Le financement a été fourni par le National Institutes of Health (EB 001 013), en sciences naturelles et en génie du Canada et les Instituts canadiens de recherche en santé. Nous remercions le Dr AP McGuigan pour son expertise et son aide dans l&#39;élaboration de modules.

1) Préparation des tubes <ol><li> Couper 2 à 3 m de longueur de tuyaux en polyéthylène (0,76 mm ID x 1,22 mm de diamètre extérieur). </li><li> Enfilez une aiguille 20 G1 à une extrémité du tube. </li><li> Enrouler le tube et le placer dans une auto-joint convertisseurs poche (7 1 / 2 &quot;x 13&quot;). Les deux extrémités de la tubulure doit être placé à la fin descellé du sac et scellés en place avec du ruban stérilisation au gaz. </li><li> Sceller le sac et le gaz stériliser. </li></ol> 2) Neutralisation de collagène Note: 1 ml de collagène remplit environ 2 m de tuyaux en polyéthylène. <ol><li> Placer la quantité nécessaire de collagène nécessaire à la longueur désirée du tube dans un tube de 15 ml conique et de garder sur la glace. </li><li> Ajouter 128 ul de 10x α-MEM par ml de collagène dans le tube et mélanger en pipetant plusieurs reprises la solution. Note: (1) La solution doit virer au jaune comme le α-MEM est mélangé dans le collagène acidifiés et (2) essayer de minimiser les bulles lors du mélange. </li><li> Neutraliser le collagène en ajoutant 0,8 M bicarbonate de sodium jusqu&#39;à ce que le collagène a un pH de 7,4. Pour une estimation générale du bicarbonate de sodium ajouter du pH jusqu&#39;à ce que la solution de collagène jaune vire au rose pâle. Nota: Il faut généralement de 30 à 60 pi pour chaque ml de collagène. </li><li> Gardez collagène neutralisé sur la glace jusqu&#39;au moment de l&#39;utiliser comme le collagène neutralisée sera un gel s&#39;il n&#39;est pas conservé à 4 ° C. </li></ol> 3) Intégrer des cellules (en option) Remarque: En fonction du type cellulaire, les cellules peuvent être intégrées à une concentration comprise entre 1 million de cellules par mL à 20 millions de cellules par ml. Utilisez le support requis pour les cellules que vous souhaitez intégrer. <ol><li> Retirez le support à partir des cellules et laver avec 5 ml de PBS. </li><li> Retirer du PBS et ajouter 3 ml de trypsine-EDTA. </li><li> Incuber les cellules dans de la trypsine-EDTA pendant 5 min à 37 ° C. </li><li> Resuspendre les cellules dans les médias ml 7. </li><li> Compter les cellules. </li><li> Transférer le nombre requis de cellules pour l&#39;intégration à un tube de 15 ml conique. </li><li> Pellet les cellules à 300 xg pendant 5 min. </li><li> Aspirer les médias hors du culot cellulaire. </li><li> Resuspendre les cellules dans le collagène neutralisé et de garder sur la glace. </li></ol> 4) Gélification du collagène <ol><li> Dans une enceinte de sécurité biologique couper l&#39;extrémité scellée du sac contenant les tuyaux en polyéthylène. </li><li> Attacher une seringue de 3 ml à l&#39;aiguille 20G dans l&#39;une des extrémités du tube en utilisant une pince à épiler stérilisée. Gardez le tuyau dans le sac. </li><li> Insérez l&#39;autre extrémité du tuyau dans le collagène neutralisé jusqu&#39;à la pointe est au fond du tube conique en utilisant une pince à épiler stérilisée. Garder le tube conique sur la glace. </li><li> Tirez le collagène dans les tuyaux en polyéthylène en rétractant le piston de la seringue lentement. Remarque: Si le collagène est tiré dans le tube à bulles rapidement puis se forme dans le collagène. </li><li> Après avoir tiré le collagène par le tube, retirez l&#39;aiguille 20G sur de tubes et de mettre les deux extrémités dans le sac. Tape le sac fermé. </li><li> Incuber le sac à 37 ° C pendant 60 min. Si le collagène contient des cellules, puis retournez le sac sur toutes les 15 min. afin que les cellules ne sont pas réglées sur un côté du module. </li></ol> 5) Découpe de tubes contenant du collagène gélifié <ol><li> Têtes autoclave et la lame pour le dispositif de coupe et le bac à tenir tube. </li><li> Assemblez le dispositif de coupe. </li><li> Placer dans un tube de plateau stérile en face du dispositif de coupe et de charger le tube dans le dispositif de coupe. </li><li> Mettre en place un tube conique de 50 ml contenant 25 ml de médias à la sortie du dispositif de coupe pour recueillir les modules de coupe. Remarque: Utilisez les médias contenant FBS même pour le collagène seule modules comme les modules ne se séparent pas de la tubulure sans FBS dans les médias. Si les modules contiennent des cellules utiliser le support requis pour les cellules embarquées. S&#39;il n&#39;ya pas de cellules intégrées dans les modules utilisent les médias pour les cellules endothéliales. </li><li> Réglez le dispositif de coupe pour couper 2 mm de longueur de tube et couper le tuyau. </li><li> Incuber coupé modules à 37 ° C pendant 60 min dans le tube conique de 50 ml. </li></ol> 6) Suppression des modules du collagène à partir de tubes <ol><li> Vortex du tube conique de 50 ml contenant des modules dans le tube pendant 10 sec. Note: Soyez doux lorsque la séparation avec les modules intégrés à haute densité cellulaire pour éviter les bris. </li><li> Laissez les modules se déposent au fond du tube. Cela prend environ 5 min. </li><li> En utilisant une large bouche pipette de 10 ml sérologique transférer les modules installés dans un tube de 15 ml conique. </li><li> Répétez les étapes 6.1 à 6.3 fois. </li><li> Passez à l&#39;étape 7 à des modules de revêtement avec des cellules endothéliales ou des modules de transfert à une plaque de culture tissulaire non traitée. </li></ol> 7) Revêtement des modules avec des cellules endothéliales <ol><li> Settle modules dans le fond d&#39;un tube 15 ml conique avec 5 mL de médias pour ee intégré cellules. </li><li> Retirez le support à partir des cellules endothéliales et les laver avec 5 ml de PBS. </li><li> Retirer du PBS et ajouter 3 ml de trypsine-EDTA. </li><li> Incuber les cellules dans de la trypsine-EDTA pendant 5 min à 37 ° C. </li><li> Resuspendre les cellules dans les médias ml 7. </li><li> Compter les cellules. Besoin 5x10 6 