This work was supported by the Belgian National Foundation for Scientific Research (F.R.S.-FNRS) through &#8220;MIS Confocal Microscopy&#8221;, &#8220;Cr&#233;dit aux Chercheurs&#8221; grants and the &#8220;Nanomotility FRFC project&#8221; (no. 2.4622.11). T.G. doctoral fellowship is supported by the Foundation for Training in Industrial and Agricultural Research (FRIA). The authors gratefully acknowledge Sylvain Desprez for mechanical characterization and G&#233;raldine Circelli for confocal imaging.

1 Activation de la surface de verre Lamelles <ol><li> Lieu lamelles de verre circulaires (diamètre 25 mm) dans une boîte de Pétri et frottis de 0,1 M NaOH solution sur elle pendant 5 min (hotte pour émanations chimiques recommandé). </li><li> Retirer la solution de NaOH et une immersion totale lamelles avec le trou DDH 2 O stérile pendant 20 minutes tout en balançant doucement sur ​​une plaque à bascule dans une hotte de culture stérile. </li><li> Égoutter stérile ddH 2 O et répéter l&#39;étape 1.2. </li><li> Retirez les lamelles avec des pincettes stériles et les placer dans une nouvelle boîte de Pétri avec le visage activée vers le haut. </li><li> Lamelles sec sous un flux constant de haute pureté de l&#39;azote gazeux. </li><li> Dans une hotte de culture stérile, appliquer une mince couche de l&#39;acrylate de 3 (triméthoxysilyl) propyle (92%) sur le côté actif de la lamelle couvre-objet pendant 1 heure. </li><li> Laver abondamment lamelles de verre avec 3 lavages de ddH stérile 2 O et les plonger dans le trou DDH 2 O stérile dans une nouvelle boîte de Pétri. </li><li> Appuyez sur la boîte de Pétriavec du parafilm et placer sur une plaque à bascule sous agitation douce pendant 10 min. </li><li> Retirez les lamelles de ddH 2 O avec des pincettes stériles avec pointes fines et placez-les dans une nouvelle boîte de Pétri avec le visage activée vers le haut. </li><li> Stocker à température ambiante dans un endroit sec avec du papier d&#39;aluminium pour éviter la poussière de coller aux lamelles. </li></ol> 2 Préparation de l&#39;hydroxy-paam hydrogels <ol><li> Préparer un poids de 65 mg de N-hydroxyéthyl acrylamide (HEA) dans un tube Eppendorf de 1,5 ml. Il est important de préparer une solution fraîche de HEA. </li><li> Ajouter 1 ml de tampon HEPES 50 mM à HEA et mélanger à l&#39;aide d&#39;un vortex jusqu&#39;à la dissolution complète de HEA. </li><li> Ajouter 400 ul de 40% en poids / poids dans une solution de HEPES acrylamide et le volume requis de 2% p / p dans de l&#39;HEPES solution de bis-acrylamide (voir tableau 1) pour atteindre la rigidité désirée de l&#39;hydrogel. Ajuster avec 50 mM de HEPES à un volume final de 5 ml. </li><li> Mélanger la solution avec un vortex et dégazerdans une chambre à vide pendant 20 minutes afin de réduire la concentration d&#39;oxygène au sein de la solution, ce qui empêche la polymérisation hydroxy-PAAm. </li><li> Sous une hotte stérile, filtrer la solution dégazée avec un filtre de 0,2 um de taille de pores, afin de la stériliser. </li><li> Activer lamelles de verre circulaires (diamètre 22 mm) dans un produit UV / ozone pendant 7 min. </li><li> Préparer 100 pi de persulfate d&#39;ammonium (APS) solution à 10%, soit 10 mg APS dans 100 ul ddH 2 O. Il est important de préparer une solution fraîche de l&#39;APS. </li><li> Ajouter 2,5 pi de la tétraméthylènediamine (TEMED) et 25 ul de solution à l&#39;APS-hydroxy PAAm solution stérilisée (étape 2.5) pour initier la polymérisation. Mélanger la solution par 3 