Cette étude a été réalisée dans le programme de recherche des matériaux biomédicaux (BMM) Institut. BMM est cofinancé par le ministère néerlandais des Affaires économiques, de l&#39;Agriculture et de l&#39;Innovation. La contribution financière de la Hartstichting Nederlandse est grandement appréciée.

1. Culture des droits de veine saphène Cells <ol><li> Isoler des cellules de la veine saphène, acquises à partir d&#39;un donneur conformément à des directives pour l&#39;utilisation de matière secondaire, selon le protocole par Schnell et al. 9 et stocker ceux-ci dans de l&#39;azote liquide. Dans le cadre de la veine saphène à une des pièces découpées de donneur de 2 x 2 mm à la culture dans une plaque à six puits. Utilisez 2 éléments par puits. Assez généralement cellules peuvent être obtenues à remplir environ 3 flacons à 0,25 x 10 6 cellules dans l&#39;azote liquide. HVSCs sont caractérisés comme myofibroblastes, en montrant l&#39;expression de la vimentine, aucune expression de la desmine et une sous-population exprimant α actine musculaire lisse 10. Suivant démarrer le protocole de décongeler les cellules de l&#39;azote liquide pour augmenter le nombre de cellules. </li><li> Placez les cellules provenant d&#39;une fiole dans un flacon de culture de tissu T75 et ajouter du milieu de croissance (GM), composé d&#39;avancée DMEM, 50 ml de sérum de veau fœtal (FBS), 5 ml de pénicilline /streptomycine et 5 ml de L-glutamax. Changer le milieu tous les 2-3 jours. </li><li> Les cellules croissent confluent environ après 14 jours. Magasin 0,5 x 10 6 cellules dans un flacon dans de l&#39;azote liquide, dénommé passage 1. </li></ol> Note: La congélation des cellules n&#39;est pas nécessaire lors de la récolte HVSCs, mais il est uniquement utilisé pour le stockage. <ol start="4"><li> Placez les HVSCs d&#39;une fiole dans un flacon de culture tissulaire et T175 ajouter GM. Changer le milieu tous les 2-3 jours. Les cellules croissent confluent après environ 7 jours. Magasin 3 x 10 6 cellules dans un flacon dans de l&#39;azote liquide, appelé passage 2. </li><li> Placez les cellules provenant d&#39;un flacon dans un rollerbottle et ajouter GM. Changer le milieu tous les 2-3 jours. Les cellules croissent confluent environ après 8 jours. Un rollerbottle contient environ 20-30 x 10 6 cellules. cellules en culture jusqu&#39;à passage 6. Ne pas utiliser des cellules d&#39;un passage supérieur à 9 passage, depuis le phénotype cellulaire peut changer. </li></ol&gt; 2. Ingénierie des constructions de tissu à base de fibrine <ol><li> Préparation de la colle silicone par le mélange de l&#39;élastomère avec l&#39;agent de durcissement (10:01). Couper 7 x 3 segments rectangulaires mm de Velcro. Coller le Velcro dans une plaque de culture 6 puits, avec des membranes flexibles pour former une croix, et à laisser un espace carré de 3 mm entre les bandes de Velcro. </li></ol> Notes: utiliser uniquement le côté doux du velcro et faire face à ce côté vers le haut. En collant le Velcro, ne couvrent que le Velcro avec colle silicone, ne se propage pas à travers la colle bien. Comme les plaques de culture ont un fond de la membrane en silicone, utiliser quelque chose en dessous de la plaque de puits pour le renforcement des membranes flexibles, afin d&#39;assurer le collage facile dans la plaque. <ol start="2"><li> Sécher la colle de silicone pendant une nuit dans une étuve à 60 ° C pour assurer le durcissement