Les auteurs reconnaissent l&#39;usage du laboratoire d&#39;analyse de surface à l&#39;Université Johns Hopkins, financé dans le cadre de la Science Materials Research and Engineering Center à travers la Fondation nationale des sciences. LED est un stagiaire et un IGERT National Science Foundation Graduate Fellow. Cette recherche a été partiellement financé par le NIH octroi U54CA143868.

1. La photolithographie standard pour fabrication de timbres Micropatterned <ol><li> Rincer une nouvelle plaquette de silicium avec de l&#39;éthanol et sécher avec un courant d&#39;air. Utilisez une pince pour manipuler plaquette et éviter les défauts de surface pendant le processus entier. </li><li> Transfert plaquette pour faire tourner coucheuse et couvrir surface de la galette avec les SU-2025 photoresist négatif. Couvrir au moins 80% de la plaquette de résine photosensible. Manteau de Spin pendant 10 secondes à 600rpm, suivie de 30 secondes à 3000rpm. En raison de la photosensibilité de la résine photosensible, éteindre les lumières Chambre à partir de ce point vers l&#39;avant. </li><li> Transfert photorésine plaquette de silicium recouvert la plaque chaude pour &quot;soft-bake&quot; à 95 ° C pendant 3 minutes. Retirer de la plaque chaude et laisser 1 minute pour refroidir. </li><li> Placer un pré-fabriqués masque avec le motif désiré sur le dessus de la plaquette de silicium photosensible couvertes et d&#39;exposer aux rayons ultraviolets (UV) (350-450 nm) pendant 20 secondes. </li><li> Plaquette de silicium Transférer dans une assiette chaude pour &quot;hard-bake&quot; à 110 ° C pendant 6 minutes. </li><li> Allumez les lumières et les retirer à partir de plaquettes plaque chauffante. Modèles distincts devraient être visibles à ce stade. </li><li> Rincer soigneusement plaquette de silicium avec des SU-8 développeur résine photosensible. Après 3 rinçages avec une solution développeur, rincer avec de l&#39;éthanol. Si aucun résidu blanc est présent, répétez rincer avec la solution de révélateur photosensible, ou tout simplement plonger la plaquette dans la solution de révélateur pendant 2 minutes et procéder à nouveau avec de l&#39;éthanol rincer. Laver à l&#39;eau et l&#39;air sec. </li><li> Mélanger polydiméthylsiloxane (PDMS) solution d&#39;élastomère et de durcisseur dans un ratio de 10:1 poids. Centrifuger à 900rpm pendant 5 minutes pour enlever les bulles d&#39;air. </li><li> Placez motifs plaquette dans une boîte de Pétri et versez PDMS sur wafer de silicium. S&#39;il ya des bulles présentes, dans dessiccateur sous vide pendant quelques minutes jusqu&#39;à ce bulles disparaissent. </li><li> Cure nuit à température ambiante (TA) pour former un tampon élastomère complémentaires. Si PDMS n&#39;est pas complètement guéri après 12 heures, mettre au four à 60 ° C pendant 30 minutes. </li><li> Retirer délicatement le PDMS à partir de plaquettes de silicium. Utilisez une lame de rasoir pour couper de timbre à la taille désirée. Soniquer dans l&#39;éthanol pendant 15 minutes. Gaufrettes peut être utilisé plusieurs fois pour créer des timbres nouveaux élastomères. </li></ol> 2. Micropatterning HA <ol><li> Plasma nettoyage des lames de verre pendant 5 minutes. Placez toutes les diapositives dans la chambre et le vide pendant 5 minutes. Puis permettent à faible débit d&#39;oxygène pour entrer dans la chambre. Tournez nettoyant plasma pendant 5 minutes. Retirer du plasma et plus propre place dans une boîte de Pétri en utilisant une pince. </li><li> Pendant le nettoyage au plasma, soniquer timbres de PDMS dans l&#39;éthanol pendant 15 minutes. </li><li> Préparer une fraîche 3% v / v solution de 3-aminopropyltriméthoxysilane (APTMS) dans 95% v / v d&#39;éthanol dans un tube à centrifuger de 15 ml. Laisser réagir pendant 5 minutes à température ambiante pour former un silanol. </li><li> Placez timbre en PDMS structure en place sur les spin coater mandrin et la couverture à motifs de surface avec une solution APTMS. Spincoat à 3500rpm pendant 30 secondes. Ne touchez pas la surface du tampon, seulement les côtés d&#39;un timbre en PDMS. </li><li> Transfert APTMS solution pour le plasma nettoyé les surfaces de verre: jeu de timbres vers le bas sur une extrémité du côté de motifs et glissent doucement en appuyant sur PDMS ainsi il est complètement en contact avec une surface en verre pendant 1 minute. </li><li> Retirez délicatement PDMS de timbre, et permettre à lame de verre à motifs à incuber en RT pendant 30 minutes. Rincer les surfaces modelées avec de l&#39;éthanol et l&#39;air sec. Soniquer PDMS timbre pour 15 minutes pour enlever l&#39;excès APTMS. </li><li> Surfaces de chaleur dans le four ou sur plaque chauffante pendant 1 heure à 115 ° C. Rincer avec de l&#39;éthanol et sécher à l&#39;air. </li><li> Préparer le polyéthylène glycol frais (PEG)-silane solution de 20% v / v 2 - [méthoxy (polyéthylèneoxy) propyl] triméthoxysilane dans le toluène. Cela servira comme une région non-adhésif entourant les modèles. </li><li> Transfert diapositives verre imprimé d&#39;un plat en verre de Pétri et incuber dans le PEG-silane solution pendant 45 minutes à 75 ° C sur une plaque chauffante. Rincer avec du toluène, l&#39;eau et sécher à l&#39;air. </li><li> Stériliser pendant 1 heure en utilisant irradiation UV germicide dans une enceinte de sécurité biologique. Procéder dans le cabinet de sécurité biologique à partir de ce point en avant pour maintenir la stérilité. </li><li> Préparer une solution aqueuse de HA stérile. 