pipettages successives sans introduction de bulles, dans des conditions stériles. </li><li> Sous une hotte stérile, déposer une goutte de 25 ul de la solution hydroxy-PAAm sur une lamelle de 25 mm (disponible à partir de l&#39;étape 1.9) et placer immédiatement un 22 mm glcul lamelle (préparé à l&#39;étape 2.5) sur le dessus de la gouttelette de serrer la solution hydroxy-PAAm. Centrer la lamelle de verre de 22 mm avec des pincettes stériles et lisser les bulles. </li><li> Permettre aux hydrogels-hydroxy Paam à polymériser à température ambiante pendant 15 min. Inverser manuellement la solution restante hydroxy-PAAm dans le tube Eppendorf de suivre la fin du processus de polymérisation. </li><li> Immerger complètement stérile avec des lamelles couvre-trou DDH 2 O et on sépare soigneusement les 22 lamelles couvre-objet en verre mm en introduisant le bord d&#39;une lame de rasoir entre les lamelles 22 mm de verre et la couche d&#39;hydrogel hydroxy-PAAm. </li><li> Lavez-hydrogels paam hydroxy avec du PBS stérile (3 échanges de PBS) et de laisser les gels totalement immergés dans du PBS stérile pour maintenir l&#39;hydratation. </li><li> Rangez-hydrogels paam hydroxy dans du PBS stérile à 4 ° C pendant 3 jours. </li></ol> 3. polydiméthylsiloxane (PDMS) Microstamp Fabrication NOTE: La fabrication d&#39;un maître de silicium est nécessaire avant de staTA PDMS fabrication microstamp. Cette microfabrication d&#39;un maître de silicium peut être fait par des techniques de lithographie, ce qui nécessite un équipement et une formation spécialisée. Collaborations avec un centre de nanofabrication sont encouragés à fabriquer le maître de silicium. Vous pouvez également contacter une entreprise qui fabrique des maîtres de silicium microstructuré sur mesure à la demande. Il est important de noter que la fabrication du maître de silicium doit seulement être effectuée une fois. En effet, les maîtres de silicium microstructurées peuvent être utilisés indéfiniment pour produire des timbres élastomères. <ol><li> Mélange PDMS et l&#39;agent de durcissement dans un rapport de 10: 1 dans un bêcher en matière plastique et homogénéiser à l&#39;aide d&#39;une pipette pendant 10 minutes. </li><li> Dégazer le mélange PDMS sous vide pour éliminer les bulles d&#39;air qui ont été formés lors de l&#39;étape 3.1. </li><li> Placez le maître de silicium microstructuré dans une boîte de Pétri et jeter une couche épaisse de 10 mm de mélange PDMS dégazé sur elle sans formation de bulles. </li><li> Laisser sécher les PDMS pendant 2 heures à 60 ° C dans unfour. </li><li> Dans un environnement exempt de poussière, peel-off de la couche de PDMS et accises 1 cm 2 microstamps avec un scalpel. </li><li> En utilisant des pinces, le lieu PDMS microstamps motif dans une boîte de Pétri. </li></ol> 4. micromodelage hydroxy-paam hydrogels <ol><li> La place PDMS microstamps dans un mélange éthanol / eau (50/50) et de solution de traitement par ultrasons pendant 15 min. </li><li> Sécher les timbres avec un courant d&#39;écoulement d&#39;azote et les mettre en place dans le modèle d&#39;un nettoyeur UV / ozone (λ &lt;200 nm) pendant 7 min. </li><li> Sous une hotte stérile, déposer une goutte de 150 pl d&#39;une solution de protéine souhaitée (par exemple, 100 pg / ml de laminine dans du PBS ou 25 pg / ml de fibronectine dans du PBS) sur la surface microstructurée de 1 cm 2 PDMS cachet. </li><li> Facultatif: modifier la concentration de la solution de protéine à moduler la densité cellulaire-ligand. </li><li> Répartir