Os autores reconhecem o uso do laboratório de análise de superfície na Universidade Johns Hopkins, financiado, como parte da Pesquisa em Ciências Materiais e Centro de Engenharia através da National Science Foundation. LED é um estagiário e um IGERT National Science Foundation Graduate Fellow. Esta pesquisa foi parcialmente financiado pelo NIH conceder U54CA143868.

1. Fotolitos padrão para micropatterned Fabricação Stamp <ol><li> Enxágüe um wafer de silício novo com etanol e seco com fluxo de ar. Use uma pinça para manusear wafer e prevenir defeitos de superfície durante todo o processo. </li><li> Transferência de wafer para girar aplicador e tampa superfície do wafer com SU-2025 fotorresiste negativo. Cobrir pelo menos 80% de wafer com fotorresiste. Casaco giro por 10 segundos a 600rpm, seguido por 30 segundos a 3000rpm. Devido à fotossensibilidade do fotorresiste, desligar luzes da sala a partir deste ponto em diante. </li><li> Transferência de fotorresiste wafer de silício revestido de chapa quente para &quot;soft-assar&quot; a 95 ° C por 3 minutos. Remover da placa quente e permitir que um minuto para esfriar. </li><li> Coloque uma máscara pré-fabricado com o padrão desejado na parte superior do wafer de silício coberta de fotorresiste e expor à radiação ultravioleta (UV) irradiação (350-450 nm) por 20 segundos. </li><li> Transferência de wafer de silício para a chapa quente para &quot;hard-bake&quot; a 110 ° C por 6 minutos. </li><li> Ligue as luzes e remover wafer de chapa quente. Padrões distintos devem ser visíveis neste momento. </li><li> Lavar cuidadosamente wafer de silício com SU-8 desenvolvedor fotorresiste. Após 3 lavagens com solução de desenvolvedor, lavar com etanol. Se algum resíduo branco está presente, repita enxaguar com solução desenvolvedor fotorresiste, ou simplesmente submergir o wafer em solução desenvolvedor por 2 minutos e proceda novamente com o etanol enxaguar. Lavar com água e ar seco. </li><li> Mix PDMS (polidimetilsiloxano) solução de elastômero e agente de cura em uma proporção de peso de 10:1. Centrifugar a 900rpm por 5 minutos para remover bolhas de ar. </li><li> Lugar padronizados wafer em placa de Petri e despeje sobre PDMS wafer de silício. Se houver bolhas presente, lugar em desicator e vácuo por alguns minutos até que as bolhas desaparecem. </li><li> Cura de noite à temperatura ambiente (RT) para formar um selo elastomérico complementares. Se PDMS não está completamente curada depois de 12 horas, coloque no forno a 60 ° C por 30 minutos. </li><li> Remova cuidadosamente PDMS de wafer de silício. Use um fio da navalha para cortar selo para tamanho desejado. Sonicado em etanol por 15 minutos. Wafers pode ser utilizado várias vezes para criar selos elastoméricos novo. </li></ol> 2. Micropatterning HA <ol><li> Plasma vidro limpo slides por 5 minutos. Coloque todos os slides na câmara de vácuo e por 5 minutos. Em seguida, permitir baixo fluxo de oxigênio para entrar na câmara. Por sua vez limpa plasma por 5 minutos. Retire do limpador de plasma e coloque em um prato de Petri usando uma pinça. </li><li> Durante a limpeza de plasma, sonicate selos PDMS em etanol por 15 minutos. </li><li> Prepare uma nova solução de 3% v / v de 3-aminopropyltrimethoxysilane (APTMS) em 95% etanol v / v em um tubo de centrífuga de 15 mL. Deixar reagir por 5 minutos em temperatura ambiente para formar um silanol. </li><li> Coloque selo padrão de PDMS-up na rotação coater chuck e cubra a superfície com solução padronizada APTMS. Spincoat a 3500rpm por 30 segundos. Não toque na superfície de selo, somente os lados do carimbo de PDMS. </li><li> Transferir a solução para o plasma APTMS limpas as superfícies de vidro: conjunto de selo voltado para baixo em uma extremidade do lado estampados e desliza delicadamente pressionando PDMS por isso é completamente em contato com a superfície do vidro por 1 minuto. </li><li> Remover PDMS selo suavemente, e permitir lâmina de vidro decorado para incubar no RT por 30 minutos. Enxaguar superfícies com padrões com etanol e ar seco. Sonicate PDMS selo por 15 minutos para remover o excesso APTMS. </li><li> Superfícies de calor no forno ou na chapa quente por 1 hora a 115 ° C. Lavar com álcool etílico e secar com o ar. </li><li> Prepare polietileno glicol fresco (PEG)-silano solução de 20% v / v 2 - [metoxi (polyethyleneoxy) propil] trimetoxissilano em