de la colle. Stériliser en ajoutant 70% de EtOH dans les puits et incuber pendant 30 min. Rincer 3x avec PBS et retirez tous PBS à partir du wells et le Velcro. Mettez sous UV pendant 30 min et garder stérile jusqu&#39;à l&#39;utilisation. </li><li> Préparer moyen Tissue Engineering (TM), composé de GM et de 130 mg d&#39;acide L-ascorbique 2-phosphate. </li><li> Ajouter 1 mg / ml caproïque ε-amino (ACA) à la TM. ACA est utilisée pendant les 7 premiers jours de culture pour empêcher la fibrine à partir dégradants 11. Alternativement, l&#39;aprotinine peut être utilisé. </li><li> Pour éviter la formation de touffe, laissez fibrinogène à atteindre la température ambiante avant ouverture. Sans touffes fibrinogène va se dissoudre plus facilement. Dissoudre le fibrinogène à une concentration de 10 mg réelle protéine / ml de TM 7 supplémenté avec ACA. Filtre stérile la solution de fibrinogène, de multiples filtres pourrait être nécessaire. Stocker sur la glace jusqu&#39;à utilisation. </li></ol> Note: Pour dissoudre mélange de fibrinogène doucement pour éviter la formation de mousse trop. <ol start="6"><li> Dissoudre la thrombine à une concentration de 10 UI / ml de TM 7 supplémenté avec ACA. Stocker sur la glace jusqu&#39;à utilisation. </li> &lt;/ Ol&gt; Note: Stockage sur la glace est nécessaire pour éviter la gélification début de la thrombine et fibrinogène. <ol start="7"><li> Trypsiniser les cellules, remettre en suspension dans GM et le nombre. </li><li> Utilisez 15 x 10 6 cellules / ml pour ensemencer les constructions de tissu à base de fibrine (concentration basée sur les méthodes de valves cardiaques de l&#39;ingénierie tissulaire 7). Un seul gel est constitué de 100 pi GOF el, et ainsi de 1,5 x 10 6 cellules. Mettez 1,5 x 10 6 cellules dans un tube de centrifugeuse, résultant en autant de tubes de centrifugeuses que le nombre de gels qui seront faites. </li><li> Centrifuger les cellules à 350 g pendant 7 min et jeter le surnageant. Reprendre les cellules dans 50 ul de thrombine. Ajouter 4 pi de microsphères de polystyrène bleu fluorescent à cette suspension. Mettez 50 pl de fibrinogène dans un flacon. Utiliser une pipette pour prendre la thrombine 50 pi avec des cellules. Augmenter le volume de la pipette à 100 pi. Introduire à la pipette la solution de 50 pi avec de la thrombinecellules dans le flacon de fibrinogène pour mélanger les thrombine et le fibrinogène, et prennent le mélange de 100 pi. </li></ol> Remarque: Les microsphères de polystyrène fluorescentes sont utilisés en tant que marqueurs de référence interne pour l&#39;analyse de l&#39;image. Lorsque la thrombine et le fibrinogène mélange, éviter la formation de bulles d&#39;air par aspiration soigneusement le mélange. Les bulles d&#39;air se traduira par des trous dans le gel de fibrine. <ol start="10"><li> Pipeter le mélange de gel dans et entre les bandes de Velcro. Le temps de gélification typique des gels de fibrine, une fois que les composants sont mélangés, est de l&#39;ordre de 20 s. </li></ol> Notes: Pour ce faire, le plus rapidement possible pour éviter la gélification avant que le mélange est introduit à la pipette dans la plaque bien. Pratique avant d&#39;utiliser des cellules et des perles. <ol start="11"><li> Incuber suspensions pendant 30 min à 37 ° C dans une atmosphère humidifiée à 95% d&#39;air / 5% de CO 2 incubateur pour permettre la gélification avant d&#39;ajouter la culture TM avec