cellules endothéliales par ml de modules emballé au fond du tube conique. </li><li> Pellet les cellules à 300 xg pendant 5 min et remettre en suspension dans 5 ml de cellules endothéliales des médias. </li><li> Ajouter les 5 ml du support contenant les cellules endothéliales des modules. Note: Ceci donne un ratio de 50:50 de la deux types de média que nous avons trouvé à travailler pour la survie et la fonction des deux types de cellules. Test de l&#39;utilisation d&#39;un moyen de co-culture formulés pour assurer le bon entretien des phénotypes des cellules deux types avant de l&#39;utiliser. </li><li> Incuber les modules avec les cellules endothéliales à la fois statique et dynamique. </li><li> Pour statiques modules mélange d&#39;incubation avec des cellules endothéliales par inversion et placer le tube en position verticale à 37 ° C pendant 15 min. Répéter. </li><li> Dynamiquement semences les cellules endothéliales en secouant délicatement le tube à 37 ° C pendant 60 min. </li><li> Répétez incubation statique à deux reprises. </li><li> Placez les modules dans une plaque de culture de tissus non traités et incuber une nuit à 37 ° C. </li><li> Le lieu le lendemain des modules dans une plaque de culture non de nouveaux tissus traités par du milieu frais (mélange 50:50 si vous utilisez le système de co-culture) pour enlever les seules cellules endothéliales. </li><li> Incuber les modules qui sont revêtus de cellules endothéliales jusqu&#39;à ce qu&#39;ils couvrent 100% de la surface des modules. Cela prend habituellement environ 7 jours. </li></ol> 8) Les résultats représentatifs: Les modules cylindriques se penchera la première fois qu&#39;ils sont retirés du tube. Si les modules contiennent des cellules intégrées et / ou sont revêtues de cellules endothéliales, ils peuvent contracter jusqu&#39;à 50% en volume et de développer une forme ovale (figure 1). Les modules seront également devenue plus dense et plus opaque lorsqu&#39;il est vu par microscopie optique (figure 1). Aussi, quand les cellules endothéliales sont confluentes sur la surface des modules il ya une formation de jonctions serrées qui peuvent être vus par immunofluorescence de la VE-cadhérine (Figure 2). L&#39;analyse de transfert de masse a montré que les modules sont capables de supporter de fortes densités cellulaires (8x10 7 cellules / cm 3) sans développer un noyau nécrotique due à transporter l&#39;oxygène insuffisante, ce qui est souvent problématique dans les grandes tissus (par exemple des modules de grand diamètre, D = 1,4 mm) (figure 3). <img src="/files/ftp_upload/2177/2177fig1.jpg" alt="Figure 1" /> Figure 1: Module de fabrication et de contraction. Module de contraction s&#39;est produite pendant les trois jours suivants HUVEC-C (ligne de cellules endothéliales) ensemencement des résultats de diamètre nettement plus petit module de et de la longueur (p &lt;0,001) (A). Embarqué cellules HepG2 ont été uniformément réparties dans les modules au moment de la fabrication et conservé une viabilité élevée (B). [Live cellules vertes; les cellules mortes rouge] [adapté de Corstorphine 2010] <img src="/files/ftp_upload/2177/2177fig2.jpg" alt="Figure 2" /> Figure 2: VE-cadhérine en immunofluorescence des jonctions cellulaires serrées CE 10 jours après RAEC où enduit sur ​​la surface d&#39;un module. Barre d&#39;échelle = 50 microns. <img src="/files/ftp_upload/2177/2177fig3.jpg" alt="Figure 3" /> Figure 3: coloration trichrome de Masson de modules typiques (0,76 mm de diamètre initial) et des modules de grande taille (1,4 mm de diamètre initial) démontrent l&#39;effet des restrictions de diffusion d&#39;oxygène. Sept jours après la fabrication, un grand nombre de cellules mortes avaient formé dans le noyau des modules de grande taille (panneau en bas à droite), ne laissant qu&#39;une mince bordure (~ 200μm d&#39;épaisseur) de cellules viables. Inversement, les petits modules conservé une distribution uniforme et élevé de cellules vivantes. [Modules intégrés aux cellules HepG2 et recouvert de cellules endothéliales.] [Tiré de Corstorphine 2010] <img src="/files/ftp_upload/2177/2177fig4.jpg" alt="Figure 4" /> Figure 4: (A) HMEC-1 ensemencées sur poloxamine - Modules de collagène. Après une journée de l&#39;ensemencement des modules poloxamine-collagène conservent leur forme cylindrique et il est limité propriétés de fixation cellulaire par rapport à des modules de collagène seulement. Barre d&#39;échelle = 200 pm. (B) image par microscopie lumineuse d&#39;un module de collagène contenant PLGA basée sur des microsphères biodégradables. Barre d&#39;échelle = 500 pm. <img src="/files/ftp_upload/2177/2177fig5.jpg" alt="Figure 5" /> Figure 5: Exemples d&#39;essais in vitro. Dosage de l&#39;angiogenèse (A): capillaire comme des formations sur le module ensemencées avec des cellules endothéliales peuvent être facilement détectés et quantifiés après 5 jours d&#39;incubation avec des cellules souches adipeuses. (B) des images de microscopie confocale d&#39;un dosage de vivants / morts sur les modules où le scontenant des cellules vivantes est vert et pour les cellules mortes est rouge. <img src="/files/ftp_upload/2177/2177fig6.jpg" alt="Figure 6" /> Figure 6: Les modules intégrés de collagène contenant les cellules souches mésenchymateuses et une couche de surface des cellules endothéliales. Les modules ont été exposés à un stress de cisaillement (~ 0,64 dyn / cm 2) pendant 7 jours dans une chambre microfluidique et commencent à montrer de fusion à des points de contact. Les cellules endothéliales sont colorées avec le vWF (marron) et mésenchymateuses noyaux de cellules souches apparaissent en bleu (H &amp; E). Barre d&#39;échelle = 100 microns. <img src="/files/ftp_upload/2177/2177fig7.jpg" alt="Figure 7" /> Figure 7: des centaines de modules de collagène contenant des cellules souches adipeuses (ASC) et recouvert de cellules endothéliales microvasculaires humaines (HMEC) sont implantés sous la peau de souris pour étudier ASC / HMEC interaction in vivo pour les applications de la régénération de graisse.