10mm de 1-éthyl-3-(3-diméthylaminopropyl) carbodiimide (EDC) 5mM de N-hydroxysuccinimide 50μg / ml marquée à la fluorescéine HA </li><li> Appliquer la solution HA à des substrats de verre à motifs dans des conditions stériles et protégés de la lumière. Des substrats doit être au repos pendant 16-24 heures. </li><li> Aspirer solution HA, laver avec du tampon phosphate salin (PBS), et couvrir la surface avec 1% d&#39;albumine sérique bovine (BSA) dans du PBS pendant 1 heure. La surface est maintenant prête pour la culture cellulaire. </li></ol> 3. Culture cellulaire sur HA Surfaces Micropatterned <ol><li> Développer MDA-MB-231 cellules humaines de cancer du sein et LS174T cellules carcinome colorectal, selon les instructions du fabricant. </li><li> Suspensions semences cellule unique de cellules cancéreuses avec des densités comprises entre 0,4 et 1x10 <sup&gt; Les cellules du cancer 6 par 18,75 cm 2 de surface de verre et sur ​​HA immobilisé surfaces. Incuber dans incubateur humidifié (37 ° C) dans des atmosphères maintenue avec 5% de CO 2. L&#39;adhérence cellulaire doit se produire dans les 24 heures et peut être observé en utilisant un microscope à lumière inversée </li><li> Surfaces de culture cellulaire peut être maintenue jusqu&#39;à 5 jours dans des conditions incubateur standard. Changer les médias tous les jours. La morphologie cellulaire et la croissance peut être observé en utilisant un microscope à lumière inversée. D&#39;autres dosages cellulaires peuvent être appliquées pour analyser les réponses à la surface HA. </li></ol> 4. Les résultats représentatifs: La chimie carbodiimide utilisé pour immobiliser de façon covalente à des diapositives HA APTMS verre imprimé est montré dans la figure 1A. EDC est un agent de réticulation de longueur nulle qui réagit avec HA groupes carboxyle pour former une amine réactive intermédiaires. Comme cet intermédiaire est sensible à l&#39;hydrolyse, le NHS est ajouté pour augmenter l&#39;efficacité de réaction carbodiimide. Comme solution de HA interagit avec micropatterns APTMS, une des formes stables liaison amide entre HA intermédiaires et amine primaire de ATPMS. Un exemple de motifs de surface HA visualisés en utilisant un microscope à fluorescence et une analyse ImageJ intensité de fluorescence de démontrer l&#39;immobilisation motifs de HA sont représentées (figure 1B-C). Les surfaces HA micropatterned permettent l&#39;étude des interactions des cellules cancéreuses avec HA expgenous. Les deux MDA-MB-231 maternel et LS174T humaine lignes cellulaires du cancer du côlon préférentiellement adhérer à HA régions micropatterned (figure 2). Imagerie à haute résolution en utilisant la microscopie électronique à balayage peuvent être utilisés pour analyser les interactions des cellules cultivées avec des surfaces HA immobilisé (figure 3). <img src="/files/ftp_upload/2413/2413fig1.jpg" alt="Figure 1" /> Figure 1 HA surfaces micropatterned (A) Schéma décrivant le développement des surfaces fonctionnelles HA, (B) image de fluorescence de la fluorescéine (vert)-étiquetées HA micropatterned surface. (C) carte 3D répartition des pixels en utilisant le logiciel ImageJ démontrant les modèles HA discret. Barre d&#39;échelle = 100 microns. Modifié à partir de 9 avec la permission d&#39;Elsevier. <img src="/files/ftp_upload/2413/2413fig2.jpg" alt="Figure 2" /> Figure 2. Adhérence des cellules du cancer sur les surfaces micropatterned HA. Les deux points (A) et du sein (B) le carcinome de préférence collé sur plaques HA avec 24 heures de culture. Modifié à partir de 9 avec la permission d&#39;Elsevier. <img src="/files/ftp_upload/2413/2413fig3.jpg" alt="Figure 3" /> Figure 3. Interactions cellulaires du cancer avec HA. (A) Numérisation d&#39;images de microscopie électronique de cellules du cancer du côlon (A) cultivées sur des surfaces HA montré à faible grossissement (à gauche) et à fort grossissement (à droite; de ​​carrés à gauche) et des cellules du cancer du sein (B) cultivées sur des surfaces HA montré à faible grossissement (à gauche) et à fort grossissement (à droite; de ​​carrés sur la gauche).