tolueno. Isto irá servir como uma região não-adesiva em torno de padrões. </li><li> Transferência de lâmina de vidro decorado para um prato de vidro Petri e incubar em PEG-silano solução por 45 minutos a 75 ° C em uma chapa quente. Enxágüe com tolueno, água e seca com o ar. </li><li> Esterilizar por 1 hora utilizando irradiação germicida ultravioleta em uma câmara de segurança biológica. Proceder em cabine de segurança biológica a partir deste ponto para a frente para manter a esterilidade. </li><li> Preparar a solução aquosa estéril HA. 10mM de 1-etil-3-(3-dimetilaminopropil) carbodiimida (EDC) 5mM de N-hydroxysuccinimide 50μg / mL fluoresceína marcado HA </li><li> Aplicar a solução para o HA substratos de vidro modelado em condições estéreis, protegidas da luz. Padronizada substratos devem ser perturbado durante 16-24 horas. </li><li> Aspirar off solução HA, lavar com tampão fosfato (PBS), e cobrir a superfície com 1% de albumina bovina sérica (BSA) em PBS por 1 hora. A superfície está pronta para cultura de células. </li></ol> 3. Cultura de células em HA micropatterned Superfícies <ol><li> Expandir MDA-MB-231 células de câncer de mama humano e células LS174T carcinoma colorretal, de acordo com as instruções do fabricante. </li><li> Suspensões única semente de células cancerígenas com densidades entre 0,4 e 1x10 <sup&gt; 6 células cancerosas por 18,75 centímetros 2 de superfície de vidro e em HA imobilizado superfícies. Incubar em incubadora umidificada (37 ° C) em atmosferas mantido com 5% de CO 2. Adesão celular deve ocorrer dentro de 24 horas e pode ser observado através do microscópio de luz invertida </li><li> Superfícies de cultura de células pode ser mantida até 5 dias em condições-padrão incubadora. Mudança de mídia todos os dias. Morfologia das células eo crescimento pode ser observado usando microscópio de luz invertida. Ensaios adicionais celular pode ser aplicado para analisar as respostas à superfície HA. </li></ol> 4. Resultados representativos: A química carbodiimida utilizada para imobilizar covalentemente HA para lâminas de vidro APTMS padronizada é mostrado na Figura 1A. EDC é um agente de reticulação de comprimento zero que reage com grupos carboxila HA para formar aminas reativas intermediários. Como este intermediário é suscetível à hidrólise, NHS é adicionado para aumentar a eficiência da reação carbodiimida. Como solução HA interage com APTMS micropadrões, uma forma amida estável vínculo entre HA intermediários e amina primária de ATPMS. Um exemplo de HA superfície modelada visualizada usando um microscópio de fluorescência e uma análise de intensidade de fluorescência ImageJ demonstrar a imobilização de HA padronizadas são mostradas (Figura 1B-C). As superfícies HA micropatterned permitir o estudo das interações das células de câncer com HA expgenous. Ambos MDA-MB-231 de mama humano e do cólon humano LS174t linhas celulares de cancro preferencialmente aderem às regiões micropatterned HA (Figura 2). Imagens de alta resolução usando microscopia eletrônica de varredura pode ser usado para analisar as interações das células em cultura com superfícies HA imobilizada (Figura 3). <img src="/files/ftp_upload/2413/2413fig1.jpg" alt="Figura 1" /> Figura 1 HA superfícies micropatterned (A) Esquema descrevendo o desenvolvimento de superfícies funcionais HA, (B) imagem da fluorescência da fluoresceína (verde) com etiqueta HA superfície micropatterned. (C) mapa de distribuição de pixels 3D usando software ImageJ demonstrando os padrões HA discreta. Barra de escala = 100μm. Modificado a partir do 9 com permissão da Elsevier. <img src="/files/ftp_upload/2413/2413fig2.jpg" alt="Figura 2" /> Figura 2. Adesão celular em superfícies Cancer HA micropatterned. Ambos os dois pontos (A) e mama (B) carcinoma preferencialmente aderiu em manchas HA com 24 horas de cultura. Modificado a partir do 9 com permissão da Elsevier. <img src="/files/ftp_upload/2413/2413fig3.jpg" alt="Figura 3" /> Figura 3. Interação das células cancerosas com HA. (A) de digitalização de imagens de microscopia eletrônica de células de câncer de cólon (A) cultivadas em superfícies HA mostrado em baixa ampliação (esquerda) e alta ampliação (à direita; de quadrados à esquerda) e de células de câncer de mama (B) cultivadas em superfícies HA mostrado na baixa ampliação (esquerda) e alta ampliação (à direita; de quadrados à esquerda).