<ol>
	<li>Chamberlain, M. D., Gupta, R., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+vessel+tissue+engineering+driven+by+endothelialized+modules.">Chimeric vessel tissue engineering driven by endothelialized modules.</a> , (2010).</li><li>Corstorphine, L. E., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+factor+and+diffusion+limitations+in+collagen+gel+modules+containing+HepG2+cells.">Effectiveness factor and diffusion limitations in collagen gel modules containing HepG2 cells.</a> J Tissue Eng Regen Med. , (2010).</li><li>Gupta, R., Van Rooijen, N., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+of+endothelialized+modular+constructs+implanted+in+an+omental+pouch+in+nude+rats.">Fate of endothelialized modular constructs implanted in an omental pouch in nude rats.</a> Tissue Eng. Part A 15, 2875-2887 (2009).</li><li>Leung, B. M., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modular+tissue+engineering+construct+containing+smooth+muscle+cells+and+endothelial+cells.">A modular tissue engineering construct containing smooth muscle cells and endothelial cells.</a> Ann Biomed Eng. 35, 2039-2049 (2007).</li><li>McGuigan, A. P., Leung, B., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+cell-containing+gel+modules+to+assemble+modular+tissue-engineered+constructs+[corrected].">Fabrication of cell-containing gel modules to assemble modular tissue-engineered constructs [corrected].</a> Nat Protoc. 1, 2963-2969 (2006).</li><li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularized+organoid+engineered+by+modular+assembly+enables+blood+perfusion.">Vascularized organoid engineered by modular assembly enables blood perfusion.</a> Proc Natl Acad Sci U S A. 103, 11461-11466 (2006).</li><li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular+tissue+engineering:+fabrication+of+a+gelatin-based+construct.">Modular tissue engineering: fabrication of a gelatin-based construct.</a> J Tissue Eng Regen Med. 1, 136-145 (2007).</li><li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+fabrication+of+sub-mm-sized+modules+containing+encapsulated+cells+for+modular+tissue+engineering.">Design and fabrication of sub-mm-sized modules containing encapsulated cells for modular tissue engineering.</a> Tissue Eng. 13, 1069-1078 (2007).</li><li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+criteria+for+a+modular+tissue-engineered+construct.">Design criteria for a modular tissue-engineered construct.</a> Tissue Eng. 13, 1079-1089 (2007).</li><li>McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+thrombogenicity+of+human+umbilical+vein+endothelial+cell+seeded+collagen+modules.">The thrombogenicity of human umbilical vein endothelial cell seeded collagen modules.</a> Biomaterials. 29, 2453-2463 (2008).</li><li>Sosnik, A., Leung, B., McGuigan, A. P., Sefton, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen/poloxamine+hydrogels:+cytocompatibility+of+embedded+HepG2+cells+and+surface-attached+endothelial+cells.">Collagen/poloxamine hydrogels: cytocompatibility of embedded HepG2 cells and surface-attached endothelial cells.</a> Tissue Eng. 11, 1807-1816 (2005).</li></ol>