la solution de protéine à travers la surface de tampon en le déplaçant avec une pointe d&#39;une pipette stérile de l&#39;un vers l&#39;angle dele timbre. </li><li> Laisser la solution de protéine à adsorber sur le timbre de PDMS 60 min sous une hotte stérile. Éteindre les lampes pour éviter d&#39;endommager des protéines. </li><li> Sous une hotte stérile, transférer des lamelles recouvertes hydroxy-paam (disponibles à partir de l&#39;étape 2.13) dans une boîte de Pétri. </li><li> Enlever l&#39;excès de PBS de la surface de substrats hydroxy-paam avec un flux d&#39;azote faible dans des conditions stériles. Arrêter la procédure dès que aucune preuve de l&#39;eau stagnante sur la surface du gel est observé. Le gel ne doit pas être complètement séchée à ce stade. </li><li> Sécher soigneusement la surface structurée du PDMS timbre avec un flux constant de haute pureté de l&#39;azote gazeux. </li><li> Saisir le poinçon revêtu de protéine avec une pince de tissu de chambre et placer la surface structurée en contact avec la surface de l&#39;hydrogel séché. Appliquer brefs points de pression avec le bout de pinces sur le dessus du timbre de PDMS pour assurer un bon contact entre microcaractéristiques de timbre et la surface de l&#39;hydrogel. </li><li> Laissez le timbre de PDMS sur ee surface d&#39;hydrogel pendant 1 heure à température ambiante. </li><li> Retirez délicatement PDMS timbres de hydrogels hydroxy-paam avec une pince de tissus d&#39;habillage et de suivre l&#39;étape 4.1 pour nettoyer le timbre. </li><li> Laver abondamment les hydrogels-paam hydroxy estampillés par 3 échanges de PBS (pH = 7,4) dans des conditions stériles pendant 10 minutes par l&#39;échange. </li><li> Micropatterns supplémentaires optionnels: d&#39;autres protéines de la MEC peut être ajouté à la surface hydroxy-PAAm en suivant les étapes 4.5 à 4.11. </li><li> Des zones non-imprimées passiver avec une solution stérile de BSA à 5 mg / ml dans du PBS pendant une nuit à 4 ° C sous une agitation douce sur un plateau à bascule. </li><li> Laver abondamment par 3 échanges de PBS (pH = 7,4) dans des conditions stériles pendant 10 minutes par l&#39;échange. A ce stade, les hydrogels Paam-hydroxy embouties peuvent être stockés à 4 ° C jusqu&#39;à une semaine. </li></ol> 5. cellulaire dépôt sur microélectrodes hydroxy-paam hydrogels <ol><li> Incuber lamelles dans les médias de la cellule pendant 30-45 min avant le placage caunes. </li><li> Laver les cellules adhérentes en culture dans un flacon de 75 cm 2 de culture avec de la PBS stérile à 37 ° C et la détacher avec 3 ml de trypsine-EDTA ou Accutase pendant 10 min. </li><li> Transférer la quantité désirée de milieu complet de croissance approprié préchauffé à votre lignée de cellules dans le flacon contenant les cellules détachées et centrifuger la suspension de cellules pendant 3 minutes à 650 x g. </li><li> Retirer le surnageant avec une micropipette et remettre en suspension les cellules dans du milieu de culture complet à 15-20,000 cellules / ml. </li><li> Ajouter 4 ml de la solution de cellules à une lamelle microélectrodes (obtenu à l&#39;étape 5.1) et placer la lamelle couvre-objet couvert de cellules dans un incubateur de culture à 37 ° C et 5% d&#39;humidité pendant 1-2 heures. </li><li> Aspirer délicatement les cellules seules et remplacer le milieu de culture. Remettre les cellules fixées à l&#39;incubateur et de les laisser entièrement répartis (3-6 heures, en fonction du type de cellule). </li></ol>

