Tracy C. Grikscheit, Erik R. Barthel, et Frédéric G. Sala sont pris en charge par le California Institute for Regenerative Medicine (CIRM), numéros de subvention RN2-00946-1 (TCG) et TG2-01168 (ERB, FGS). Allison L. Speer est une Société de l&#39;Université chirurgiens savant Ethicon. Yashuhiro Torashima est financé par un Hôpital pour enfants de Los Angeles Saban Bourse de recherche Institut de développement de carrière.

1. Organoïde Préparation unités <ol><li> Instruments appropriés pour la dissection de la souris (ciseaux et des pinces) doivent être stérilisés par autoclave. </li><li> Euthanasier sans douleur la souris donneuse selon les protocoles locaux IACUC. Assurez-vous que l&#39;animal est mort avant de poursuivre. </li><li> Faire une incision médiane pour accéder à la cavité péritonéale. Lambeaux cutanés peuvent être présentées comme nécessaires pour améliorer l&#39;exposition. </li><li> Eviscération l&#39;intestin grêle et le diviser juste en aval du ligament de Treitz. Séparer l&#39;intestin grêle de son mésentère utilisant nette et douce dissection. Identifier la jonction iléo-colique et de diviser l&#39;intestin grêle proximal 5 mm à cela. </li><li> Avec des ciseaux, ouvrez l&#39;intestin sens de la longueur le long de la frontière antimésentérique dans une boîte de Pétri avec 10 ml 4 ° C, une solution saline tamponnée stérile de Hanks (HBSS, Invitrogen, Carlsbad CA) / 1X antibiotique antifongique-(Anti-Anti, Invitrogen) solution. Effacer les matières fécales dans l&#39;intestin ouvert avecagitation douce puis de transférer l&#39;intestin a ouvert un tube à centrifuger 15ml avec 10 ml de 4 ° C, HBSS stérile / 1X anti-anti. </li><li> Laver l&#39;intestin ouvert trois fois dans 10 ml de 4 ° C, HBSS stérile / 1X anti-anti-dans un tube à essai. Chaque lavage peut être effectué avec une agitation douce du tube de 15 ml pendant 30 sec. Après agitation, le tissu intestinal coule au fond du tube. Jeter les matériels flottants qui est des débris mésenchymateuse. Retirez soigneusement la solution de lavage avec une pipette. </li><li> Hachez l&#39;intestin lavé dans une boîte de Pétri avec 10 ml de 4 ° C, HBSS stérile / 1X anti-anti-moins de 1 mm morceaux carrés en utilisant une paire de ciseaux. Rassemblez le matériel hachée avec une pipette automatique et le placer dans un tube à essai. </li><li> Centrifuger le tube à 500 tours par minute pendant 8 min. Jeter le surnageant, qui contient de la graisse et le mésenchyme. </li><li> Digérer le hachée, matériau lavé avec 10 ml de HBSS stérile / 1X anti-anti majoré de 0,125 mg / ml dispase (Invitrogen) et 800 unités / ml collagénase de type 1 (Worthington, Lakewood NJ). Pour préparer 40 ml de la solution de digestion, peser 5 mg de dispase, 142 mg de collagénase, et ajouter HBSS stérile jusqu&#39;à un volume de 40 ml. Préparer cette solution fraîchement chacune des unités organoïdes temps sont préparés, et conserve à 4 ° C jusqu&#39;au moment de servir. Ajouter la solution de digestion directement à l&#39;étape de 1,8 à granulés. </li><li> Incuber le tube à essai contenant le hachée, la matière lavée avec la solution de digestion à 37 ° C pendant 20 min. </li><li> Récupérer le tube à essai et perturber encore davantage le tissu digéré par trituration avec une pipette de 10 ml. Répéter entre 20 à 50 fois jusqu&#39;à ce qu&#39;une apparence uniforme. </li><li> Centrifuger le tube à essai pendant 5 min à 800 rpm. Jeter le surnageant, qui contient des cellules individuelles. </li><li> Arrêter la réaction de digestion avec 10 ml de 4 ° C, stérile Eagle modifié par Dulbecco (DMEM, Invitrogen) majoré de 10% v / v inactivé par la chaleur du sérum fœtal bovin (FBS-HI, Invitrogen). Reprendre le pellet et agiter le tube. </li><li> Centrifuger le tube à essai pendant 5 min à 800 rpm. Retirez soigneusement le surnageant avec une pipette automatique jusqu&#39;à ce que les dernières gouttes. Utiliser une pipette en plastique jetable pour les dernières gouttes d&#39;éviter la remise en suspension du culot. </li></ol> 2. Chargement de l&#39;acide polyglycolique échafaudage <ol><li> Formulaire de 2 mm de long, de 5 mm de diamètre échafaudages cylindriques extérieures de l&#39;acide glycolique non-tissé (épaisseur de la tôle de 2 mm, 60 mg densité en vrac cm-3; porosité&gt; 95%, Fibres Concordia, RI Coventry) tel que décrit dans la référence. 