<ol>
	<li>Toole, B. P., Wight, T. N., Tammi, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronan-cell+interactions+in+cancer+and+vascular+disease.">Hyaluronan-cell interactions in cancer and vascular disease.</a> Journal of Biological Chemistry. 277, 4593-4596 (2002).</li><li>Hamilton, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hyaluronan+Receptors+CD44+and+Rhamm+(CD168)+Form+Complexes+with+ERK1,2+That+Sustain+High+Basal+Motility+in+Breast+Cancer+Cells.">The Hyaluronan Receptors CD44 and Rhamm (CD168) Form Complexes with ERK1,2 That Sustain High Basal Motility in Breast Cancer Cells.</a> Journal of Biological Chemistry. 282, 16667-16680 (2007).</li><li>G&#246;tte, M., Yip, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparanase,+Hyaluronan,+and+CD44+in+Cancers:+A+Breast+Carcinoma+Perspective.">Heparanase, Hyaluronan, and CD44 in Cancers: A Breast Carcinoma Perspective.</a> Cancer Research. 66, 10233-10237 (2006).</li><li>Lord, M. S., Pasqui, D., Barbucci, R., Milthorpe, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+adsorption+on+derivatives+of+hyaluronic+acid+and+subsequent+cellular+response.">Protein adsorption on derivatives of hyaluronic acid and subsequent cellular response.</a> Journal of Biomedical Materials Research. Part A 91, 635-646 (2009).</li><li>Barbucci, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+of+hyaluronic+acid+by+sulphate+groups+insertion+to+obtain+a+heparin-like+molecule.">Modification of hyaluronic acid by sulphate groups insertion to obtain a heparin-like molecule.</a> Gazz. Chim. Ital. 125, 169-180 (1995).</li><li>Morra, M., Cassinelli, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-fouling+properties+of+polysaccharide-coated+surfaces.">Non-fouling properties of polysaccharide-coated surfaces.</a> Journal of Biomaterials Science, Polymer Edition. 10, 1107-1124 (1999).</li><li>Khademhosseini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Layer-by-layer+deposition+of+hyaluronic+acid+and+poly-L-lysine+for+patterned+cell+co-cultures.">Layer-by-layer deposition of hyaluronic acid and poly-L-lysine for patterned cell co-cultures.</a> Biomaterials. 25, 3583-3592 (2004).</li><li>Ibrahim, S., Joddar, B., Craps, M., Ramamurthi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+surface-tethered+model+to+assess+size-specific+effects+of+hyaluronan+(HA)+on+endothelial+cells.">A surface-tethered model to assess size-specific effects of hyaluronan (HA) on endothelial cells.</a> Biomaterials. 28, 825-835 (2007).</li><li>Dickinson, L. E., Ho, C. C., Wang, G. M., Stebe, K. J., Gerecht, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+surfaces+for+high-resolution+analysis+of+cancer+cell+interactions+on+exogenous+hyaluronic+acid.">Functional surfaces for high-resolution analysis of cancer cell interactions on exogenous hyaluronic acid.</a> Biomaterials. 31, 5472-5478 (2010).</li><li>Gao, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronan+oligosaccharides+promote+excisional+wound+healing+through+enhanced+angiogenesis.">Hyaluronan oligosaccharides promote excisional wound healing through enhanced angiogenesis.</a> Matrix Biology. 29, 107-116 (2010).</li><li>Slevin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronan-mediated+angiogenesis+in+vascular+disease:+Uncovering+RHAMM+and+CD44+receptor+signaling+pathways.">Hyaluronan-mediated angiogenesis in vascular disease: Uncovering RHAMM and CD44 receptor signaling pathways.</a> Matrix Biology. 26, 58-68 (2007).</li></ol>

