>
		<tr>
			<td>1N sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>38215</td>
			<td>Sodium hydroxide concentrate</td>
		</tr>
		<tr>
			<td>Collagen I, rat tail</td>
			<td>BD Biosciences</td>
			<td>354236</td>
			<td>Rat tail&#160;</td>
		</tr>
		<tr>
			<td>Micro centrifuge tube</td>
			<td>Fisher Scientific</td>
			<td>02-681-332</td>
			<td>Volume: 2.0 ml; O.D. x L: 13 x 40 mm; sterile; single-wrapped</td>
		</tr>
		<tr>
			<td>Carboxylate-modified microspheres</td>
			<td>Invitrogen</td>
			<td>&#160;F-8813</td>
			<td>Carboxylate-modified microspheres, 0.5 &#181;m, yellow-green fluorescent (505/515), 2% solids</td>
		</tr>
		<tr>
			<td>Rhodamine</td>
			<td>Sigma-Aldrich</td>
			<td>83689</td>
			<td>Rhodamine B for fluorescence</td>
		</tr>
	</tbody>
</table>

planar gradient diffusion system to investigate chemotaxis in a 3d collagen matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

A capacidade do presente ensaio para avaliar com precisão a migração da célula depende de uma boa configuração do sistema. Portanto, é crucial para certificar-se de projetar o molde sistema de difusão de forma precisa e tomar muito cuidado na colocação de lamelas de ambos hidrofóbicos e hidrofílicos, como ilustrado na figura 1. Se o sistema for projetado corretamente e durante a etapa de modelagem difusão garantindo para encontrar um muito boa linha de partida linear, uma é capaz de alcan?...

Watch this Scientific Journal Video about Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix at JoVE.com

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

Gradiente planar do sistema de difusão para investigar Quimiotaxia numa Matriz de Colagénio 3D

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

planar-gradient-diffusion-system-to-investigate-chemotaxis-3d

Research

JoVE Journal

Bioengineering

8.4K visualizações.  California State University, Long Beach. Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

Vídeo: Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Engenharia de Tecidos de um 3D Humano<em&gt; In vitro</em&gt; Sistema de Teste de Tumor

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Bioengenharia

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

Análise da migração celular dentro de uma matriz de colagénio tridimensional

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to tumor and cancer metastasis. Despite intense research efforts, the basic biochemical and biophysical principles of cell migration are still not fully understood, especially in the physiologically relevant three-dimensional (3D) microenvironments. Here, we describe an in vitro assay designed to allow quantitative examination of 3D cell migration behaviors. The method exploits the cell&#8217;s mechanosensing ability and propensity to migrate into previously unoccupied extracellular matrix (ECM). We use the invasion of highly invasive breast cancer cells, MDA-MB-231, in collagen gels as a model system. The spread of cell population and the migration dynamics of individual cells over weeks of culture can be monitored using live-cell imaging and analyzed to extract spatiotemporally-resolved data. Furthermore, the method is easily adaptable for diverse extracellular matrices, thus offering a simple yet powerful way to investigate the role of biophysical factors in the microenvironment on cell migration.

The mechanical properties and microstructure of the extracellular matrix strongly affect 3D migration of cells. An in vitro method to study the spatiotemporal cell migration behavior in biophysically variable environments, at both population and individual cell levels, is described.

Sistema de Gel Concentric para estudar o papel Biofísica da Matrix Microenvironment sobre a migração celular 3D

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Engenharia 3D celularizados Colágeno Gel para Vascular Regeneração Tecidual

