Ce travail a été réalisé en utilisant les installations du Département de génie biomédical à l&#39;Université internationale de Floride.

1. Configuration Électrofilage Collector <ol><li> Couper la feuille d&#39;aluminium dans une pièce rectangulaire. </li><li> Pliez le morceau rectangulaire dans une bande rectangulaire, et l&#39;attacher perpendiculaire à une plaque de métal plat avec du ruban adhésif (figure 1). Remarque: La taille de la natte de fibre dépend de la longueur et de la largeur de la bande. Ainsi, les dimensions de la bande peut être ajustée selon les besoins. </li></ol> 2. Préparation de la solution polymérique <ol><li> Mélanger le glycérol et l&#39;acide dodécanedioïque (DDA) dans un rapport molaire 1:1 dans un bécher à 120 ° C pendant 100 heures pour obtenir un polymère de DPI. </li><li> Dissoudre du poly (oxyde d&#39;éthylène) (PEO) et de la gélatine dans 65% d&#39;éthanol avec un rapport en poids de 1.5:3:95.5 dans un tube de 15 ml, serrer le bouchon et chauffer le mélange dans une étuve à 60 ° C pendant 1 h sous agitation jusqu&#39;à ce qu&#39;il devienne une solution homogène (solution de fond). </li><li> Pour électrofilature, mélanger le polymère de DPI et la solution de base à 04h06 ratio de poids. Remarque: concentration DPI a un big effet sur le diamètre des fibres. 30% -50% pour cent DPI est acceptable pour produire des fibres de plus de 5 cm de diamètre de la fibre a augmenté. </li><li> Ajouter 0,1% de la riboflavine à la solution de polymère et bien mélanger. </li></ol> 3. Électrofilage <ol><li> Nourrir la solution de polymère dans une seringue de 5 ml en standard avec un 18 G émoussé aiguille en acier inoxydable. </li><li> Insérer la seringue dans une pompe à seringue. </li><li> Fixer le fil de mise à la terre d&#39;une source de puissance à haute tension à la plaque métallique et le conducteur chargé positivement à l&#39;aiguille. </li><li> Ajuster la distance entre l&#39;aiguille et la bande de feuille d&#39;aluminium à 15 cm. </li><li> Placer la pompe à seringue dans un angle d&#39;environ 15 ° avec l&#39;horizontale pour empêcher l&#39;agrégation des fibres à l&#39;avant de la bande. </li><li> Mettre en marche la pompe à seringue et d&#39;ajuster le débit de la pompe d&#39;écoulement de 0,6 ml / h. </li><li> Mettre en marche la source d&#39;alimentation à haute tension et régler la tension de fonctionnement à 14,6 kV. </li></ol> 4.Traitement de la fibre <ol><li> Après la collecte est terminée, exposer le mat de fibres à une lumière UV pendant 60 min pour la réticulation. </li><li> Transférer la nappe fibreuse de la bande de papier d&#39;aluminium pour une boîte de Pétri de 100 mm, et de l&#39;exposer à une lumière UV pendant 20 minutes pour la stérilisation. </li><li> Dans une enceinte de biosécurité, couper la natte en fibres en morceaux ronds de la même taille avec une lame chirurgicale et placer les morceaux dans une plaque de 24 puits. </li><li> Pour la cellule de traitement pré-semis, plonger échantillons de fibres dans 1 ml de tampon phosphate salin (PBS) et incuber à 37 ° C pendant la nuit. </li><li> Le lendemain, aspirer soigneusement PBS, ajouter 1 ml de milieu de différenciation (DMEM/F12, N2, et FGF2) (voir la recette dans le tableau Matériaux) à chaque puits et incuber à 37 ° C pendant 3 heures. </li><li> milieu de différenciation de Aspirer soigneusement, ajouter 0,2 ml de matrigel pour chaque échantillon de fibre et incuber à 37 ° C pendant 30 min. Remarque: La laminine peut également être utilisé pour le revêtement de la