4. </li><li> Coupez l&#39;extrémité distale de 2 mm d&#39;une pipette jetable 1000 microlitre pointe avec des ciseaux préparés avec de l&#39;éthanol à 70% dans de l&#39;eau distillée. </li><li> Placez l&#39;échafaud dans une plaque de culture de 4-bien. Chargez les unités organoïdes sur l&#39;échafaud avec la pipette microlitre 1000, d&#39;abord dans la lumière, puis sur la surface extérieure. Utiliser une pince pour assurer revêtement de la lumière. Ne pas perturber ou briser la forme cylindrique du polymère. </li&gt; </ol> 3. Implantation dans la souris hôte <ol><li> Utiliser une souris hôte syngénique sur le fond même du donateur si disponible. Sinon, utiliser un diabétique non obèse immunodéprimés / grave déficit immunitaire combiné ou NOD / SCID animal (Jackson Laboratories, Sacramento CA). </li><li> Induire une anesthésie générale à l&#39;isoflurane. Shave, la préparation et le drapé abdomen de la souris. </li><li> Faire une incision médiane de 5 mm pour pouvoir entrer dans la cavité péritonéale. Identifier et soigneusement vider le grand épiploon. Placer le polymère chargé sur l&#39;épiploon et l&#39;envelopper avec le tissu. Ne déchirez pas l&#39;épiploon. </li><li> Obtenir le polymère à l&#39;épiploon avec une suture 5-0 monocryl. Replacez doucement l&#39;épiploon avec le polymère enveloppé dans sa position anatomique. </li><li> Fermer l&#39;incision abdominale en couches en utilisant des sutures 4-0 vicryl. Exécutez la fermeture des muscles et prendre soin de ne pas blesser les viscères abdominaux dessous de l&#39;incision. Utilisez des points séparés pour la peau. </li><li> Administrer une analgésie postopératoire de 2 mg / kg de kétoprofène (Ketofen, Fort Dodge Animal Health) dans de l&#39;eau stérile comme une papule cutanée à côté de l&#39;incision. Les animaux doivent être évalués quotidiennement et si l&#39;animal montre des signes de douleur ou de détresse, une dose supplémentaire de kétoprofène peut être administrée au jour post-opératoire 2. Le troisième jour postopératoire, l&#39;animal doit être complètement récupéré sans signes de douleur ou de détresse. Si la douleur ou de la détresse continue le jour post-opératoire 3, ceci est considéré comme anormal et devrait être traitée en conformité avec les protocoles IACUC et de soins pour animaux. </li><li> Laissez la souris pour récupérer et l&#39;intestin par génie tissulaire à croître pendant quatre semaines. Offrir l&#39;accès à volonté des animaux annonce à rongeur (Laboratoire Diet 5001, PMI Nutrition, St. Louis MO) et de l&#39;eau avec Septra 200 mg / 40 mg pour 5 ml (Salut-Tech Pharmacal, Amityville NY) à 1:100 dilution. </li></ol> 4. Récolte <ol><li> Humanely euthanasier l&#39;animal hôte quatre semaines après l&#39;implantation. </li><li> Rouvrir l&#39;incision initiale et refléter la peau céphalique pour faciliter l&#39;accès à la cavité péritonéale. </li><li> Ouvrez la couche musculaire et d&#39;identifier la construction par génie tissulaire comme un globe de tissu. </li><li> Prenez les adhérences à la construction de viscères intra-abdominale à l&#39;aide dissection. </li><li> Fixer la construction dans le formol pour le montage ultérieur de paraffine, ou d&#39;utiliser le tissu frais pour les tests biochimiques tels que PCR en temps réel ou l&#39;isolement des protéines. </li></ol>