<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>

Figure schématique vidéo reproduit avec la permission; auteur Wiley-VCH Verlag GmbH &amp; Co. KGaA. Dickinson LE, S Kusuma, Gerecht S. Reconstruire la niche la différenciation des cellules souches embryonnaires utilisant des biomatériaux. Macromol. Biosci. 2010; le 21 octobre. [Epub ahead of print].

La méthode présentée permet micropatterning HA l&#39;étude des interactions cellulaires avec exogène HA. HA est connue pour jouer un rôle clé dans une progression du cancer mais il ya eu des études limitées étudie l&#39;interaction des cellules de cancer sur deux dimensions surfaces texturées HA. Une étude contrôlables sur micropatterns HA exogène permet la visualisation haute résolution de l&#39;adhérence des cellules cancéreuses, la croissance et la migration et peut élucider les fondament...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<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>
		</table>
		</div>

micropatterned surfaces to study hyaluronic acid interactions with cancer cells

L&#39;invasion et la progression du cancer implique un phénotype cellulaire mobiles, qui est sous réglementation complexe de facteurs de croissance / cytokines et la matrice extracellulaire (ECM) des composants dans le microenvironnement de la tumeur. L&#39;acide hyaluronique (HA) est une composante stromale ECM qui est connue pour faciliter la progression de la tumeur par l&#39;invasion amélioration, la croissance et l&#39;angiogénèse 1. Interaction des HA avec son récepteur cellulaire CD44 de surface induit événements de signalisation qui favorisent la croissance des cellules tumorales, la survie et la migration, augmentant ainsi la dissémination métastatique 2-3. HA est une anioniques, glycosaminoglycane nonsulfated composé d&#39;unités répétitives de l&#39;acide D-glucuronique et de DN-acétylglucosamine. En raison de la présence de groupes carboxyle et hydroxyle sur les unités disaccharide répéter, HA natif est largement hydrophiles et se prêtent à des modifications chimiques qui introduisent des groupes sulfate d&#39;immobilisation photoreative 4-5. Des études précédentes impliquant le immobilisations de HA sur des surfaces d&#39;utiliser le comportement bioresistant de HA et de son dérivé sulfaté de contrôler l&#39;adhésion cellulaire sur des surfaces 6-7. Dans ces études de l&#39;adhésion cellulaire survient préférentiellement sur les régions à motifs non-HA. Pour analyser les interactions cellulaires avec HA exogène, nous avons développé motifs surfaces fonctionnalisées qui permettent une étude contrôlable et la visualisation haute résolution des interactions des cellules cancéreuses avec HA. Nous avons utilisé l&#39;impression par microcontact (RUU) pour définir des motifs discrets régions HA sur les surfaces en verre. Un &quot;tethering&quot; approche qui s&#39;applique la chimie carbodiimide reliant à immobiliser HA a été utilisé 8. Les surfaces vitrées ont été imprimés avec une microcontact aminosilane et réagir avec une solution HA des ratios d&#39;EDC et optimisé afin de permettre l&#39;immobilisation du NHS HA dans des tableaux à motifs. Incorporer la chimie carbodiimide avec MCP a permis l&#39;immobilisation de l&#39;HA à des régions définies, créant des surfaces appropriées pour les applications in vitro. Les deux cellules de cancer du côlon et de cellules du cancer du sein implicitement interagi avec les surfaces micropatterned HA. L&#39;adhésion des cellules du cancer survenu dans les 24 heures avec la prolifération de 48 heures. L&#39;utilisation de surfaces HA micropatterned, nous avons démontré que l&#39;adhésion des cellules du cancer se fait par le CD44 récepteur HA. Par ailleurs, HA surfaces texturées étaient compatibles avec microscopie électronique à balayage (MEB) et a permis d&#39;imagerie haute résolution des protubérances cellulaires du cancer adhésif et répandre sur les habitudes de HA pour analyser la motilité des cellules cancéreuses sur HA exogène.

