This work was funded by a New Faculty Award II from the California Institute of Regenerative Medicine (CIRM; RN2-00921-1), NIH-funded National Research Award (F32-HL104924), and CIRM Training Grant (TG21163). Materials were provided by: Glycosan Biosystems Inc / BioTime and Dr. Stephen Badylak (University of Pittsburgh)

1 Création de plaques PNIPAAm revêtues <ol><li> Dissoudre 2,6 g de PNIPAAm dans 2 ml d&#39;une solution / 40% d&#39;hexane à 60% dans le toluène. </li><li> Chauffer le mélange à 60 ° C pendant 10 min sous agitation, jusqu&#39;à ce que l&#39;on dissout PNIPAAm. </li><li> Couper le papier filtre dans un cercle de 60 mm de diamètre et placez le papier dans l&#39;entonnoir de Büchner. </li><li> Filtrer la solution à travers un entonnoir de Büchner dans le bécher pré-pesée verre (ne pas utiliser des matières plastiques, comme l&#39;hexane fera fondre les plastiques). </li><li> Placer le bécher et contenu dans un vide de cloche (24 psi) O / N (16 h). Note: Jusqu&#39;à ce que le résidu est traité avec isopropylique il s&#39;oxyde alors assurez-vous qu&#39;il n&#39;entre pas en contact avec l&#39;oxygène. </li><li> Peser le bécher pour déterminer la masse de la PNIPAAm. </li><li> Ajouter de l&#39;alcool isopropylique à la PNIPAAm, la création d&#39;un 50/50 p / p de la solution. </li><li> Placer 2 ml de la solution sur la surface de la plaque de culture de tissu, et le pelage pendant 5 min sous une lampe UV. </li><li> Laver la plaque avec 2 ml de PBS chaud deux foisavant de l&#39;utiliser pour la culture cellulaire. </li></ol> 2 Création de feuilles cellulaires Remarque: feuilles cellulaires de cellules primaires pour l&#39;organe cible peuvent être créés en utilisant un certain nombre de méthodes différentes, ou par le revêtement de surfaces de culture de tissu de polymère thermo-sensible comme décrit ici. Plaques thermo-sensible pré-enrobées sont également offerts par un certain nombre de fournisseurs. Remarque: Ce protocole est de culture en utilisant une boîte de 35 mm. Brièvement, les cellules sont d&#39;abord mises en incubation à 37 ° C pendant un minimum de 24 h à confluence pour établir des connexions latérales entre les cellules adjacentes. Pour détacher les feuilles de cellules, les plaques sont soumises à des températures inférieures à 32 ° C. Le tapis cellulaire est ensuite transféré dans la matrice fibreuse de base forte contenant un hydrogel permissive néovasculaire avec des cellules endotheliales vasculaires. <ol><li> Isoler la population de cellules. Remarque: cette méthode dépend des procédures de dérivation individuels et le type de cellules. Rat mu lisse aortiquedes cellules sub-aigu (RASMC) sont utilisés dans cet exemple. Ce sont des primaires de cellules musculaires lisses isolées de l&#39;aorte abdominale d&#39;un rat. </li><li> Laver les cellules avec 2 ml de PBS chaud. </li><li> Ajouter 3 ml de trypsine (ou autre / solution dissocier de clivage) sur les cellules pendant 5 min. </li><li> Inhibition de la trypsine par addition de 3 ml de milieu de culture, ou une solution tampon phosphate (PBS) contenant 10% de sérum bovin fœtal (FBS). </li><li> Les cellules dans un tube conique et compter une aliquote. </li><li> Faites tourner les cellules à 1000 rpm (228 xg) pendant 5 min. </li><li> Aspirer le surnageant et remettre en suspension les cellules dans leur milieu de croissance (milieu de culture SmGM2 plus kit balle est utilisé pour RASMC). </li><li> Placer le support contenant les cellules sur une plaque thermo-sensible 35 mm - PNIPAAm plaque revêtue à une concentration qui permettra d&#39;atteindre 100% de confluence. Remarque: Pour CMLAR ce nombre a été déterminé que 100 000 cellules / cm 2. Toutefois, en raison de la perte de cellules lors de la transmission, que 120% de VA<html> lue est utilisée. </li><li> Placer dans un incubateur à 37 ° CO / N. Remarque: il est important de maintenir les cellules à 37 ° C pour maintenir l&#39;adhérence cellulaire de la plaque. </li></ol> 3 Préparation de Matrix Foundational Remarque: Plusieurs matrices fibreuses 3D peuvent être utilisés pour la couche solide matrice fibreuse entre les feuilles de cellules délicates. Voici quelques exemples: gelfoam, bioverre, matériaux naturels acellularized 