Nous avons fabriqué plusieurs tissus différents de micro-utilisant les modules intégrés aux différents types de cellules 1-11. Nous avons réussi à intégré les cardiomyocytes primaires, les îlots, les cellules souches adipeuses et les cellules stromales mésenchymateuses ainsi que plusieurs lignées cellulaires, y compris HepG2, NIH 3T3 et clone 9 cellules du foie. Nous avons recouvert les modules avec plusieurs types de cellules endothéliales aortiques de rat dont les cellules endothéliales, les c...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Intramedic tubing PE60</td>
<td>&nbsp;</td>
<td>Becton Dickson</td>
<td>427416</td>
<td>Different diameter of tubing can be used to change the diameter of the modules</td>
</tr>
<tr>
<td>Phosphate-buffered saline (PBS)</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>20012-027</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25200-072</td>
<td>&nbsp;</td>
<tr>
<td>Purcol acidificed collagen, 3 mg/mL</td>
<td>&nbsp;</td>
<td>Cedarlane</td>
<td>5005-B</td>
<td>&nbsp;</td>
<tr>
<td>Sodium bicarbonate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S5761</td>
<td>&nbsp;</td>
<tr>
<td>20G needle</td>
<td>&nbsp;</td>
<td>Becton Dickson</td>
<td>305175</td>
<td>Diameter of the needle needs to be similar to the diameter of the tubing</td>
</tr>
<tr>
<td>3 mL syringe</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>309585</td>
<td>&nbsp;</td>
<tr>
<td>15 mL tube</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>352096</td>
<td>&nbsp;</td>
<tr>
<td>50 mL tube</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>352070</td>
<td>&nbsp;</td>
<tr>
<td>Convertors Self-Seal Pouch 7 1/2&#x201D; x 13&#x201D;</td>
<td>&nbsp;</td>
<td>Cardinal Health</td>
<td>92713</td>
<td>&nbsp;</td>
<tr>
<td>10 ml Wide Tip serological pipet</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>357504</td>
<td>&nbsp;</td>
<tr>
<td>10 ml serological pipet</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>357551</td>
<td>&nbsp;</td>
<tr>
<td>5 ml serological pipet</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>357543</td>
<td>&nbsp;</td>		</tbody>
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fabrication of micro-tissues using modules of collagen gel containing cells