l&#39;ACA. </li><li> Remplacer TM avec l&#39;ACA tous les 2-3 jours pendant les 7 premiers jours. Les gels sont assez stable après une semaine à être cultivé sans ACA. Après la première semaine TM remplacer tous les 2-3 jours. Ajouter 6 ml de TM par puits. Le jour 12 cellules ont produit suffisamment de collagène pour la visualisation. </li></ol> 3. Application de souche et induisant des changements dans la souche et contraintes <ol><li> Allongement statique est appliquée directement par les cellules, dues à l&#39;accélération de la cellule et de compactage 12. Pour induire une réorganisation du collagène, couper le tissu construire lâche de deux bandes Velcro dans un seul sens. Il en résulte des contraintes unidirectionnelles (figure 1A). Effectuez cette 12e jour, puisque le concept est alors assez stable et le collagène a été déposé. </li><li> Appliquer déformation cyclique en appliquant un vide à la partie inférieure des plaques de culture avec des membranes flexibles, avec l&#39;utilisation du système FX4000T flexcell. Placez les plaques sur une plaque de base, en plus de postes de chargement (qui sont une partie de ee dispositif de chargement cyclique). La pompe applique un vide sur la membrane et tire ainsi la membrane sur le poteau rectangulaire se trouvant en dessous. En raison des fixations en forme de croix du tissu construit sur ​​la membrane (via le Velcro) et le poste rectangulaire la souche cyclique appliqué est uniaxe (figure 1B). </li><li> Initialement, utilisez culture statique pendant 5 jours pour atteindre l&#39;intégrité mécanique initial, avant l&#39;application de la contrainte cyclique à partir du jour 6. Programme un protocole tache cyclique dans l&#39;unité de commande pour la pompe à vide. Par exemple en utilisant un protocole de déformation cyclique intermittente établi précédemment, à l&#39;orientation uniaxiale 13. Il est composé d&#39;une souche intermittent d&#39;une onde sinusoïdale à 1 Hz, l&#39;effort de 0 à 5% de déformation, pour des périodes de 3 heures, alternant avec des périodes de repos de 3 heures. Effectuez ce protocole de déformation cyclique pendant 7 jours, induisant une organisation de collagène alignés. </li><li> Typiquement, après 12 jours HVSCs ont produit une organizatio de collagène alignésn. Après une organisation de collagène alignée est atteinte, changer la direction de déformation cyclique uniaxiale, à être perpendiculaire à la direction de la souche originale. Pour ce faire, en tournant les messages rectangulaires de 90 °. </li></ol> 4. Cellules visualisation et de collagène <ol><li> Pour visualiser, le remodelage du collagène active en temps réel, des échantillons d&#39;étiquettes avec des sondes qui ne gênent pas la viabilité cellulaire ou de la formation de collagène. Utiliser des sondes pour colorer fluorescence du cytoplasme des cellules et de collagène. </li><li> Alternativement génération de second harmonique en utilisant la microscopie confocale à balayage laser peut être utilisé pour visualiser les structures de collagène à l&#39;aide autofluorescence 14, avec une excitation à 780 nm et la détection entre 500-550 nm. </li><li> Retirez les plaques de culture à partir de la configuration et de l&#39;incubateur, pour les échantillons de manière cyclique tendues au cours de la période de repos de 3 heures, et de les transporter à un microscope confocal à balayage laser pour visualiser les cellules et de collagène. </li></ol>