Figura esquemática de vídeo reproduzido com permissão; Direitos Autorais Wiley-VCH Verlag GmbH &amp; Co. KGaA. Dickinson LE, Kusuma S, Gerecht S. Reconstruindo o nicho de diferenciação das células-tronco embrionárias usando biomateriais. Macromol. Biosci. 2010; 21 de outubro. [Epub ahead of print].

O método apresentado micropatterning HA permite o estudo das interações celulares com HA exógeno. HA é conhecida por desempenhar um papel fundamental na progressão do câncer 1 no entanto tem havido estudos limitados a investigar a interação de células de câncer em duas dimensões superfícies HA padronizadas. Um estudo controlado sobre micropadrões exógenos HA permite a visualização de alta resolução de adesão de células cancerosas, crescimento e migração e pode elucidar ainda mais os fun...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>SU-2025 photoresist</td>
<td>&nbsp;</td>
<td>MicroChem Corp.</td>
<td>Y111069</td>
<td>&nbsp;</td>
<tr>
<td>SU-8 developer</td>
<td>&nbsp;</td>
<td>MicroChem Corp.</td>
<td>Y020100</td>
<td>&nbsp;</td>
<tr>
<td>Sylgard 184</td>
<td>&nbsp;</td>
<td>Dow Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>3-aminopropyltrimethoxysilane (APTMS)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>281778</td>
<td>&nbsp;</td>
<tr>
<td>2- [methoxy(polyethyleneoxy) propyl] trimethoxysilane (Peg-silane)</td>
<td>&nbsp;</td>
<td>Gelest Inc</td>
<td>SIM6492.7</td>
<td>&nbsp;</td>
<tr>
<td>1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>22980</td>
<td>&nbsp;</td>
<tr>
<td>N-hydroxysuccinimide (NHS)</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>24500</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescein labeled hyaluronic acid (FL-HA)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrick</td>
<td>F1177</td>
<td>Reconstitute with 10ml of DI water </td>
</tr>
<tr>
<td>MDA-MB-231 breast carcinoma cells</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>HTB-26</td>
<td>&nbsp;</td>
<tr>
<td>LS174t colon carcinoma cells</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>Cl-188</td>
<td>&nbsp;</td>		</tbody>
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micropatterned surfaces to study hyaluronic acid interactions with cancer cells