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

Une nouvelle approche qui permet l&#39;analyse de la haute résolution des interactions cellulaires du cancer avec l&#39;acide hyaluronique exogène (HA) est décrite. Surfaces texturées sont fabriqués en combinant la chimie carbodiimide et l&#39;impression par microcontact.

Surfaces Micropatterned pour étudier les interactions acide hyaluronique avec les cellules cancéreuses

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 vues.  Johns Hopkins University. Une nouvelle approche qui permet l'analyse de la haute résolution des interactions cellulaires du cancer avec l'acide hyaluronique exogène (HA) est décrite. Surfaces texturées sont fabriqués en combinant la chimie carbodiimide et l'impression par microcontact.

Vidéo: 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.

Amélioration de la visualisation et l&#39;analyse quantitative des effets des drogues utilisant des cellules 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 ...

Recherche

Bio-ingénierie

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.

Conception d&#39;un bioréacteur pour la pression cyclique Ex Vivo Étude des valves cardiaques aortiques

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

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.

Microfluidique endothélialisée pour étudier les interactions microvasculaires dans Hémopathies

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.

Stretching cellules Micropatterned sur une membrane PDMS

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.

Ingénierie tissulaire de la tumeur stromale Microenvironnement avec application de 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 ...

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.

Transfection génique vers les cellules Spheroid sur microélectrodes plaques de culture destinés à la transplantation de cellules génétiquement modifiées

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.

Préparation de thermosensible surfaces nanostructurées pour l&#39;ingénierie tissulaire

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.

Préparation de la 3D collagène Gels et microcanaux pour l&#39;étude des interactions 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 et écologique Route pour fabriquer poly (acide lactique) Échafaudages avec Graded Pore Taille

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

Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present several limitations that have led to the use of biomaterials in combination with osteogenic growth factors, such as bone morphogenetic proteins (BMPs). Commonly used absorption or encapsulation methods require supra-physiological amounts of BMP2, typically resulting in a so-called initial burst release effect that provokes several severe adverse side effects. A possible strategy to overcome these problems would be to covalently couple the protein to the scaffold. Moreover, coupling should be performed in a site-specific manner in order to guarantee a reproducible product outcome. Therefore, we created a BMP2 variant, in which an artificial amino acid (propargyl-L-lysine) was introduced into the mature part of the BMP2 protein by codon usage expansion (BMP2-K3Plk). BMP2-K3Plk was coupled to functionalized beads through copper catalyzed azide-alkyne cycloaddition (CuAAC). The biological activity of the coupled BMP2-K3Plk was proven in vitro and the osteogenic activity of the BMP2-K3Plk-functionalized beads was proven in cell based assays. The functionalized beads in contact with C2C12 cells were able to induce alkaline phosphatase (ALP) expression in locally restricted proximity of the bead. Thus, by this technique, functionalized scaffolds can be produced that can trigger cell differentiation towards an osteogenic lineage. Additionally, lower BMP2 doses are sufficient due to the controlled orientation of site-directed coupled BMP2. With this method, BMPs are always exposed to their receptors on the cell surface in the appropriate orientation, which is not the case if the factors are coupled via non-site-directed coupling techniques. The product outcome is highly controllable and, thus, results in materials with homogeneous properties, improving their applicability for the repair of critical size bone defects.

Biomaterials doped with Bone Morphogenetic Protein 2 (BMP2) have been used as a new therapeutic strategy to heal non-union bone fractures. To overcome side effects resulting from an uncontrollable release of the factor, we propose a new strategy to site-directly immobilize the factor, thus creating materials with improved osteogenic capabilities.

Immobilisation dirigée de protéine morphogénétique osseuse 2 aux Surfaces solides par Click Chemistry

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present ...