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Les auteurs déclarent qu&#39;ils n&#39;ont aucun intérêt financier concurrents.

Le système modèle décrit des constructions de fibrine cellule de population a un grand potentiel pour l&#39;étude des cellules et de collagène (ré) organisation (de Jonge et al. 15), par exemple, être utilisé à des fins d&#39;ingénierie tissulaire. En utilisant la fibrine en tant que support de cellule initiale, après la dégradation de la fibrine, d&#39;un tissu est créé avec des cellules et de la matrice endogène seulement. De cette façon, les cellules sont stimulées à ré...

Les tissus cardiovasculaires ont une fonction de charge importante. En particulier, le contenu et l&#39;organisation des fibres de collagène dans la matrice extracellulaire contribuer aux propriétés porteuses et dominer la force globale de tissus 1. Dans l&#39;ingénierie tissulaire conditionnement mécanique de la construction est utilisée - généralement composé d&#39;(cyclique) des régimes rude épreuve - afin d&#39;améliorer l&#39;organisation des tissus et des propriétés mécaniques 2,3. Une bonne compréhension de l&#39;organisation du collagène induite par contrainte dans des géométries complexes de tissus pour créer des tissus à l&#39;architecture de collagène prédéfinie n&#39;a pas encore été atteint. Ceci est principalement dû à notre connaissance limitée de remodelage du collagène dans les tissus en voie de développement. Les modèles existants donnent surtout des informations sur le résultat net final de remodelage du collagène avec l&#39;utilisation des contraintes statiques 4-6. Ici nous fournissons un système modèle très configurable qui permet l&#39;étude de collagène (ré) organisation d&#39;une manière en temps réel, en 3D, sous l&#39;influencede contrainte statique ou cyclique. Les constructions de tissus sont à base de fibrine, s&#39;assurer que tous collagène dans la construction est endogène. organisation de cellules et de collagène dans les constructions est visualisée, et un système de référence interne nous permet de relocaliser les cellules et les structures de collagène pour l&#39;analyse time-lapse. Dans ce protocole, nous allons décrire l&#39;utilisation du système de modèle pour les cellules Saphena Vena humaines (HVSCs), puisque ces cellules sont connus pour leur meilleure production supplémentaire cellulaire de la matrice et la capacité de remodeler la matrice et notre usage établi dans les tissus cardiovasculaires ingénierie 7, sur la base sur les travaux de de Jonge et al. 8

<table border="1">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Culture plastic</td>
			<td>Greiner</td>
			<td></td>
			<td>Includes culture flasks and pipettes</td>
		</tr>
		<tr>
			<td>Advanced DMEM</td>
			<td>Gibco</td>
			<td>12491</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Greiner</td>
			<td>758075</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Gibco</td>
			<td>10378016</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMax</td>
			<td>Gibco</td>
			<td>35050-079</td>
			<td></td>
		</tr>
		<tr>
			<td>Elastomer and curing agent</td>
			<td>Dow Corning Corporation</td>
			<td>3097358-1004</td>
			<td>Silastic MDX 4-4210#</td>
		</tr>
		<tr>
			<td>Velcro</td>
			<td>Regular store</td>
			<td></td>
			<td>You can buy this at a regular store, only use the soft side</td>
		</tr>
		<tr>
			<td>Bioflex culture plates</td>
			<td>Flexcell Int</td>
			<td>BF-3001U</td>
			<td>Untreated</td>
		</tr>
		<tr>
			<td>L-Ascorbic Acid 2-phosphatase</td>
			<td>Sigma</td>
			<td>A8960</td>
			<td></td>
		</tr>
		<tr>
			<td>&#949;-Amino Caproic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>D7754</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine thrombin</td>
			<td>Sigma</td>
			<td>T4648</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine fibrinogen</td>
			<td>Sigma</td>
			<td>F8630</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 syringe filter</td>
			<td>Whatmann (Schleicher and Scheul)</td>
			<td>10462100</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene microspheres</td>
			<td>Invitrogen</td>
			<td>F-8829</td>
			<td>Blue fluorescent, 10 &#956;m diameter</td>
		</tr>
		<tr>
			<td>Flexcell FX-4000T</td>
			<td>Flexcell Int</td>
			<td></td>
			<td>Includes rectangular loading posts</td>
		</tr>
		<tr>
			<td>Cell Tracker Orange</td>
			<td>Invitrogen Molecular Probes</td>
			<td>C2927</td>
			<td></td>
		</tr>
		<tr>
			<td>CNA35-OG488</td>
			<td></td>
			<td></td>
			<td>Cordially provided by the Laboratory for Macromolecular and Organic Chemistry, Department of Biomedical Engineering, Eindhoven University of Technology</td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>LSM 510 Meta laser scanning microscope and Two-Photon-LSM mode</td>
		</tr>
		<tr>
			<td>Amphotericin</td>
			<td>Gibco</td>
			<td>15290-018</td>
			<td>Needed for cell isolation</td>
		</tr>
	</tbody>
</table>