Invasão e progressão do câncer envolve um fenótipo de células móveis, que está sob regulamentação complexa por fatores de crescimento / citocinas e da matriz extracelular (ECM) componentes dentro do microambiente do tumor. Ácido hialurônico (AH) é um componente ECM estroma que é conhecido para facilitar a progressão do tumor pela invasão melhoria, crescimento e angiogênese 1. Interação da HA com o seu celular CD44 receptor de superfície induz sinalização eventos que promovem o crescimento de células tumorais, sobrevivência e migração, aumentando assim a propagação metastática 2-3. HA é um glicosaminoglicano, aniônicos nonsulfated composto por unidades repetitivas de ácido D-glucurônico e DN-acetilglucosamina. Devido à presença de grupos carboxila e hidroxila em unidades de dissacarídeo repetindo, HA nativa é em grande parte hidrofílica e passíveis de modificações químicas que introduzem grupos sulfato para imobilização photoreative 4-5. Estudos anteriores envolvendo a imobilização de HA em superfícies utilizam o comportamento bioresistant da HA e seu derivado sulfatado para controlar a adesão celular em superfícies 6-7. Nestes estudos de adesão celular ocorre preferencialmente em regiões não-HA padronizadas. Para analisar as interacções celulares com HA exógeno, desenvolvemos padrões superfícies funcionalizadas que permitam um estudo controlado e de alta resolução de visualização das interações de células cancerosas com HA. Utilizamos a impressão microcontact (UCP) para definir padrões discretos regiões de HA em superfícies de vidro. A &quot;tethering&quot; abordagem que se aplica a química carbodiimida ligando para imobilizar o AH foi utilizado 8. Superfícies de vidro foram microcontact impressos com uma aminosilane e reagiu com uma solução de HA de rácios otimizada de EDC e NHS para habilitar imobilização HA em matrizes padronizadas. Incorporando a química carbodiimida com MCP permitiu a imobilização de HA para regiões definidas, criando superfícies adequadas para aplicações em vitro. Ambas as células de câncer de cólon e células de câncer de mama implicitamente interagiram com as superfícies HA micropatterned. Adesão célula cancerosa ocorreu dentro de 24 horas com a proliferação por 48 horas. Utilizando superfícies HA micropatterned, demonstramos que a adesão de células cancerosas ocorre através do receptor CD44 HA. Além disso, as superfícies HA padronizadas eram compatíveis com microscopia eletrônica de varredura (MEV) e resolução de imagem permitiu elevado de câncer de saliências adesivas celular e espalhar sobre os padrões de HA para analisar a motilidade de células cancerosas em HA exógeno.

Watch this Scientific Journal Video about Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells at JoVE.com

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Uma nova abordagem que permite a resolução de alta análise das interações de células de câncer com ácido hialurônico exógeno (HA) é descrita. Superfícies padronizadas são fabricados através da combinação química carbodiimida e impressão microcontact.

Superfícies micropatterned para estudar as interações ácido hialurônico com células de câncer

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

micropatterned-surfaces-to-study-hyaluronic-acid-interactions-with

Research

JoVE Journal

Bioengineering

15.0K visualizações.  Johns Hopkins University. Uma nova abordagem que permite a resolução de alta análise das interações de células de câncer com ácido hialurônico exógeno (HA) é descrita. Superfícies padronizadas são fabricados através da combinação química carbodiimida e impressão microcontact.

Vídeo: Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

Visualização melhorada e Análise Quantitativa dos Efeitos de Drogas Usando Células micropatterned

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

Bioengenharia

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

Projeto de um biorreator de pressão cíclica para o Ex Vivo Estudo de válvulas cardíacas Aórtica

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

An Experimental System to Study Mechanotransduction in Fetal Lung Cells

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key component of lung development is the differentiation of alveolar type II epithelial cells, the major source of pulmonary surfactant. These cells also participate in fluid homeostasis in the alveolar lumen, host defense, and injury repair. In addition, distal lung parenchyma cells can be directly exposed to exaggerated stretch during mechanical ventilation after birth. However, the precise molecular and cellular mechanisms by which lung cells sense mechanical stimuli to influence lung development and to promote lung injury are not completely understood. Here, we provide a simple and high purity method to isolate type II cells and fibroblasts from rodent fetal lungs. Then, we describe an in vitro system, The Flexcell Strain Unit, to provide mechanical stimulation to fetal cells, simulating mechanical forces in fetal lung development or lung injury. This experimental system provides an excellent tool to investigate molecular and cellular mechanisms in fetal lung cells exposed to stretch. Using this approach, our laboratory has identified several receptors and signaling proteins that participate in mechanotransduction in fetal lung development and lung injury.