Ce protocole décrit la fabrication d&#39;un type de micro-tissus appelés modules. L&#39;approche module génère uniforme, les tissus extensibles et vascularisé. Les modules peuvent être faites de collagène ainsi que d&#39;autres matériaux gélifiable ou réticulables. Ils sont environ 2 mm de longueur et 0,7 mm de diamètre à la fabrication, mais diminuer en taille avec des cellules ou lorsque les modules sont recouverts avec des cellules endothéliales. Les modules individuellement sont suffisamment petits pour que les cellules sont intégrées dans la limite de diffusion de l&#39;oxygène et autres nutriments, mais les modules peuvent être emballés ensemble pour former de grands tissus qui sont perfusable. Ces tissus sont de conception modulaire, car différents types de cellules peuvent être intégrés dans ou enduit sur les modules avant qu&#39;ils ne soient emballés ensemble pour former des tissus complexes. Il ya trois étapes principales pour rendre les modules: (1) la neutralisation du collagène et d&#39;incorporation des cellules en elle, (2) gélification du collagène dans le tube et couper les modules et (3) revêtement des modules avec des cellules endothéliales.

Watch this Scientific Journal Video about Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells at JoVE.com

Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

Création de micro-tissus en utilisant des gels de collagène cylindriques, appelés modules, qui contiennent des cellules embarqués et dont la surface est recouverte de cellules endothéliales.

Fabrication de micro-tissus en utilisant des modules de gel de collagène cellules contenant

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized ...

fabrication-micro-tissues-using-modules-collagen-gel-containing

Research

JoVE Journal

Bioengineering

12.6K vues.  University of Toronto. Création de micro-tissus en utilisant des gels de collagène cylindriques, appelés modules, qui contiennent des cellules embarqués et dont la surface est recouverte de cellules endothéliales.

Vidéo: Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

Fabrication d&#39;hydrogels pour les systèmes de culture Micropatterned neuronaux utilisant la photolithographie dynamique projection de masque

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

Recherche

Bio-ingénierie

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i)&#160;to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

This procedure demonstrates in vivo near IR fluorescence imaging of collagen remodeling activities in mice as well as ex vivo staining of collagens in tissue sections using caged collagen mimetic peptides that can be photo-triggered to hybridize with denatured collagen strands.