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No conflicts of interest declared.

Plusieurs observations in vitro dans des cellules en biologie moderne ont été réalisées sur des lamelles de verre rigide, souvent recouvertes d&#39;une fine couche de protéines de la MEC ou des peptides synthétiques contenant la séquence RGD. Cependant, ces substrats de culture de base ne récapitulent toute la complexité physico-chimique de l&#39;ECM et n&#39;offrent donc pas un modèle précis pour étudier les processus de mécano-cellulaires. Pour résoudre ce problème, nous proposons une alternati...

De nombreux aspects du microenvironnement cellulaire local (par exemple, la rigidité, la taille des pores, la nature des protéines, ou la densité cellulaire-ligand) fournir un ensemble de coordonnées de signaux de régulation qui contrôlent les processus cellulaires tels que la motilité, la prolifération cellulaire, la différenciation et l&#39;expression des gènes. Modifications des propriétés physico-chimiques du milieu extracellulaire peuvent être perçus par les cellules et provoquent différentes conséquences physiologiques, y compris la déformation de la polarisation cellulaire, la migration et la différenciation. On ne sait pas, cependant, comment les cellules se traduisent modifications ECM en signaux biochimiques cellulaires. Il est donc d&#39;une importance majeure à l&#39;ingénieur contrôlée dans des micro-environnements in vitro qui peuvent reproduire les interactions entre les cellules et leur microenvironnement pour étudier les voies d&#39;mécano. Pour résoudre ce problème, nous avons récemment mis en place une nouvelle méthode 1, appelé hydrogels-paam hydroxy, de générer facilement des deux sounsional matrices souples qui permettent de contrôler de manière indépendante des signaux mécano importantes: la rigidité de la matrice, la géométrie de la cellule et de confinement, de la nature de la protéine et de la densité cellulaire-ligand. ECM dirige les processus cellulaires à travers des gradients de morphogènes (chimiotaxie), des protéines adhésives (haptotaxis), et la rigidité (durotaxis). Au cours des dernières décennies, avancée dans les plates-formes in vitro ont été mis au point pour isoler ces signaux extracellulaires afin de démêler la façon dont les cellules sont capables de traduire les caractéristiques biochimiques et biophysiques dans les processus physiologiques 2-5. Faisceau d&#39;électrons 6, 7 photolithographie, l&#39;immobilisation photochimique 8 ou techniques assistés par plasma 9 ont été élaborés pour orienter la croissance de cellules vivantes sur des substrats microélectrodes. Bien que ces techniques ont donné des résultats importants, la plupart d&#39;entre eux ne permettent pas de discrimination entre l&#39;influence individuelle des différents indices sur le comportement des celluleset ils nécessitent des installations techniques que peu de laboratoires peuvent se permettre. Parmi ces techniques, microcontact impression (uCP), a émergé comme une méthode robuste et accessible pour créer des micro-îles de cellules-adhésif 10. Plus récemment, des efforts considérables 11-14 ont été faits pour développer uCP d&#39;hydrogels avec rigidités accordables afin de reproduire la vaste gamme de rigidités observées dans les tissus vivants. Parmi ces œuvres, le polyacrylamide (PAAm) est devenu populaire 15 et est déjà l&#39;une des matrices les base de polymères les plus couramment utilisés pour la biomécanique de cellules tests. Paam surfaces fonctionnalisées sont communément avec l&#39;agent de réticulation hétérobifonctionnel N-sulfosuccinimidyle-6-protéines [4&#39;-azido-2&#39;-nitrophenylamido] (sulfo-SANPAH) et ECM sont reliés à la surface par activation d&#39;UV de la nitrophényl sulfo-SANPAH groupes azide 16. Une autre technique consiste à couplage de l&#39;hydrazine à des protéines qui ont été fortement oxydéesavec du periodate 17. Hynd et ses collaborateurs ont introduit une technique de structuration des surfaces d&#39;hydrogel biomimétiques avec la protéine et des peptides exige que la photopolymérisation en présence d&#39;un monomère d&#39;acroyl-18 streptavidine. Plus récemment, Tseng et al. Ont rapporté une nouvelle méthode de micromodelage 19 sur la base de l&#39;exposition aux UV profond de PAAm à travers un masque de quartz optique qui oblige à incuber gels PA activés avec [3-diméthylaminopropyl] carbodiimide chlorhydrate de 1-éthyl-3 (EDC) et le N-hydroxysuccinimide (NHS) des solutions d&#39;eau avant d&#39;ajouter la protéine. Malgré la capacité de ces techniques pour créer homogènes et reproductibles protéines micropatterns, la plupart d&#39;entre eux souffrent des limites importantes: processus de longue de synthèse (par exemple, la dialyse, la lyophilisation, etc), des composés chimiques coûteux (par exemple, l&#39;acide hyaluronique, sulfo-SANPAH) de profondeur UV ou irradiation. De plus, ces techniques ne permettent pas de modulation indépendante de la rigidité du substrat, motif finla géométrie, la protéine ECM nature et de la densité cellulaire-ligand. Compte tenu de ces limitations en compte, nous avons développé une approche à base d&#39;acrylamide roman et simple qui permet l&#39;immobilisation d&#39;une variété de protéines et de biomolécules sur des hydrogels mous et permet le réglage indépendant de mécano repères pour décrypter leur rôle sur les fonctions cellulaires. Au lieu de traiter hydrogels paam avec des composés chimiques agressifs, nous introduisons un monomère d&#39;acrylamide commercial avec des groupes hydroxyles lors de la polymérisation PAAm. Cette simple opération permet de surmonter la propriété anti-adhésive intrinsèque d&#39;hydrogels paam sans autres exigences techniques. La présence de groupes hydroxyles conduit à une forte affinité d&#39;hydrogels hydroxy-Paam pour des protéines et des biomolécules qui forment des interactions de liaison hydrogène. En combinaison avec uCP, hydrogels-paam hydroxy permettent une production rapide de deux dimensions plate-forme de culture avec un contrôle indépendantsur la matrice de rigidité, le type de protéines de la MEC, la densité cellulaire-ligand et l&#39;adhérence limitée, qui sont prévu pour être une plate-forme puissante pour étudier la mécanotransduction. Le but de ce protocole est de fournir les informations nécessaires pour faire facilement des hydrogels-paam hydroxy sans aucune expertise en sciences des matériaux. Le but ultime est de fournir un moyen pour les chercheurs de poser des questions physiologiquement pertinents aux niveaux cellulaires et tissulaires qui peuvent conduire à une meilleure compréhension des voies de mécanotransduction impliqués dans les mécanismes physiopathologiques.