fibre. Ajouter 0,2 ml de 20 pg / ml de lamininepour la fibre et incuber à température ambiante pendant 3 heures. </li><li> Retirez délicatement l&#39;excès matrigel de chaque puits. Rincer avec 2 ml de milieu de différenciation fois. Note: Les échantillons de fibres sont prêtes pour la culture cellulaire. </li></ol> 5. Cellule de semis sur fibres <ol><li> Ajouter 1 ml d&#39;Accutase à la boîte de culture cellulaire MES et incuber pendant 10 min à 37 ° C. </li><li> Après 10 minutes, les cellules MES sont détachées de la boîte de culture. Ajouter 4 ml de milieu de différenciation de la plaque. Recueillir les cellules MES flottant dans un tube de 15 ml et les cellules de pipette et pour briser colonies (environ 15x). </li><li> Centrifuger à 400 g pendant 5 min et les cellules re-suspension dans 4 ml de milieu de différenciation. </li><li> Compter les cellules en utilisant un hématimètre. Transférer 200 pl de la suspension cellulaire dans un tube de 15 ml et diluer 10 fois avec du milieu de différenciation. Transférer 15 ul de la suspension cellulaire diluée à une chambre sur la hémocytomètre avec une lamelle couvre-objet en place. Compterles cellules de la place centrale de 1 mm et les quatre cases de coins de la hémocytomètre sous le microscope. Note: cellule par ml = le nombre moyen par mètre carré x le facteur de dilution x 10 4 </li><li> Placez environ 5 x 10 4 cellules MES sur chaque puits. Déposer lentement la suspension cellulaire sur le milieu des échantillons de fibres, au lieu de glisser sur le côté du puits, pour empêcher la solution de s&#39;écouler hors du tapis de fibres. </li><li> Ajouter 1 ml de milieu de différenciation à chaque puits, et maintenir la plaque dans un incubateur à 37 ° C et 5% de CO 2, afin de permettre la fixation des cellules, la croissance et la différenciation. </li><li> Aspirer le vieux milieu de chaque puits et les remplacer par 1 ml de milieu de différenciation frais tous les deux jours. </li></ol> 6. Viabilité cellulaire <ol><li> Aspirer l&#39;ancien milieu de chaque puits. </li><li> Mélanger 1/10 ème volume de résazurine réactif de fluorescence avec du milieu de culture et ajouter 1 ml dans chaque puits. </li><li> Incubmangé à 37 ° C et 5% de CO 2 pendant 4 heures, à l&#39;abri de la lumière directe. </li><li> Après 4 heures, transférer 100 pl 3x du réactif de chaque puits à une plaque de 96 puits, puis les échantillons sont prêts à être mesurée sur un spectrophotomètre de fluorescence à l&#39;aide des paramètres de filtre 560EX nm/590EM. </li></ol> 7. PCR en temps réel <ol><li> Après 14 jours de culture, ajouter un tampon de lyse pour les échantillons et d&#39;isoler l&#39;ARN total à partir des cellules sur des échantillons de fibres. </li><li> Utilisez 4 ul d&#39;ARN total pour synthétiser l&#39;ADNc dans 20 échelles de réaction ul par transcription inverse. </li><li> Préparer le mélange PCR de réaction dans chaque tube optique: 10 pi Master Mix (2x), 0,2 pi colorant, 8 pi d&#39;eau de nucléase libre, 1 ul d&#39;ADNc, et 1 pi amorce (amorces utilisées dans cette étude sont présentés au tableau 1). </li><li> Mettre en place le programme de PCR: a. 95 ° C 02:20 min, 1 cycle b. 95 ° C 3 sec → 60 ° C 1 min, 40 cycles c. 95 ° C 15 sec, 1 cycle d. 60 ° C 1min, 1 cycle e. 95 ° C 15 sec, 1 cycle. Choisissez comparative méthode C T pour déterminer les niveaux d&#39;expression des gènes relative. </li><li> Après PCR est terminée, analyser les résultats PCR en temps réel avec le logiciel StepOne et exporter les résultats au format Excel. </li></ol>