engineering fibrin-based tissue constructs from myofibroblasts and application of constraints and strain to induce cell and collagen reorganization

teneur en collagène et de l&#39;organisation dans le développement de tissus collagènes peuvent être influencés par des souches de tissus locaux et des contraintes de tissu. ingénieurs de tissus visent à utiliser ces principes pour créer des tissus avec des architectures de collagène prédéfinis. Une pleine compréhension des processus sous-jacents exactes de remodelage du collagène de contrôler l&#39;architecture finale de tissu, cependant, est manquant. En particulier, on sait peu au sujet de l&#39;orientation (re) des fibres de collagène en réponse aux changements des conditions de chargement mécaniques des tissus. Nous avons développé un système modèle in vitro, composé de bi-contraints myofibroblastes tête de série fibrine constructions, à élucider collagène orientation (re) en réponse à i) revenant biaxial à des conditions de chargement statique uniaxiaux et ii) chargement cyclique uniaxial de la bi-contraint constructions avant et après un changement de direction de chargement, avec l&#39;utilisation du dispositif de chargement FX4000T flexcell. Imagerie confocale time-lapse est utilisé pour visualize collagène orientation (re) d&#39;une manière non-destructive. organisation de cellules et de collagène dans les constructions peuvent être visualisées en temps réel et un système de référence interne nous permet de délocaliser les cellules et les structures de collagène pour l&#39;analyse time-lapse. Divers aspects du système de modèle peuvent être ajustés, comme source ou l&#39;utilisation de cellules saines et malades cellule. Des additifs peuvent être utilisés pour élucider les mécanismes remodelage du collagène sous-jacent, par exemple en ajoutant MMP ou le blocage des intégrines. Forme et la taille de la construction peuvent être facilement adaptés à des besoins spécifiques, résultant en un système modèle très configurable pour étudier la cellule et du collagène (ré) organisation.

Ce système modèle permet pour la culture des myofibroblastes ensemencées gels de fibrine. Figure 1A montre un tissu cultivé d&#39;abord sous contraintes biaxiales statiques. contraintes de tissus sont libérés en coupant le gel de fibrine à partir de deux contraintes, à créer des contraintes statiques uniaxiaux et compacte de tissus et remodèle la suite (figure 1A). Par déformation cyclique, le tissu est cultivé dans des contraintes statiques biaxiales ainsi. Après 5 jours s...

Watch this Scientific Journal Video about Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization at JoVE.co

Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization

Ce système de modèle commence à partir d&#39;un gel de fibrine myofibroblastes peuplée qui peut être utilisé pour étudier collagène endogène (ré) organisation en temps réel de façon non destructive. Le système de modèle est très accordable, comme il peut être utilisé avec des sources différentes de cellules, des additifs moyen, et peut être adapté facilement à des besoins spécifiques.

Ingénierie à base de fibrine constructions de tissu de myofibroblastes et application des contraintes et de déformation à Provoquer Organisation cellulaire et collagène (Re)

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim ...

engineering-fibrin-based-tissue-constructs-from-myofibroblasts

Research

JoVE Journal

Bioengineering

10.8K vues.  Eindhoven University of Technology.  Ce système de modèle commence à partir d'un gel de fibrine myofibroblastes peuplée qui peut être utilisé pour étudier collagène endogène (ré) organisation en temps réel de façon non destructive. Le système de modèle est très accordable, comme il peut être utilisé avec des sources différentes de cellules, des additifs moyen, et peut être adapté facilement à des besoins spécifiques. 