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	<li>Grikscheit, T. C., Siddique, A., Ochoa, E. R., 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=Tissue-engineered+small+intestine+improves+recovery+after+massive+small+bowel+resection.">Tissue-engineered small intestine improves recovery after massive small bowel resection.</a> Ann. Surg. 240, 748-754 (2004).</li><li>Wales, P. W., Christison-Lagay, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+bowel+syndrome:+epidemiology+and+etiology.">Short bowel syndrome: epidemiology and etiology.</a> Sem. Ped. Surg. 19, 3-9 (2010).</li><li>Evans, G. S., Flint, N., Somers, A. 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=The+development+of+a+method+for+the+preparation+of+rat+intestinal+epithelial+cell+primary+cultures.">The development of a method for the preparation of rat intestinal epithelial cell primary cultures.</a> J. Cell Sci. 101, 219-231 (1992).</li><li>Sala, F. G., Matthews, J. A., Speer, A. L., 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=A+multicellular+approach+forms+a+significant+amount+of+tissue-engineered+small+intestine+in+the+mouse.">A multicellular approach forms a significant amount of tissue-engineered small intestine in the mouse.</a> Tiss. Eng. Part A. 17, 1841-1850 (2011).</li><li>Speer, A. L., Sala, F. G., Matthews, J. A., Grikscheit, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+tissue-engineered+stomach+demonstrates+epithelial+differentiation.">Murine tissue-engineered stomach demonstrates epithelial differentiation.</a> J. Surg. Res. 171, 6-14 (2011).</li><li>Haxhija, E. Q., Yang, H., Spencer, A. U., 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=Intestinal+epithelial+cell+proliferation+is+dependent+on+the+site+of+massive+small+bowel+resection.">Intestinal epithelial cell proliferation is dependent on the site of massive small bowel resection.</a> Pediatr. Surg. Int. 23, 379-390 (2007).</li><li>Zhao, L., Cheng, Z., Dhall, 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=A+novel+corrective+pullthrough+surgery+in+a+mouse+model+of+Hirschsprung's+disease.">A novel corrective pullthrough surgery in a mouse model of Hirschsprung's disease.</a> J. Pediatr. Surg. 44, 759-766 (2009).</li><li>Petrosyan, M., Guner, Y. S., Williams, M., 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=Current+concepts+regarding+the+pathogenesis+of+necrotizing+enterocolitis.">Current concepts regarding the pathogenesis of necrotizing enterocolitis.</a> Ped. Surg. Int. 25, 309-318 (2009).</li><li>Shew, S. 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Hepatol. 7, 183-191 (2009).</li><li>Klempnauer, J., Grothues, F., Bektas, H., Pichlmayr, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+results+after+surgery+for+acute+mesenteric+ischemia.">Long-term results after surgery for acute mesenteric ischemia.</a> Surgery. , 121-239 (1997).</li><li>Fitzgibbons, S. C., Jones, B. A., Hull, M. A., 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=Relationship+between+biopsy-proven+parenteral+nutrition-associated+liver+fibrosis+and+biochemical+cholestasis+in+children+with+short+bowel+syndrome.">Relationship between biopsy-proven parenteral nutrition-associated liver fibrosis and biochemical cholestasis in children with short bowel syndrome.</a> J. Ped. Surg. 45, 95-99 (2010).</li><li>Spencer, A. U., Kovacevich, D., McKinney-Barnett, M., 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=Pediatric+short+bowel+syndrome:+the+cost+of+comprehensive+care.">Pediatric short bowel syndrome: the cost of comprehensive care.</a> Am. J. Clin. Nutr. 88, 1552-1559 (2008).</li><li>Kato, T., Tzakis, A. G., Selvaggi, G., 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=Intestinal+and+multivisceral+transplantation+in+children.">Intestinal and multivisceral transplantation in children.</a> Ann. Surg. 243, 756-766 (2006).</li><li>Reyes, J., Bueno, J., Kocoshis, 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=Current+status+of+intestinal+transplantation+in+children.">Current status of intestinal transplantation in children.</a> J. Ped. Surg. 33, 243-254 (1998).</li></ol>