<ol>
	<li>Migneco, F., Huang, Y. -. C., Birla, R. K., Hollister, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly+(glycerol-dodecanoate),+a+biodegradable+polyester+for+medical+devices+and+tissue+engineering+scaffolds.">Poly (glycerol-dodecanoate), a biodegradable polyester for medical devices and tissue engineering scaffolds.</a> Biomaterials. 30, 6479-6484 (2009).</li><li>Reneker, D. H., Yarin, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning+jets+and+polymer+nanofibers.">Electrospinning jets and polymer nanofibers.</a> Polymer. 49, 2387-2425 (2008).</li><li>Wang, Y., Ameer, G. A., Sheppard, B. J., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tough+biodegradable+elastomer.">A tough biodegradable elastomer.</a> Nature biotechnology. 20, 602-606 (2002).</li><li>Panunzi, S., De Gaetano, A., Mingrone, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approximate+linear+confidence+and+curvature+of+a+kinetic+model+of+dodecanedioic+acid+in+humans.">Approximate linear confidence and curvature of a kinetic model of dodecanedioic acid in humans.</a> American Journal of Physiology-Endocrinology And Metabolism. 289, (2005).</li><li>Park, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apparatus+for+preparing+electrospun+nanofibers:+designing+an+electrospinning+process+for+nanofiber+fabrication.">Apparatus for preparing electrospun nanofibers: designing an electrospinning process for nanofiber fabrication.</a> Polymer Internationa l. 56, 1361-1366 (2007).</li><li>Barnes, C. P., Sell, S. A., Boland, E. D., Simpson, D. G., Bowlin, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanofiber+technology:+designing+the+next+generation+of+tissue+engineering+scaffolds.">Nanofiber technology: designing the next generation of tissue engineering scaffolds.</a> Advanced drug delivery reviews. 59, 1413-1433 (2007).</li><li>Li, W. -. J., Mauck, R. L., Tuan, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+nanofibrous+scaffolds:+production,+characterization,+and+applications+for+tissue+engineering+and+drug+delivery.">Electrospun nanofibrous scaffolds: production, characterization, and applications for tissue engineering and drug delivery.</a> Journal of Biomedical Nanotechnology. 1, 259-275 (2005).</li><li>Pham, Q. P., Sharma, U., Mikos, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning+of+polymeric+nanofibers+for+tissue+engineering+applications:+a+review.">Electrospinning of polymeric nanofibers for tissue engineering applications: a review.</a> Tissue engineering. 12, 1197-1211 (2006).</li><li>Lim, S. H., Mao, H. -. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+scaffolds+for+stem+cell+engineering.">Electrospun scaffolds for stem cell engineering.</a> Advanced drug delivery reviews. 61, 1084-1096 (2009).</li><li>Lowery, J. L., Datta, N., Rutledge, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+fiber+diameter,+pore+size+and+seeding+method+on+growth+of+human+dermal+fibroblasts+in+electrospun+poly+(epsilon-caprolactone)+fibrous+mats.">Effect of fiber diameter, pore size and seeding method on growth of human dermal fibroblasts in electrospun poly (epsilon-caprolactone) fibrous mats.</a> Biomaterials. 31, 491-504 (2010).</li><li>Tillman, B. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+in+vivo+stability+of+electrospun+polycaprolactone-collagen+scaffolds+in+vascular+reconstruction.">The in vivo stability of electrospun polycaprolactone-collagen scaffolds in vascular reconstruction.</a> Biomaterials. 30, 583-588 (2009).</li><li>Ju, Y. M., Choi, J. S., Atala, A., Yoo, J. J., Lee, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bilayered+scaffold+for+engineering+cellularized+blood+vessels.">Bilayered scaffold for engineering cellularized blood vessels.</a> Biomaterials. 31, 4313-4321 (2010).</li><li>McCullen, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+collagen+polymerization+of+layered+cell-seeded+electrospun+scaffolds+for+bone+tissue+engineering+applications.">In situ collagen polymerization of layered cell-seeded electrospun scaffolds for bone tissue engineering applications.</a> Tissue Engineering Part C: Methods. 16, 1095-1105 (2010).</li></ol>

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Les limitations de collecteurs simples ou les complexités de collecteurs qui sont actuellement utilisés pour augmenter la restriction électrofilage d&#39;obtention de la longueur désirée et la taille de matelas de fibres pour certaines applications tournant. En outre, le transfert des fibres à partir du collecteur de masse à la boîte de culture ou d&#39;autres substrats est un défi 5. Dans ce rapport, un collecteur nouvellement conçus, fabriqués en attachant une bande de papier d&#39;aluminium pour...

Électrofilage est l&#39;un des procédés de traitement efficaces pour produire des échafaudages de fibres de taille de micropore de nanomètres. Le principe de base de électrofilature implique un cône de Taylor de la solution qui se tient à l&#39;orifice d&#39;une aiguille par application d&#39;une haute tension entre la pointe de l&#39;aiguille et un collecteur de terre. Lorsque la répulsion électrostatique dans la solution dépasse la tension de surface, un jet de fluide chargé est éjecté hors de la pointe de l&#39;aiguille, se déplace à travers l&#39;air par évaporation de solvant, et est finalement déposé sur le collecteur à la masse. La pompe de seringue fournit un flux continu de solution sortant de la filière et par conséquent des copies multiples des fibres électrofilées peut être fabriqué à l&#39;intérieur d&#39;une courte période de temps. Au cours de sortie de la filière d&#39;arriver au niveau du collecteur, le jet chargé va subir des étirements et fouetter selon un certain nombre de paramètres qui comprennent la viscosité et la tension superficielle de la solution de polymère, les électrostatiques!force de c dans la solution, et l&#39;interaction du champ électrique externe, etc 2. Dans le procédé d&#39;électrofilage, un collecteur servant de substrat conducteur, où les fibres à micro-nanomètres pourraient être déposées. Dans cette étude, un nouveau type de collecteur de fibres a été conçue pour obtenir des mats de fibres avec la taille souhaitée (longueur x largeur). Traditionnellement, la feuille d&#39;aluminium est utilisée en tant que collecteur, mais il est difficile de transférer les fibres à partir de la surface plane à un autre substrat. La difficulté de la récolte d&#39;une natte de fibres intactes à partir d&#39;un collecteur traditionnel était principalement dû au fait que les fibres électrofilées fixent fermement à la surface du collecteur. Par conséquent, nous avons modifié le collecteur par pliage d&#39;un morceau de feuille d&#39;aluminium en une bande rectangulaire et sa fixation perpendiculairement à une plaque de métal plate. Les fibres électrofilées sont étirées à travers la zone entre l&#39;extrémité de la bande et la plaque métallique, qui peut être facilement transféré à un autre Substrate. L&#39;intérêt pour les polymères élastomères thermiquement réticulés est en pleine expansion en raison du travail de pionnier du groupe Robert Langer, qui a introduit le poly (glycérol sebacate) (PGS), un polyester qui est analogue au caoutchouc vulcanisé en 2002 3. Similaires à PGS, nous avons développé avec succès poly (glycérol-dodécanoate) (DPI) par condensation thermique de glycérol et l&#39;acide dodécanedioïque et démontré sa propriété unique à mémoire de forme 1. Contrairement rigide matériaux synthétiques poly (hydroxy butyrate) ou poly (L-lactide) (le modules de Young de 250 MPa et 660 MPa respectivement), le DPI présente une propriété élastomère comme du caoutchouc, avec un module de Young de 1,08 MPa lorsque la température est supérieure à 37 ° C, qui est un match serré au nerf périphérique in-situ (0,45 MPa). En outre, le DPI est biodégradable et le temps de dégradation peut être affiné en faisant varier le rapport de glycérol et l&#39;acide dodécanedioïque. Dodécanedioïque est un sous-douze carboneposition avec deux groupes carboxyliques terminaux, HOOC (CH 2) 10 COOH. Acides dicarboxyliques même numérotées comme l&#39;acide sébacique et l&#39;acide dodécanedioïque peuvent être métabolisés en acétyl-CoA et entrez le Krebs (TCA) / (acide citrique) cycle. Le produit métabolique des acides dicarboxyliques, le succinyl-CoA, est un précurseur intermédiaire gluconeogenetic et de cycle TCA 4. Ainsi, certaines études ont suggéré qu&#39;ils pourraient être utilisés en tant que substrat de carburant alternatif pour la nutrition entérale et parentérale, en particulier dans les états pathologiques. En outre, le DPI présente une mémoire de forme unique, car sa température de transition vitreuse est de 31 ° C, donc il montre des propriétés mécaniques distinctes, à température ambiante et à la température corporelle. En somme, le DPI est biodégradable, biocompatible, présentant des propriétés élastiques uniques avec des propriétés mécaniques similaires aux tissus nerveux; Par conséquent, il s&#39;agit d&#39;un matériau approprié pour des applications nerf d&#39;ingénierie tissulaire. Dans ce protocole, la électrofiléfibres longues couvrant une vaste zone de dépôt ont été fabriqués par le collecteur nouvellement conçu de DPI. Les échafaudages de fibres peuvent soutenir la croissance des cellules souches pluripotentes de souris et de la différenciation.

<table border="0" cellpadding="0" cellspacing="0" width="729">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Glycerol</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">G7757</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Dodecanedioic acid</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">D1009</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Gelatin</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">D1890</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Poly (ehtylene oxide) (PEO)</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">182028</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Riboflavin</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">132350250</td>
			<td style="width:167px;">0.10%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Mouse embryonic stem cells</td>
			<td style="width:175px;">GlobalStem</td>
			<td style="width:119px;">GSC-5002</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Matrigel</td>
			<td style="width:175px;">Becton Dickinson</td>
			<td style="width:119px;">356234</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">DMEM/F12</td>
			<td style="width:175px;">Thermo Scientific</td>
			<td style="width:119px;">SH30272.02</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">N2 supplement&#160;</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:119px;">17502048</td>
			<td style="width:167px;">1%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">FGF2</td>
			<td style="width:175px;">Stemgent</td>
			<td style="width:119px;">03-0002</td>
			<td style="width:167px;">10ng/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Accutase</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:119px;">A11105-01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Phosphate buffered saline (PBS)</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:119px;">10010-031&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Resazurin fluorescence dye&#160;</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">62758-13-8&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">SV Total RNA Isolation System</td>
			<td style="width:175px;">Promega</td>
			<td style="width:119px;">Z3100</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">GoScript Reverse Transcription System</td>
			<td style="width:175px;">Promega</td>
			<td style="width:119px;">A5000</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">GoTaq&#129; qPCR Master Mix</td>
			<td style="width:175px;">Promega</td>
			<td style="width:119px;">A6001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Syringe pump&#160;</td>
			<td style="width:175px;">Fisher scientific</td>
			<td style="width:119px;">14-831-200</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:268px;">High voltage power source&#160;</td>
			<td style="width:175px;">Spellman High Voltage Electronics Corporation</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">SL30</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">UV light</td>
			<td style="width:175px;">Philips</td>
			<td style="width:119px;">308643</td>
			<td style="width:167px;">15W/G15T8</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Synergy HT Multi-Mode Microplate Reader</td>
			<td style="width:175px;">BioTek</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Perkin Elmer GeneAmp PCR System 9600</td>
			<td style="width:175px;">Perkin Elmer</td>
			<td style="width:119px;">8488</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">StepOne Real-time PCR System</td>
			<td style="width:175px;">Applied Biosystems</td>
			<td style="width:119px;">4376357</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

electrospun fibrous scaffolds of poly(glycerol-dodecanedioate) for engineering neural tissues from mouse embryonic stem cells

Pour les applications de génie tissulaire, la préparation d&#39;échafaudages biodégradables et biocompatibles est la tâche la plus souhaitable, mais difficile. Parmi les différents procédés de fabrication, électrofilage est le plus intéressant en raison de sa simplicité et sa polyvalence. En outre, les nanofibres électrofilées imitent la taille de la matrice extracellulaire naturelle assurant un soutien supplémentaire pour la survie et la croissance cellulaire. Cette étude a montré la viabilité de la fabrication de fibres longues couvrant une plus grande surface de dépôt pour un polymère biodégradable et biocompatible roman nommé poly (glycérol-dodécanoate) (DPI) 1 en utilisant un collecteur nouvellement conçu pour électrofilature. DPI présente des propriétés élastiques uniques avec des propriétés mécaniques similaires aux tissus nerveux, il est donc approprié pour des applications de génie tissulaire de neurones. La synthèse et la fabrication mis en place pour la fabrication de matériaux d&#39;échafaudage fibreux était simple, hautement reproductible et peu coûteux. Dans biocompatibilitéles essais, les cellules dérivées de cellules souches embryonnaires de souris pourraient adhérer à et se développer sur les fibres électrofilées de DPI. En résumé, ce protocole fournit un procédé de fabrication polyvalent de fabrication de fibres DPI électrofilé pour soutenir la croissance des cellules de la lignée de cellules souches embryonnaires de souris dérivées de neurones.

Les principaux composants de l&#39;électrofilage sont présentés dans la figure 1. Un grand matelas de fibres de taille a été généralement obtenue à travers la bande de feuille d&#39;aluminium perpendiculairement ci-joint et d&#39;une plaque métallique plane. Figure 2 montre la conception de collecteur et le mat de fibres d&#39;électrofilage. La largeur et la longueur peut être ajustée pour différentes applications. La longueur de la fibre à base de polymère de DPI et mél...