Mechanical forces play a key role in lung development and lung injury. Here, we describe a method to isolate rodent fetal lung type II epithelial cells and fibroblasts and to expose them to mechanical stimulation using an in vitro system.

Um sistema experimental para estudar Mecanotransdução em células do pulmão fetal

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key ...

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales 1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.
Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 &mu;m) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

A microfluídica Endothelialized para estudo das interacções microvasculares em Doenças Hematológicas

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

Stretching Micropatterned Cells on a PDMS Membrane

Mechanical forces exerted on cells and/or tissues play a major role in numerous processes. We have developed a device to stretch cells plated on a PolyDiMethylSiloxane (PDMS) membrane, compatible with imaging. This technique is reproducible and versatile. The PDMS membrane can be micropatterned in order to confine cells or tissues to a specific geometry. The first step is to print micropatterns onto the PDMS membrane with a deep UV technique. The PDMS membrane is then mounted on a mechanical stretcher. A chamber is bound on top of the membrane with biocompatible grease to allow gliding during the stretch. The cells are seeded and allowed to spread for several hours on the micropatterns. The sample can be stretched and unstretched multiple times with the use of a micrometric screw. It takes less than a minute to apply the stretch to its full extent (around 30%). The technique presented here does not include a motorized device, which is necessary for applying repeated stretch cycles quickly and/or computer controlled stretching, but this can be implemented. Stretching of cells or tissue can be of interest for questions related to cell forces, cell response to mechanical stress or tissue morphogenesis. This video presentation will show how to avoid typical problems that might arise when doing this type of seemingly simple experiment.

This manuscript presents a technique to apply or release forces on adherent cells or tissues using unidirectional stretching.

Alongamento Células micropatterned num PDMS Membrana

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

Engenharia de Tecidos de tumor estromal Microenvironment com Aplicação a invasão Cancer Cell

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

A transfecção Gene direção Células Spheroid em placas micropadronadas Cultura para Transplante de Células geneticamente modificados

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

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

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

Preparação de Thermoresponsive nanoestruturados Surfaces para Engenharia de Tecidos

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

Preparation of 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 ...

A Facile and Eco-friendly Route to Fabricate Poly(Lactic Acid) Scaffolds with Graded Pore Size

Over the recent years, functionally graded scaffolds (FGS) gaineda crucial role for manufacturing of devices for tissue engineering. The importance of this new field of biomaterials research is due to the necessity to develop implants capable of mimicking the complex functionality of the various tissues, including a continuous change from one structure or composition to another. In this latter context, one topic of main interest concerns the design of appropriate scaffolds for bone-cartilage interface tissue. In this study, three-layered scaffolds with graded pore size were achieved by melt mixing poly(lactic acid) (PLA), sodium chloride (NaCl) and polyethylene glycol (PEG). Pore size distributions were controlled by NaCl granulometry and PEG solvation. Scaffolds were characterized from a morphological and mechanical point of view. A correlation between the preparation method, the pore architecture and compressive mechanical behavior was found. The interface adhesion strength was quantitatively evaluated by using a custom-designed interfacial strength test. Furthermore, in order to imitate the human physiology, mechanical tests were also performed in phosphate buffered saline (PBS) solution at 37 &#176;C. The method herein presented provides a high control of porosity, pore size distribution and mechanical performance, thus offering the possibility to fabricate three-layered scaffolds with tailored properties by following a simple and eco-friendly route.

A Facile and Eco-friendly Route to Fabricate PolyLactic Acid Scaffolds with Graded Pore Size

In this work, poly(lactic acid)/polyethylene glycol (PLA/PEG) scaffolds were prepared by using a combination of melt mixing and selective leaching. The method herein discussed permitted to develop three-layer scaffolds by highly controlling both porosity and pore size. The mechanical properties were also evaluated in a physiological environment.

A Facile e Eco-friendly rota para fabricar poli (ácido láctico) Scaffolds com Graded Pore Tamanho

Over the recent years, functionally graded scaffolds (FGS) gaineda crucial role for manufacturing of devices for tissue engineering. The importance of ...