26 ou nanospun matériaux 27,28 Le porc urinaire matrice de la vessie (UBM) utilisée dans ces études a été généreusement fourni par notre collaborateur, le Dr Badylak 29. <ol><li> Avant d&#39;utiliser, de déterminer les caractéristiques de la matrice, y compris le manque de contenu cellulaire si matrice décellularisé est utilisée, 27,28 viabilité cellulaire spécifique, et un espace vide. 22 </li><li> Couper la matrice de pré-stérilisé dans une taille et une forme souhaitées. Note: Ici, un poinçon est utilisé pour découper un cercle de 4 mm de diamètre. </li></ol>ve_title &quot;&gt; 4. Cellules endothéliales ensemencement dans un néovasculaire permissive hydrogel Remarque: Les cellules endotheliales peuvent être obtenus à partir d&#39;une variété de sources, y compris la différenciation de cellules souches ou progénitrices. Ici, on utilise des cellules HUVEC. <ol><li> Utilisation tout hydrogel permissive (de fibrine, des gels de collagène) aussi longtemps que le temps de reticulation est suffisamment courte pour que les cellules restent viables. Remarque: ici, un gel acide hyaluronique (HA) réticulé à base d&#39;un pont disulfure est utilisé. </li><li> Préparer l&#39;hydrogel HA en conformité avec le protocole de la société. </li><li> Recueillir les cellules endothéliales et les disperser dans une solution à une seule cellule à l&#39;aide de la trypsine 1x. Remarque: Accutase ou tampon de dissociation cellulaire peuvent également être utilisés pour la dispersion de la cellule unique. </li><li> Désactiver l&#39;enzyme de la trypsine en utilisant une quantité égale d&#39;inhibiteur de trypsine de soja (si il est important que les cellules ne sont pas en contact avec le sérum) ou 10% de FBS dans du PBS, la collecte de la solution / cellules dans un tube conique de 15 ml. </li><li> Contant les cellules, et de calculer le volume nécessaire pour les dimensions de raccordement (auparavant quantifiés). Remarque: Pour un patch de 4 mm, ici de 2 millions de cellules endothéliales sont utilisés. </li><li> Extrait 2 millions de cellules, et la placer dans un nouveau tube de 15 ml conique. </li><li> Spin au (228 xg) pendant 5 min. </li><li> Aspirer le surnageant, ce qui laisse les cellules en culot dans un tube conique. </li><li> Mélangez les matières liquides de HA et de la gélatine dans un rapport 1: 1 ration. Ensuite, ajouter 80% du volume total dans le tube conique contenant le culot. </li><li> Remettre en suspension les cellules endothéliales dans le mélange 1: 1 HA / Gélatine </li><li> Mettre les cellules en suspension dans le mélange HA / gélatine dans la matrice fibreuse de base à partir de l&#39;étape 2. </li><li> Ajouter cinquième (20 pour cent) du volume total souhaité de l&#39;agent de réticulation </li><li> Incuber pendant 1 heure à 37 ° C. </li></ol> 5 Isolation de feuilles cellulaires <ol><li> Retirez les plaques de 35 mm PNIPAAm traités contenant les cellules de l&#39;incubateur et le placer dans une hotte de culture cellulaire àRT. </li><li> Aspirer rapidement les médias à partir des cellules, et ajouter 2 ml de 6% de gélatine normale qui a été chauffée à 37 ° C. </li><li> Bien que la gélatine est encore chaud, placer le treillis métallique dans la gélatine, la plongeant sous la surface de la gélatine normale (Film 1). </li><li> Placer la totalité de la plaque sur de la glace pendant 5 à 7 minutes, ce qui permet la gélatine à durcir. </li><li> Après 7 minutes, utiliser une spatule pour bien séparer les bords de gélatine sur le côté de la plaque, puis utilisez une pince pour soulever le treillis métallique de la plaque Remarque: Le 6% de gélatine, et la feuille de cellules devrait soulever avec le réseau. </li><li> Déplacer la feuille de cellules dans le plat et placer sur le dessus de la combinaison de matrice hydrogel base fibreuse, définissant avec soin le réseau au-dessus de la construction. Remarque: Le côté apical de la feuille de cellule sera toujours en position haute. </li><li> Ajouter 2 ml de milieu chaud (37 ° C). </li><li> Incuber O / N permettre à la feuille de cellules d&#39;adhérer à la surface de l&#39;hydrogel. </li><li> Retirer til treillis métallique après que la solution se réchauffe (environ 1 heure), ou le jour suivant. </li></ol>