<table border="0" cellpadding="0" cellspacing="0" width="573">
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	<tbody>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">UV/Ozone Photoreactor</td>
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			<td style="width:119px;">Model PR-100</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Rocking plate</td>
			<td style="width:71px;">IKAcWerke</td>
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			<td style="width:167px;"></td>
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		</tr>
		<tr height="85">
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			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Parafilm</td>
			<td style="width:71px;">Sigma-Aldrich</td>
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			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Stainless steel forceps with fine tip</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z225304-1EA</td>
			<td style="width:167px;"></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Dressing tissue forceps</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">F4392-1EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Petri dishes in polystyrene</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">P5731-500EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Aluminium foil, thickness 0.5&#160;mm</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">266574-3.4G</td>
			<td style="width:167px;"></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Isopore membrane filter (0,2 &#181;m pore size)</td>
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		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Variable volume micropipette</td>
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			<td style="width:119px;">Z114820</td>
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		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Protein microcentrifuge tubes</td>
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			<td style="width:119px;">Z666505-100EA</td>
			<td style="width:167px;"></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Scalpel handles</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">S2896-1EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Scalpel blades</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">S2771-100EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Cell culture flasks (75 cm2)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">CLS430641</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Ultrasonic bath tray, solid (stainless steel)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z613983-1EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;"></td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Name of the Reagent</td>
			<td style="width:71px;">Company</td>
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		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Polydimethylsiloxane&#160;</td>
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		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Acrylamide (powder)</td>
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			<td style="width:119px;">A3553</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">N,N&#8217;-Methylenebis(acrylamide)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">146072</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">N-Hydroxyethylacrylamide</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">697931</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">N,N,N&#8217;,N&#8217;-Tetramethylethylenediamine</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">T9281</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Amonium PerSulfate (APS)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">A3678</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">3-(Trimetoxysilyl)propyle acrylate</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">1805</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Human Plasma Fibronectin</td>
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		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Laminin from EHS</td>
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		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Sodium hydroxyde</td>
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		<tr height="26">
			<td height="26" style="height:26px;width:216px;">Double-distilled water (ddH2O)</td>
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			<td style="width:167px;"></td>
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		<tr height="64">
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			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Human Umbilical Vein Endothelial Cells (HUVEC)</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">C-003-5C</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Accutase</td>
			<td style="width:71px;">PAA laboratories</td>
			<td style="width:119px;">L11-007</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">HEPES buffer solution 1M in H20</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">83264-500ML-F</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Antibiotics-antimycotics</td>
			<td style="width:71px;">PAA laboratories</td>
			<td style="width:119px;">P11-002</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Phosphate Buffer Saline solution</td>
			<td style="width:71px;">PAA laboratories</td>
			<td style="width:119px;">H15-002</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Alexa Fluor 488 Phaloidin</td>
			<td style="width:71px;">Molecular Probes</td>
			<td style="width:119px;">A12379</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-vinculin antibody produced in mouse</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">V9131</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Goat anti-mouse antibody-tetramethylrhodamine</td>
			<td style="width:71px;">Molecular Probes</td>
			<td style="width:119px;">T-2762</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-Fibronectin</td>
			<td rowspan="2" style="width:71px;">Sigma-Aldrich</td>
			<td rowspan="2" style="width:119px;">F3648</td>
			<td rowspan="2" style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">(rabbit)</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Streptavidin&#160;</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">41469</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-Laminin antibody</td>
			<td rowspan="2" style="width:71px;">Sigma-Aldrich</td>
			<td rowspan="2" style="width:119px;">L9393</td>
			<td rowspan="2" style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">(rabbit)</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-rabbit IgG-FITC</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">F7512</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Trypsin-EDTA solution</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">T3924-100ML</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Absolute ethanol</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">459844-2.5L</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

preparation of hydroxy-paam hydrogels for decoupling the effects of mechanotransduction cues

Il est maintenant bien établi que de nombreuses fonctions cellulaires sont régis par des interactions de cellules avec des indices physico-chimiques et mécaniques de leur matrice extracellulaire (MEC) environnement. Les cellules eucaryotes détectent constamment leur micro-environnement local à travers les mécanorécepteurs de surface pour transduire les changements physiques de l&#39;ECM en signaux biochimiques, et à intégrer ces signaux pour obtenir des modifications spécifiques de l&#39;expression génique. Fait intéressant, les paramètres physico-chimiques et mécaniques de l&#39;ECM peut se coupler avec l&#39;autre pour réguler le destin des cellules. Par conséquent, une clé pour comprendre la mécanotransduction est de dissocier la contribution relative de l&#39;ECM indices sur les fonctions cellulaires. Nous présentons ici un protocole expérimental détaillé pour générer rapidement et facilement des hydrogels biologiquement pertinentes pour le réglage indépendant de mécano indices in vitro. Nous hydrogels de polyacrylamide chimiquement modifiées (paam) à surmonter leurs intrinsèquement non-ADHESive propriétés par l&#39;incorporation de monomères d&#39;acrylamide fonctionnalisé par des groupes hydroxy au cours de la polymérisation. Nous avons obtenu un roman PAAm hydrogel, appelé hydroxy-PAAm, qui permet l&#39;immobilisation de toute nature souhaitée de protéines de la MEC. La combinaison de l&#39;hydroxy-PAAm hydrogels avec l&#39;impression par microcontact permet de contrôler de façon indépendante la morphologie des cellules individuelles, la rigidité de la matrice, la nature et la densité de protéines de la MEC. Nous fournissons une méthode simple et rapide qui peut être mis en place dans tous les laboratoires de biologie pour étudier dans les processus de mécanotransduction cellulaire in vitro. Nous validons cette nouvelle plate-forme à deux dimensions en menant des expériences sur les cellules endothéliales qui démontrent un couplage mécanique entre la rigidité de l&#39;ECM et le noyau.

La figure 1A présente la co-polymérisation de l&#39;acrylamide (AAM) et de bisacrylamide (bis-AAM) avec du N-hydroxyethylacrylamide (HEA) des monomères contenant un groupe hydroxyle primaire formé par polymérisation radicalaire aléatoire d&#39;un réseau hydrophile de polyacrylamide avec des groupes hydroxyle incorporés (hydroxy-PAAm) . Dans ce protocole, un poids de 65 mg de HEA doit être dilué dans un volume de 1 ml d&#39;HEPES. Sachant que la densité de HEA est à peu près égal à un, nou...

Watch this Scientific Journal Video about Preparation of Hydroxy-PAAm Hydrogels for Decoupling the Effects of Mechanotransduction Cues at JoVE.com

Preparation of Hydroxy-PAAm Hydrogels for Decoupling the Effects of Mechanotransduction Cues

Nous présentons une nouvelle hydrogel de polyacrylamide, appelé hydroxy-PAAm, qui permet une liaison directe de protéines de la MEC avec un coût ou une expertise minimale. La combinaison de l&#39;hydroxy-PAAm hydrogels avec l&#39;impression microcontact facilite le contrôle indépendant de nombreux indices du microenvironnement cellulaire naturel pour l&#39;étude mechanostransduction cellulaire.

Préparation des hydroxy-PAAm hydrogels pour découplage des effets de la mécanotransduction Cues

It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their ...

preparation-hydroxy-paam-hydrogels-for-decoupling-effects

Research

JoVE Journal

Bioengineering

13.2K vues.  Université de Mons.  Nous présentons une nouvelle hydrogel de polyacrylamide, appelé hydroxy-PAAm, qui permet une liaison directe de protéines de la MEC avec un coût ou une expertise minimale. La combinaison de l'hydroxy-PAAm hydrogels avec l'impression microcontact facilite le contrôle indépendant de nombreux indices du microenvironnement cellulaire naturel pour l'étude mechanostransduction cellulaire. 

Vidéo: Preparation of Hydroxy-PAAm Hydrogels for Decoupling the Effects of Mechanotransduction Cues

Axon Stretch Growth: The Mechanotransduction of Neuronal Growth

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata are separated from their axon terminals due to skeletal growth of the enlarging organism (Weiss 1941; Gray, Hukkanen et al. 1992). This mechanotransduction induces a secondary mode of neuronal growth capable of accommodating continual elongation of the axon (Bray 1984; Heidemann and Buxbaum 1994; Heidemann, Lamoureux et al. 1995; Pfister, Iwata et al. 2004).
Axon Stretch Growth (ASG) is conceivably a central factor in the maturation of short embryonic processes into the long nerves and white matter tracts characteristic of the adult nervous system. To study ASG in vitro, we engineered bioreactors to apply tension to the short axonal processes of neuronal cultures (Loverde, Ozoka et al. 2011). Here, we detail the methods we use to prepare bioreactors and conduct ASG. First, within each stretching lane of the bioreactor, neurons are plated upon a micro-manipulated towing substrate. Next, neurons regenerate their axonal processes, via growth cone extension, onto a stationary substrate. Finally, stretch growth is performed by towing the plated cell bodies away from the axon terminals adhered to the stationary substrate; recapitulating skeletal growth after growth cone extension.
Previous work has shown that ASG of embryonic rat dorsal root ganglia neurons are capable of unprecedented growth rates up to 10mm/day, reaching lengths of up to 10cm; while concurrently resulting in increased axonal diameters (Smith, Wolf et al. 2001; Pfister, Iwata et al. 2004; Pfister, Bonislawski et al. 2006; Pfister, Iwata et al. 2006; Smith 2009). This is in dramatic contrast to regenerative growth cone extension (in absence of mechanical stimuli) where growth rates average 1mm/day with successful regeneration limited to lengths of less than 3cm (Fu and Gordon 1997; Pfister, Gordon et al. 2011). Accordingly, further study of ASG may help to reveal dysregulated growth mechanisms that limit regeneration in the absence of mechanical stimuli.

A unique tissue engineering method was developed to elongate numerous nerve fibers in culture by recapitulating axon stretch growth; a form of nervous system growth whereby nerves elongate in conjunction with growth of the enlarging body.

Croissance stretch Axon: La mécanotransduction de croissance neuronale

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata ...

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Bio-ingénierie

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

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

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

Les hydrogels d&#39;alginate de culture d&#39;organe en trois dimensions des ovaires et des trompes

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

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

Faible enrichissement moléculaire des protéines de poids sur les films minces de silice mésoporeuse de recherche de biomarqueurs

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and functionalize polymer chains with compounds that are able to improve biocompatibility, mechanical properties, or cell viability allows the design of novel materials to meet new challenges in the biomedical field. With the polymer functionalization strategies, click chemistry is a powerful tool to improve cell-compatibility and drug delivery properties of polymeric devices. Similarly, the fundamental need of biomedicine to use sterile tools to avoid potential adverse-side effects, such as toxicity or contamination of the biological environment, gives rise to increasing interest in the microwave-assisted strategy.
The combination of click chemistry and the microwave-assisted method is suitable to produce biocompatible hydrogels with desired functionalities and improved performances in biomedical applications. This work aims to synthesize RGD-functionalized hydrogels. RGD (arginylglycylaspartic acid) is a tripeptide that can mimic cell adhesion proteins and bind to cell-surface receptors, creating a hospitable microenvironment for cells within the 3D polymeric network of the hydrogels. RGD functionalization occurs through Huisgen 1,3-dipolar cycloaddition. Some PAA carboxyl groups are modified with an alkyne moiety, whereas RGD is functionalized with azido acid as the terminal residue of the peptide sequence. Finally, both products are used in a copper catalyzed click reaction to permanently link the peptide to PAA. This modified polymer is used with carbomer, agarose and polyethylene glycol (PEG) to synthesize a hydrogel matrix. The 3D structure is formed due to an esterification reaction involving carboxyl groups from PAA and carbomer and hydroxyl groups from agarose and PEG through microwave-assisted polycondensation. The efficiency of the gelation mechanism ensures a high degree of RGD functionalization. In addition, the procedure to load therapeutic compounds or biological tools within this functionalized network is very simple and reproducible.

We present a protocol for the synthesis of RGD-functionalized hydrogels as devices for cell and drug delivery. The procedure involves copper catalyzed alkyne-azide cycloaddition (CuAAC) between alkyne-modified polyacrylic acid (PAA) and a RGD-azide derivative. The hydrogels are formed using microwave-assisted polycondensation and their physicochemical properties are investigated.