Aucun conflit d&#39;intérêt déclaré.

Nous présentons un protocole pour la production par génie tissulaire intestin chez la souris en utilisant un organoïde unités-sur-échafaudage approche. Les étapes les plus importantes sont celles de la préparation organoïde unités. Il faut prendre soin de bien nettoyer et traiter mécaniquement le tissu, mais le même soin doit être pris pour ne pas overdigest ou overtriturate les unités organoïdes après la digestion est effectuée (étape 1.11). Si cela est fait, les unités organoïdes peut être réduit ...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires (optionnel) </td></tr><tr><td> HBSS </td><td> Gibco </td><td> 114170-112 </td><td></td></tr><tr><td> Antibiotique-antimycotique 100X </td><td> Invitrogen </td><td> 15240-062 </td><td></td></tr><tr><td> Dispase </td><td> Gibco </td><td> 17105-041 </td><td></td></tr><tr><td> Type de collagénase 1 </td><td> Worthington </td><td> LS004194 </td><td></td></tr><tr><td> DMEM glycémie élevée 1X </td><td> Gibco </td><td> 11995-065 </td><td></td></tr><tr><td> La chaleur de FBS inactivé </td><td> Invitrogen </td><td> 16140-071 </td><td></td></tr><tr><td> Biofelt 100% PGA </td><td> Concordia médicale </td><td> FELT01-1005 </td><td> Pour la préparation de polymères selon la Réf.4 </td></tr><tr><td> La poly-L-lactique </td><td> DURECT </td><td> B6002-1 </td><td> Pour la préparation de polymères selon la Réf. 4 </td></tr><tr><td> Type I collagène de queue de rat, </td><td> Sigma-Aldrich </td><td> C3867-1VL </td><td> Pour la préparation de polymères selon la Réf. 4 </td></tr><tr><td> Kétoprofène 100 mg / ml </td><td> Fort Dodge Animal Health </td><td> 71-KETOI-100-50 </td><td></td></tr><tr><td> LabDiet 5001 chow rongeurs </td><td> LabDiet </td><td> 5001 </td><td></td></tr><tr><td> Septra 200 mg / 40 mg pour 5 ml, USP </td><td> Salut-Tech Pharmacal </td><td> 50383-824-16 </td><td></td></tr><tr><td> Isoflurane, USP </td><td> Phoenix Pharma </td><td> 57319-507-06 </td><td></td></tr></tbody></table>