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

Baseada em Silk-colágeno Engineered 3D Model of Tissue polarizada Neural

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

Gradient Strain Chip for Stimulating Cellular Behaviors in Cell-laden Hydrogel

Artificial guidance for cellular alignment is a hot topic in the field of tissue engineering. Most of the previous research has investigated single strain-induced cellular alignment on a cell-laden hydrogel by using complex experimental processes and mass controlling systems, which are usually associated with contamination issues. Thus, in this article, we propose a simple approach to building a gradient static strain using a fluidic chip with a plastic PDMS cover and a UV transparent glass substrate for the stimulation of cellular behavior in a 3D hydrogel. Overloading photo-patternable cell prepolymer in the fluidic chamber can generate a convex curved PDMS membrane on the cover. After UV crosslinking, through a concentric circular micropattern under the curved PDMS membrane, and buffer washing, a microenvironment for investigating cell behaviors under a variety of gradient strains is self-established in a single fluidic chip, without external instruments. NIH3T3 cells were demonstrated after observing the change in the cellular alignment trend under geometry guidance, in cooperation with strain stimulation, which varied from 15 - 65% on hydrogels. After a 3-day incubation, the hydrogel geometry dominated the cell alignment under low compressive strain, where cells aligned along the hydrogel elongation direction under high compressive strain. Between these, the cells showed random alignment due to the dissipation of the radical guidance of hydrogel elongation and the geometry guidance of the patterned hydrogel.

This article introduces a simple approach to providing non-continuous gradient static strains on a concentric cell-laden hydrogel to regulate cell alignment for tissue engineering.

Gradiente de pressão Chip para estimulantes comportamentos celulares em hidrogel de célula-carregado

Artificial guidance for cellular alignment is a hot topic in the field of tissue engineering. Most of the previous research has investigated single ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Preparação de 3D colágeno Géis e microcanais para o estudo das interações 3D<em&gt; In Vivo</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Sintonizável hidrogéis a partir de Pulmonar Matriz Extracelular para Cultura Celular 3D

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

A Method for Determination and Simulation of Permeability and Diffusion in a 3D Tissue Model in a Membrane Insert System for Multi-well Plates

In vitro cultivated skin models have become increasingly relevant for pharmaceutical and cosmetic applications, and are also used in drug development as well as substance testing. These models are mostly cultivated in membrane-insert systems, their permeability toward different substances being an essential factor. Typically, applied methods for determination of these parameters usually require large sample sizes (e.g., Franz diffusion cell) or laborious equipment (e.g., fluorescence recovery after photobleaching (FRAP)). This study presents a method for determining permeability coefficients directly in membrane-insert systems with diameter sizes of 4.26 mm and 12.2 mm (cultivation area). The method was validated with agarose and collagen gels as well as a collagen cell model representing skin models. The permeation processes of substances with different molecular sizes and permeation through different cell models (consisting of collagen gel, fibroblast, and HaCaT) were accurately described.
Moreover, to support the above experimental method, a simulation was established. The simulation fits the experimental data well for substances with small molecular size, up to 14 x 10-10 m Stokes radius (4,000 MW), and is therefore a promising tool to describe the system. Furthermore, the simulation can considerably reduce experimental efforts and is robust enough to be extended or adapted to more complex setups.

A method for determination of permeability in a membrane insert system for multi-well plates and in silico parameter optimization for the calculation of diffusion coefficients using simulation are presented.

Um método para a determinação e simulação da permeabilidade e difusão em um modelo 3D do tecido em uma membrana inserir sistema para placas multi bem

In vitro cultivated skin models have become increasingly relevant for pharmaceutical and cosmetic applications, and are also used in drug development as ...

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily identified and used. Currently, this gap is filled with tendon auto-, allo-, xeno-, or artificial grafts, but clinical methods to secure them are not necessarily translatable to animals because of the scale. In order to evaluate new biomaterials or study a tendon graft made up of collagen type 1, we have developed a modified suture technique to help maintain the engineered tendon in alignment with the tendon ends. Mechanical properties of these grafts are inferior to the native tendon. To incorporate engineered tendon into clinically relevant models of loaded repair, a strategy was adopted to offload the tissue engineered tendon graft and allow for the maturation and integration of the engineered tendon in vivo until a mechanically sound neo-tendon was formed. We describe this technique using incorporation of the collagen type 1 tissue engineered tendon construct.

In this paper, we present an in vitro and in situ protocol to repair a tendon gap of up to 1.5 cm by filling it with engineered collagen graft. This was performed by developing a modified suture technique to take the mechanical load until the graft matures into the host tissue.