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We have nothing to disclose.

Les étapes critiques du protocole comprennent: le revêtement des surfaces de la plaque avec le polymère thermosensible et la manipulation des feuilles de cellules après refroidissement des plaques. Parce que les cellules présentent différentes propriétés physiques différentes, comme l&#39;adhésivité, le temps de levée doit être optimisée pour chaque type de cellule différent. La seconde, et la plus significative difficile composante de ce protocole, se concentre sur la manipulation de la feuille de cellul...

Injection of cells and/or single materials alone has shown variable success in other organ systems and limited success in cardiac regeneration.5,7-12 Currently, stem cell-derived cells are delivered to damaged tissue using a variety of delivery methods including: direct cell injection into tissue and perfusion into the blood supply.13-17 Others have implanted cells alone, materials alone and/or in combination with material carriers to help regenerate damaged organs.18-21 This design combines multiple strategies that provide material strength, patterning in multiple materials and multiple cell types. 
Specifically, the base acellularized fibrous matrix provides the foundational physical strength to the construct, making it suitable for suturing in into the patient, if necessary. The void spaces in the base matrix are filled with endothelial cells in a neovascular permissive hydrogel22 for rapidly establishing vascularization of the implanted construct. This composite is then integrated with pre-patterned cell sheets that allow enhanced cell-to-cell communication, more closely mimic the native tissue.1,23-25 The overall production process for the layered cellular patch is outlined by the flowchart in Figure 1.

<table border="0" cellpadding="0" cellspacing="0" width="695">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:207px;">Table of Reagents:</td>
			<td style="width:128px;"></td>
			<td style="width:140px;"></td>
			<td style="width:221px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Reagent</td>
			<td style="width:128px;">Company</td>
			<td style="width:140px;">Catalogue number</td>
			<td style="width:221px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Calcein-AM</td>
			<td style="width:128px;">Invitrogen</td>
			<td style="width:140px;">C3099</td>
			<td style="width:221px;">Cell tracker / live dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Lysotracker Red</td>
			<td style="width:128px;">Invitrogen</td>
			<td style="width:140px;">L7528</td>
			<td style="width:221px;">Cell tracker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Neutral Red</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:140px;">N7005</td>
			<td style="width:221px;">Visible Cell dye</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:207px;">pNIPAAM</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:140px;">412780250</td>
			<td style="width:221px;">Poly(N-isopropylacrylamide)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Toluene</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:140px;">244511-1L</td>
			<td style="width:221px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Hexane</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:140px;">296090-1L</td>
			<td style="width:221px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">RAOSMC</td>
			<td style="width:128px;">Lonza</td>
			<td style="width:140px;">R-ASM-580</td>
			<td style="width:221px;">Rat Aortic Smooth Muscle Cells</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:207px;">SmGM2</td>
			<td style="width:128px;">Lonza</td>
			<td style="width:140px;">CC-4149</td>
			<td style="width:221px;">Smooth Muscle Media</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:207px;">HUVEC</td>
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tissue engineering: construction of a multicellular 3d scaffold for the delivery of layered cell sheets