tissue engineering of the intestine in a murine model

Génie tissulaire intestin grêle (TESI) a été utilisée avec succès pour sauver des rats Lewis après une résection intestinale massive de petite, ce qui entraîne en retour de poids préopératoire dans les 40 jours 1. Chez l&#39;homme, massif résection de l&#39;intestin grêle peuvent entraîner un syndrome du grêle court, une malabsorption fonctionnelle Etat qui confère une importante morbidité, la mortalité et les coûts des soins de santé, y compris la dépendance nutrition parentérale, l&#39;insuffisance hépatique et de la cirrhose, et la nécessité d&#39;une transplantation d&#39;organe multiviscérale. 2 Dans cet article, nous décrivons et documenter notre protocole pour la création par génie tissulaire intestin dans un modèle de souris avec un organoïde unités-sur-échafaudage approche multicellulaire. Unités sont organoïdes agrégats multicellulaires dérivées de l&#39;intestin qui contiennent des éléments à la fois des muqueuses et mésenchymateuses, 3 la relation entre ce qui préserve la niche tige intestinale cellule. 4 Dans la recherche en cours et à venir, la transition de notre technique dans lesouris permettra d&#39;enquête sur les processus impliqués lors de la formation TESI en utilisant les outils disponibles transgéniques chez cette espèce. 5 La disponibilité de souches de souris immunodéprimées nous permettra également d&#39;appliquer la technique de tissu intestinal humain et optimiser la formation de TESI humain comme un xénogreffe de souris avant sa transition vers les humains. Notre méthode utilise bonnes pratiques de fabrication (BPF) des réactifs et des matériaux qui ont déjà été approuvés pour une utilisation chez des patients humains, et offre donc un avantage significatif par rapport aux approches qui reposent sur des tissus animaux décellularisé. Le but ultime de cette méthode est sa traduction à l&#39;homme comme une stratégie thérapeutique pour la médecine régénérative syndrome de l&#39;intestin court.

La figure 1 montre un schéma d&#39;ensemble pour le protocole documenté ici. Le résultat final de ce protocole est une structure sphérique de globe ou par génie tissulaire intestin murin avec une lumière, muqueuse, la sous-muqueuse, musculeuse et ses environs. Figure 2A montre un globe typique par rapport à un polymère de départ échafaud. Figure 2B montre la même construction fortement bivalve de révéler sa lumière. Figure 3 montre un hém...

Watch this Scientific Journal Video about Tissue Engineering of the Intestine in a Murine Model at JoVE.com

Tissue Engineering of the Intestine in a Murine Model

Cet article et la vidéo qui l&#39;accompagne présenter notre protocole pour générer génie tissulaire intestin chez la souris, en utilisant un organoïde unités-sur-échafaudage approche.

Génie tissulaire de l&#39;intestin dans un modèle murin

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

tissue-engineering-of-the-intestine-in-a-murine-model

Research

JoVE Journal

Bioengineering

14.8K vues.  Keck School of Medicine of the University of Southern California. Cet article et la vidéo qui l'accompagne présenter notre protocole pour générer génie tissulaire intestin chez la souris, en utilisant un organoïde unités-sur-échafaudage approche.

Vidéo: Tissue Engineering of the Intestine in a Murine Model

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

L&#39;encapsulation des cardiomyocytes dans un hydrogel de fibrine pour l&#39;ingénierie du tissu cardiaque

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

Recherche

Bio-ingénierie

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 ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

Conception d&#39;un bioréacteur de chargement mécanique biaxial pour l&#39;ingénierie tissulaire

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

génie tissulaire d&#39;un être humain 3D<em&gt; In vitro</em&gt; Système de tumeur de test

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

Génération d&#39;une épaisseur en trois dimensions équivalent de peau et automatisé Blessant

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Ingénierie 3D cellularisé collagène Gels pour la régénération tissulaire vasculaire

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

Génie tissulaire par Intrinsic vascularisation dans un<em&gt; In Vivo</em&gt; Tissue Chambre Ingénierie

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.

La boucle artério (AV) dans un modèle Small Animal pour étudier l&#39;angiogenèse et du génie tissulaire vascularisé

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in ...

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been &#39;conditioned&#39; by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

We present a model of ligament tissue in which three-dimensional constructs are treated with the human exercise-conditioned serum and analyzed for collagen content, function, and cellular biochemistry.

Traitement des constructions de ligament avec un sérum conditionné par l&#39;exercice: un modèle d&#39;ingénierie des tissus de traduction

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, ...