Watch this Scientific Journal Video about Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells at JoVE.com

Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells

Synthèse et la fabrication de fibres longues électrofilé couvrant une zone de plus grand dépôt par un collecteur nouvellement conçu à partir d&#39;un polymère biodégradable roman nommé poly (glycérol-dodécanoate) (PGD) a été signalé. Les fibres sont capables de soutenir la croissance de cellules dérivées de cellules souches pluripotentes de souris.

Électrofilé Fort échafaudages de poly (glycérol-dodécanedioate) pour l&#39;ingénierie des tissus neuronaux De Souris cellules souches embryonnaires

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among ...

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Research

JoVE Journal

Bioengineering

Electrospun Fibrous Scaffolds of Poly(glycerol-dodecanedioate) for Engineering Neural Tissues From Mouse Embryonic Stem Cells

10.8K vues.  Florida International University.  Synthèse et la fabrication de fibres longues électrofilé couvrant une zone de plus grand dépôt par un collecteur nouvellement conçu à partir d'un polymère biodégradable roman nommé poly (glycérol-dodécanoate) (PGD) a été signalé. Les fibres sont capables de soutenir la croissance de cellules dérivées de cellules souches pluripotentes de souris. 

Vidéo: Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

PGS élastomère échafaudages en génie tissulaire artérielle

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

Recherche

Bio-ingénierie

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

Préparation de la 3D pour les applications de fibrine Échafaudages CULTURE DE CELLULES SOUCHES

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

Traitement post-production des fibres électrofilées de génie tissulaire

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

Les tissus d&#39;ingénierie muscle squelettique de myoblastes murins cellules progénitrices et application d&#39;une stimulation électrique

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

Le développement, l&#39;expansion et<em&gt; In vivo</em&gt; Suivi des cellules NK humaines à partir de cellules souches embryonnaires humaines (CSEh) et et induit des cellules souches pluripotentes (CISP)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating&#160;poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.

This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

Électrofilage facteur de croissance libération de microsphères en fibre échafaudages

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth.  This system incorporates mechanical, topographical, ...

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&#949;-caprolactone) Electrospun Yarns

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell number. While researchers commonly reference their seeding density this does not necessarily indicate the actual number of cells that have adhered to the material in question. This is particularly the case for materials, or scaffolds, that do not cover the base of standard cell culture well plates. This study investigates the initial attachment of human mesenchymal stem cells seeded at a known number onto electrospun poly(&#949;-caprolactone) yarn after 4 hr in culture. Electrospun yarns were held within several different set-ups, including bioreactor vessels rotating at 9 rpm, cell culture inserts positioned in low binding well plates and polytetrafluoroethylene (PTFE) troughs placed within petri dishes. The latter two were subjected to either static conditions or positioned on a shaker plate (30 rpm). After 4 hr incubation at 37&#160;oC, 5% CO2, the location of seeded cells was determined by cell DNA assay. Scaffolds were removed from their containers and placed in lysis buffer. The media fraction was similarly removed and centrifuged &#8211; the supernatant discarded and pellet broken up with lysis buffer. Lysis buffer was added to each receptacle, or well, and scraped to free any cells that may be present. The cell DNA assay determined the percentage of cells present within the scaffold, media and well fractions. Cell attachment was low for all experimental set-ups, with greatest attachment (30%) for yarns held within cell culture inserts and subjected to shaking motion. This study raises awareness to the actual number of cells attaching to scaffolds irrespective of the stated cell seeding density.

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&amp;#949;-caprolactone) Electrospun Yarns

This article describes a range of set-ups for seeding human mesenchymal stem cells onto materials, in this case electrospun yarns, that do not cover the base of standard culture well plates in order to maximize and quantify the number of cells that initially attach compared to the known seeding density.

Optimisation Fixation des mésenchymateuses humaines cellules souches sur Poly (ε-caprolactone) électrofilées Yarns

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.

Électrofilées nanofibres échafaudages avec des gradations dans l&#39;organisation fibre

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis.

This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

Génération de cellules de souris Airway épithéliales ESC dérivés Utilisation décellularisé Lung échafauds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

Deux méthodes de DÉCELLULARISATION de tissus végétaux pour utilisations en génie tissulaire

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...