Beaucoup de tissus, comme les adultes cœurs humains, sont incapables de se régénérer correctement après dommages. 2,3 Stratégies en ingénierie tissulaire proposent des innovations pour aider le corps dans la récupération et la réparation. Par exemple, les approches TE peuvent être en mesure d&#39;atténuer le remodelage cardiaque après un infarctus du myocarde (IM) et peut-être augmenter la fonction cardiaque totale à un niveau MI pré proche de la normale. 4 Comme avec n&#39;importe quel tissu fonctionnel, réussite de la régénération du tissu cardiaque implique la bonne exécution de plusieurs types de cellules avec des signaux environnementaux qui favorisent l&#39;intégration et la survie de la greffe de cellules / tissus implantés. Tissus d&#39;ingénierie devraient aborder plusieurs paramètres dont: signaux solubles, les interactions cellule-cellule et matériaux de matrice évalués comme les véhicules de livraison, de leurs effets sur la survie de la cellule, la résistance des matériaux, et la facilitation de l&#39;organisation de cellule à tissu. Des études utilisant l&#39;injection directe de cellules du greffon seulement ignorer ces éléments essentiels. 2,5,6Une conception de tissu combinant ces ingrédients n&#39;a pas encore été développé. Ici, nous présentons un exemple de superposition à l&#39;aide des dessins intégrés de feuilles de cellules à motifs avec deux types de matières biologiques contenant des dérivés du type cellulaire organes cibles et des cellules endothéliales pour l&#39;amélioration de la formation de nouveaux vaisseaux dans le &quot;tissu&quot;. Bien que ces études se concentrent sur la production de tissu cardiaque comme, cette conception de tissu peut être appliquée à de nombreux autres organes que le coeur de la conception et matérielles des changements minimes, et est censé être un produit hors-the-shelf pour les thérapies régénératrices. Le protocole contient cinq étapes détaillées. Une température Poly sensible (N de -isopropylacrylamide) (PNIPAAm) est utilisé pour des boîtes de culture de tissus de la robe. Ensuite, les cellules sont mises en culture tissulaire spécifique sur la surface des plaques revêtues de motif fin / des surfaces pour former des feuilles de cellules avec de fortes adhérences latérales. En troisième lieu, une matrice de base est créé pour le tissu en combinant la matrice poreuse avec Permissi néovasculaireve hydrogels et les cellules endothéliales. Finalement, les feuilles de cellules sont soulevées à partir des boîtes revêtues PNIPAAm et transférées à l&#39;élément de base, ce qui rend l&#39;assemblage complet.

Le diagramme de flux (figure 1) illustre le procédé global de fabrication de la plaque à couches multiples. les feuilles de cellules sont détachées de la plaque traitée PNIPAAm en laissant tomber la température en dessous de 32 ° C. Ensuite, la feuille de cellules est placée au-dessus de l&#39;hydrogel réticulé contenant des cellules endothéliales ensemencées sur la matrice fibreuse sous-jacente (figure 1). Les plaques thermo-sensible prétraitées peuvent également être ...

Watch this Scientific Journal Video about Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets at JoVE.com

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

Ingénierie tissulaire: Construction d&#39;un échafaudage 3D multicellulaire pour la livraison de stratifiés feuilles cellulaires

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

tissue-engineering-construction-multicellular-3d-scaffold-for

Research

JoVE Journal

Bioengineering

14.3K vues.  University of California, Merced. For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

Vidéo: Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

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  ...

Recherche

Bio-ingénierie

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 ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Les échafaudages auto-déclaration pour les 3-Dimensional Culture cellulaire

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

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. ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

Adapter le processus Électrofilage de fournir trois environnements uniques pour un Tri-couches<em&gt; In Vitro</em&gt; Modèle du mur Airway

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue geometry. The resulting 3D hydrogel-based cellular constructs have been introduced as an alternative to animal experiments for advanced biological studies, pharmacological assays and organ transplant applications. Although hydrogel-based particles and fibers can be easily fabricated, it is difficult to manipulate them for tissue reconstruction. In this video, we describe a fabrication method for micropatterned alginate hydrogel sheets, together with their assembly to form a macro-scale 3D cell culture system with a controlled cellular microenvironment. Using a mist form of the calcium gelling agent, thin hydrogel sheets are easily generated with a thickness in the range of 100 - 200 &#181;m, and with precise micropatterns. Cells can then be cultured with the geometric guidance of the hydrogel sheets in freestanding conditions. Furthermore, the hydrogel sheets can be readily manipulated using a micropipette with an end-cut tip, and can be assembled into multi-layered structures by stacking them using a patterned polydimethylsiloxane (PDMS) frame. These modular hydrogel sheets, which can be fabricated using a facile process, have potential applications of in vitro drug assays and biological studies, including functional studies of micro- and macrostructure and tissue reconstruction.

We describe the fabrication of micropatterned hydrogel sheets using a simple process, which can be assembled and manipulated in a freestanding form. Using these modular hydrogel sheets, a simple macro-scaled 3D cell culture system can be generated with a controlled cellular microenvironment.

Construction de feuilles d&#39;hydrogel modulaire pour micropatterned macro-échelle 3D Cellular architecture

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

Fabrication de Inverted colloïdal cristal Poly (éthylène glycol) Échafaudages: Une cellule plate-forme en trois dimensions de la culture pour l&#39;ingénierie tissulaire du foie

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

Fabrication de Mousses et microsupports dérivés Matrice extracellulaires culture cellulaire spécifique de tissu et plates-formes de livraison

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

Fabrication de puits d’Agarose personnalisé pour l’ensemencement de la cellule et l’anneau de tissu auto-assemblage à l’aide de moules 3D-imprimé

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also respond to neural injuries and act to protect the tissue from degenerative events. In vitro studies of astrocytes&#39; functionality are important to elucidate the mechanisms involved in such events and contribute to developing therapies to treat neurological disorders. This protocol describes a method to biofabricate a neural-like tissue structure rich in astrocytes by 3D bioprinting astrocytes-laden bioink. An extrusion-based 3D bioprinter was used in this work, and astrocytes were extracted from C57Bl/6 mice pups&#39; brain cortices. The bioink was prepared by mixing cortical astrocytes from up to passage 3 to a biomaterial solution composed of gelatin, gelatin-methacryloyl (GelMA), and fibrinogen, supplemented with laminin, which presented optimal bioprinting conditions. The 3D bioprinting conditions minimized cell stress, contributing to the high viability of the astrocytes during the process, in which 74.08% &#177; 1.33% of cells were viable right after bioprinting. After 1 week of incubation, the viability of astrocytes significantly increased to 83.54% &#177; 3.00%, indicating that the 3D construct represents a suitable microenvironment for cell growth. The biomaterial composition allowed cell attachment and stimulated astrocytic behavior, with cells expressing the specific astrocytes marker glial fibrillary acidic protein (GFAP) and possessing typical astrocytic morphology. This&#160;reproducible protocol provides a valuable method to biofabricate 3D neural-like tissue rich in astrocytes that resembles cells&#39; native microenvironment, useful to researchers that aim to understand astrocytes&#39; functionality and their relation to the mechanisms involved in neurological diseases.

Here we report a method of 3D bioprinting murine cortical astrocytes for biofabricating neural-like tissues to study the functionality of astrocytes in the central nervous system and the mechanisms involving glial cells in neurological diseases and treatments.

Bioimpression 3D d’astrocytes corticaux murins pour l’ingénierie de tissus de type neural

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also ...