La Synthèse des RGD-fonctionnalisé hydrogels comme un outil pour les applications thérapeutiques

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Formation et Patterning Light-médiée hydrogels pour la culture cellulaire Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

Synthèse des hydrogels avec des propriétés antisalissure Membranes pour la purification de l&#39;eau

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

Production de protéine élastine Hydrogels pour Encapsulation et immunomarquage des cellules en 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) culture systems. However, measurement of cell function in 3D culture is often more challenging. Many biological assays require retrieval of cellular material which can be difficult in 3D cultures. One way to address this challenge is to develop new materials that enable measurement of cell function within the material. Here, a method is presented for measurement of cellular matrix metalloproteinase (MMP) activity in 3D hydrogels in a 96-well format. In this system, a poly(ethylene glycol) (PEG) hydrogel is functionalized with a fluorogenic MMP cleavable sensor. Cellular MMP activity is proportional to fluorescence intensity and can be measured with a standard microplate reader. Miniaturization of this assay to a 96-well format reduced the time required for experimental set up by 50% and reagent usage by 80% per condition as compared to the previous 24-well version of the assay. This assay is also compatible with other measurements of cellular function. For example, a metabolic activity assay is demonstrated here, which can be conducted simultaneously with MMP activity measurements within the same hydrogel. The assay is demonstrated with human melanoma cells encapsulated across a range of cell seeding densities to determine the appropriate encapsulation density for the working range of the assay. After 24 h of cell encapsulation, MMP and metabolic activity readouts were proportional to cell seeding density. While the assay is demonstrated here with one fluorogenic degradable substrate, the assay and methodology could be adapted for a wide variety of hydrogel systems and other fluorescent sensors. Such an assay provides a practical, efficient and easily accessible 3D culturing platform for a wide variety of applications.

Here, a protocol is presented for encapsulating and culturing cells in poly(ethylene glycol) (PEG) hydrogels functionalized with a fluorogenic matrix metalloproteinase (MMP)-degradable peptide. Cellular MMP and metabolic activity are measured directly from the hydrogel cultures using a standard microplate reader.

Mesure des métalloprotéinases de la matrice cellulaire Global et l’activité métabolique des Hydrogels 3D

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

A Lab-On-A-Chip Platform for Stimulating Osteocyte Mechanotransduction and Analyzing Functional Outcomes of Bone Remodeling

Bone remodeling is a tightly regulated process that is required for skeletal growth and repair as well as adapting to changes in the mechanical environment. During this process, mechanosensitive osteocytes regulate the opposing responses between the catabolic osteoclasts and anabolic osteoblasts. To better understand the highly intricate signaling pathways that regulate this process, our lab has developed a foundationary lab-on-a-chip (LOC) platform for analyzing functional outcomes (formation and resorption) of bone remodeling within a small scale system. As bone remodeling is a lengthy process that occurs on the order of weeks to months, we developed long-term cell culturing protocols within the system. Osteoblasts and osteoclasts were grown on functional activity substrates within the LOC and maintained for up to seven weeks. Afterward, chips were disassembled to allow for the quantification of bone formation and resorption. Additionally, we have designed a 3D printed mechanical loading device that pairs with the LOC platform and can be used to induce osteocyte mechanotransduction by deforming the cellular matrix. We have optimized cell culturing protocols for osteocytes, osteoblasts, and osteoclasts within the LOC platform and have addressed concerns of sterility and cytotoxicity. Here, we present the protocols for fabricating and sterilizing the LOC, seeding cells on functional substrates, inducing mechanical load, and disassembling the LOC to quantify endpoint results. We believe that these techniques lay the groundwork for developing a true organ-on-a-chip for bone remodeling.

Here, we present protocols for analyzing bone remodeling within a lab-on-a-chip platform. A 3D printed mechanical loading device can be paired with the platform to induce osteocyte mechanostransduction by deforming the cellular matrix. The platform can also be used to quantify bone remodeling functional outcomes from osteoclasts and osteoblasts (resorption/formation).

Une plate-forme Lab-On-A-Chip pour stimuler la mécanotransduction ostéocyte et analyser les résultats fonctionnels du remodelage des os

Bone remodeling is a tightly regulated process that is required for skeletal growth and repair as well as adapting to changes in the mechanical ...

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (&#62;100 &#956;m) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel&#8217;s 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user&#8217;s needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

Souche contrôlée d’hydrogels 3D sous imagerie par microscopie dynamique

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...