Vidéo: Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do not have suitable autologous donor vessels due to disease progression or previous harvest. The aim of vascular tissue engineering is to develop a suitable alternative source for these bypass grafts. In addition, engineered vascular tissue may prove valuable as living vascular models to study cardiovascular diseases. Several promising approaches to engineering blood vessels have been explored, with many recent studies focusing on development and analysis of cell-based methods.2-5 Herein, we present a method to rapidly self-assemble cells into 3D tissue rings that can be used in vitro to model vascular tissues.
To do this, suspensions of smooth muscle cells are seeded into round-bottomed annular agarose wells. The non-adhesive properties of the agarose allow the cells to settle, aggregate and contract around a post at the center of the well to form a cohesive tissue ring.6,7 These rings can be cultured for several days prior to harvesting for mechanical, physiological, biochemical, or histological analysis. We have shown that these cell-derived tissue rings yield at 100-500 kPa ultimate tensile strength8 which exceeds the value reported for other tissue engineered vascular constructs cultured for similar durations (&lt;30 kPa).9,10 Our results demonstrate that robust cell-derived vascular tissue ring generation can be achieved within a short time period, and offers the opportunity for direct and quantitative assessment of the contributions of cells and cell-derived matrix (CDM) to vascular tissue structure and function.

This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly. Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days. Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.

Réalisé cellulaire auto-assemblage pour fabriquer des anneaux de tissus dérivés de cellules pour l&#39;analyse biomécanique et génie tissulaire

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do ...

Recherche

Bio-ingénierie

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

Sans contact, sans étiquette de surveillance des cellules et la matrice extracellulaire en utilisant la spectroscopie Raman

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

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 ...

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

Ingénierie tissulaire de la tumeur stromale Microenvironnement avec application de Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

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. ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Microenvironnements sandwich à exploiter cellulaires / Interactions matérielles

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

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, ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.

Collagène recombinant j’ai microporteurs de Peptide pour l’Expansion des cellules et leur utilisation potentielle comme système de diffusion de cellule dans un bioréacteur modèle

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily identified and used. Currently, this gap is filled with tendon auto-, allo-, xeno-, or artificial grafts, but clinical methods to secure them are not necessarily translatable to animals because of the scale. In order to evaluate new biomaterials or study a tendon graft made up of collagen type 1, we have developed a modified suture technique to help maintain the engineered tendon in alignment with the tendon ends. Mechanical properties of these grafts are inferior to the native tendon. To incorporate engineered tendon into clinically relevant models of loaded repair, a strategy was adopted to offload the tissue engineered tendon graft and allow for the maturation and integration of the engineered tendon in vivo until a mechanically sound neo-tendon was formed. We describe this technique using incorporation of the collagen type 1 tissue engineered tendon construct.

In this paper, we present an in vitro and in situ protocol to repair a tendon gap of up to 1.5 cm by filling it with engineered collagen graft. This was performed by developing a modified suture technique to take the mechanical load until the graft matures into the host tissue.

Une nouvelle technique de suture Tenorrhaphy avec greffe de collagène issue d’une ingénierie tissulaire pour réparer les gros défauts tendineux

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily ...

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary complications. However, islet transplantation still has several limitations such as the low viability of transplanted islets due to poor islet engraftment and hostile environments. In addition, the insulin-producing cells differentiated from human pluripotent stem cells have limited ability to secrete sufficient hormones that can regulate the blood glucose level; therefore, improving the maturation by culturing cells with proper microenvironmental cues is strongly required. In this article, we elucidate protocols for preparing a pancreatic tissue-derived decellularized extracellular matrix (pdECM) bioink to provide a beneficial microenvironment that can increase glucose sensitivity of pancreatic islets, followed by describing the processes for generating 3D pancreatic tissue constructs using a microextrusion-based bioprinting technique.

Decellularized extracellular matrix (dECM) can provide suitable microenvironmental cues to recapitulate the inherent functions of target tissues in an engineered construct. This article elucidates the protocols for the decellularization of pancreatic tissue, evaluation of pancreatic tissue-derived dECM bioink, and generation of 3D pancreatic tissue constructs using a bioprinting technique.

Bioink extracellulaire de matrice de tissu pancréatique-dérivé pour l'impression 3D Cell-Laden Pancréatique Tissue Constructs

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...