Imagerie collagène dénaturé Strands<em&gt; In vivo</em&gt; Et<em&gt; Ex vivo</em&gt; Par photo-déclenchée hybridation de Caged collagène Mimetic peptides

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Fabrication de tissus de cartilage humain en utilisant trois dimensions jet d&#39;encre Technologie d&#39;impression

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

Tissu haché en comprimé Collagen: Un Biotransplant contenant des cellules de réparation Reconstructive simple mise en scène

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to their intense optical absorbance in the near-infrared window, low cytotoxicity and potential to home into tumors. However, their delivery to tumors still remains an issue. An innovative approach consists of the exploitation of the tropism of tumor-associated macrophages that may be loaded with gold nanorods in vitro. Here, we describe the preparation and the photoacoustic inspection of cellular vehicles containing gold nanorods. PEGylated gold nanorods are modified with quaternary ammonium compounds, in order to achieve a cationic profile. On contact with murine macrophages in ordinary Petri dishes, these particles are found to undergo massive uptake into endocytic vesicles. Then these cells are embedded in biopolymeric hydrogels, which are used to verify that the stability of photoacoustic conversion of the particles is retained in their inclusion into cellular vehicles. We are confident that these results may provide new inspiration for the development of novel strategies to deliver plasmonic particles to tumors.

We show the preparation and address the feasibility of cellular vehicles containing gold nanorods for the photoacoustic imaging of cancer.

Préparation et Photoacoustique Analyse des véhicules cellulaires contenant de l&#39;or nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to ...

Forming Giant-sized Polymersomes Using Gel-assisted Rehydration

Polymer vesicles, or polymersomes, are being widely explored as synthetic analogs of lipid vesicles based on their stability, robustness, barrier properties, chemical versatility and tunable physical characteristics. Typical methods used to prepare giant-sized (&gt; 4 &#181;m) vesicles, however, are both time and labor intensive, yielding low numbers of intact polymersomes. Here, we present for the first time the use of gel-assisted rehydration for the rapid and high-yielding formation of giant (&gt;4 &#181;m) polymer vesicles (polymersomes). Using this method, polymersomes can be formed from a wide array of rehydration solutions including several different physiologically-compatible buffers and full cell culture media, making them readily useful for biomimicry studies. This technique is also capable of reliably producing polymersomes from different polymer compositions with far better yields and much less difficulty than traditional methods. Polymersome size is readily tunable by altering temperature during rehydration or adding membrane fluidizers to the polymer membrane, generating giant-sized polymersomes (&gt;100 &#181;m).

We present a protocol to rapidly form giant polymer vesicles (pGVs). Briefly, polymer solutions are dehydrated on dried agarose films adhered to coverslips. Rehydration of the polymer films results in rapid formation of pGVs. This method greatly advances the preparation of synthetic giant vesicles for direct applications in biomimetic studies.

Formant géants de taille polymersomes Utilisation Réhydratation Gel-assisté

Polymer vesicles, or polymersomes, are being widely explored as synthetic analogs of lipid vesicles based on their stability, robustness, barrier ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

Fabrication et caractérisation des fantômes de tissu optique contenant la Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

Préparation, Purification et l’utilisation de Liposomes contenant de l’acide gras

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

Absorption de nouvelles nanoparticules lipides enduit contenant Falcarindiol par des cellules souches mésenchymateuses humaines

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...

Microfluidic Production of Lysolipid-Containing Temperature-Sensitive Liposomes

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading chemotherapeutic drugs, such as doxorubicin (DOX). To achieve this, an ethanolic lipid mixture and ammonium sulfate solution are injected into a staggered herringbone micromixer (SHM) microfluidic device. The solutions are rapidly mixed by the SHM, providing a homogeneous solvent environment for liposomes self-assembly. Collected liposomes are first annealed, then dialyzed to remove residual ethanol. An ammonium sulfate pH-gradient is established through buffer exchange of the external solution by using size exclusion chromatography. DOX is then remotely loaded into the liposomes with high encapsulation efficiency (&#62; 80%). The liposomes obtained are homogenous in size with Z-average diameter of 100 nm. They are capable of temperature-triggered burst release of encapsulated DOX in the presence of mild hyperthermia (42 &#176;C). Indocyanine green (ICG) can also be co-loaded into the liposomes for near-infrared laser-triggered DOX release. The microfluidic approach ensures high-throughput, reproducible and scalable preparation of LTSLs.

The protocol presents the optimized parameters for preparing thermosensitive liposomes using the staggered herringbone micromixer microfluidics device. This also allows co-encapsulation of doxorubicin and indocyanine green into the liposomes and the photothermal-triggered release of doxorubicin for controlled/triggered drug release.

Production microfluidique de liposomes sensibles à la température contenant des lysolipides

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading ...