Sonja Schrepfer, Lenard Conradi et Arne Hansen a reçu un financement de la Deutsche Forschungsgemeinschaft (DFG; SCHR992/3-1, SCHR992/4-1, CO 858/1-1, HA 3423/3-1). Le travail a également été soutenue par des subventions de l&#39;Union européenne à Thomas Eschenhagen (EUGeneHeart et Angioscaff).

1. Génération d&#39;ISE et de la Culture Conditions Fabrication de base de fibrine ISE a été réalisée comme décrit par ailleurs 4. En bref, pour produire à base de fibrine ISE à des fins d&#39;implantation, un mélange maître a été préparée contenant des cellules isolées à partir néonatale ROSA / luciférase-LEW coeurs de rats transgéniques, le fibrinogène bovin et Matrigel. Agarose moules ont été préparés en utilisant des entretoises en téflon placés dans des boîtes de culture de 6 puits. Après solidification de l&#39;agarose, entretoises ont été enlevés et racks message de silicone ont été placés sur des boîtes de culture avec des messages d&#39;atteindre dans les moules. Pour générer l&#39;ISE, le Mastermix a été brièvement mélangé à un montant calculé de la thrombine et à la pipette dans le moule. Après polymérisation du fibrinogène, des supports en silicone avec HASTI adhérant à des postes ont été délicatement retirée de la coulée des moules d&#39;agarose, transférés à de nouveaux 6-même boîtes de culture cellulaire et maintenu dans un 37 ° C, 7% de CO 2 incubateur de culture cellulaire à l&#39;aide sur-mesure milieu de culture cellulaire. ISE ont été conservés dans des conditions de culture cellulaire jusqu&#39;à dix jours. Pendant ce laps de temps, les cellules de coeur de rat néonatal a commencé à allongée, d&#39;interconnexion et d&#39;aligner le long des lignes de force entre les deux poteaux de silicone. Au moment de l&#39;implantation, les contractions spontanées cohérentes ont été observées, en déviant les messages de silicone. 2. Implantation d&#39;ISE L&#39;implantation d&#39;ISE a été réalisée en soit immunocompétent brun de Norvège (BN), les rats, ou immunodéficientes rats nus pour étudier la réponse allogénique immunitaire. Rats BN et nue pesant 100 - 150 g ont été achetés auprès de Charles River Allemagne (Sandhofer Weg 7, D-97633 Sulzfeld) et logés dans des conditions classiques, nourris standards pour rats et libidum ad eau. Rats immunodéprimés sont logés dans la literie stérile, les cages, et de l&#39;eau stérilisée est utilisée. Le comité d&#39;examen éthique allemande examiné et approuvé les procédures animales. Tous les instruments chirurgicaux sont stérilisés avant leur utilisation. La procédure d&#39;implantation sont effectués comme suit: Rat sont anesthésiés à l&#39;isoflurane (2,5-3%) en utilisant une chambre d&#39;induction. L&#39;exposition humaine à l&#39;isoflurane peut être réduit en travaillant dans une table aspirante ou non-recirculation capot. La température corporelle est maintenue pendant l&#39;intervention chirurgicale. <ol><li> Rasez la zone abdominale et d&#39;appliquer une pommade oculaire pour éviter les yeux de sécher pendant l&#39;anesthésie. </li><li> Placez le rat sur son dos et placer un masque sur sa bouche et le nez pour maintenir l&#39;anesthésie. </li><li> Désinfectez la zone abdominale à l&#39;aide Bétadine puis 80% d&#39;éthanol. Swab de nettoyage de salir, de centre de la zone rasée déplaçant vers l&#39;extérieur vers la zone de cheveux. </li><li> Vérifiez réflexes pinçant les pattes postérieures pour être sûr que le rat est suffisamment anesthésiés. </li><li> Drapé de l&#39;animal et d&#39;effectuer une incision abdominale médiane séparant la peau et de muscle en deux étapes pour ouvrir le ventre. La stérilisation à chaud billes est utilisé entre la peau et l&#39;ouverture du muscle. </li><li> Placez les intestins dans un gant de poudre de sérum physiologique réchauffé hydratée libre. Pliez le gant autour de l&#39;intestin pour empêcher la perte d&#39;humidité. </li><li> Enlever les tissus adipeux et exposer la rate. </li><li> Visualisez et la propagation grand omentum avec une pince. </li><li> Découpez soigneusement l&#39;ISE de suspendre les messages de silicone et de transfert sur le grand épiploon. </li><li> Enveloppez la plus grande autour de la omentus ISE et de fixer l&#39;aide de deux simples sutures 7-0 Prolène (Ethicon, Norderstedt, Allemagne) </li><li> Suivant déplacer les intestins dans l&#39;abdomen. </li><li> Rincer l&#39;abdomen avec une solution saline stérile préchauffée. </li><li> Fermez la couche musculaire de la paroi abdominale en utilisant 6-0 prolène sutures course (Ethicon, Norderstedt, Allemagne). </li><li> Utilisez 5-0 Prolène (Ethicon, Norderstedt) fonctionnant sutures pour refermer la peau. </li><li> Alors que le rat est encore en anesthésie, injecter 5 mg / kg carprofène sous-cutanée. </li><li> Pour fournir une analgésie suffisante pour ce type de procédure, un analgésique (comme Metamizol) est ajouté à l&#39;eau potable (50 mg par 100 ml Metamizol) pour des analgésiques pour 3 jours après la transplantation. </li></ol> 3. Imagerie par bioluminescence de suite à l&#39;implantation d&#39;ISE La plate-forme bio-imagerie IVIS (Xenogen, Alameda, CA, USA) est utilisé pour l&#39;imagerie par bioluminescence non-invasif in vivo et les résultats sont analysés en utilisant l&#39;image vivante IVIS (Xenogen) logiciel. L&#39;imagerie est effectuée quotidiennement à partir de 2 heures après l&#39;opération. Anesthetize rat avec l&#39;isoflurane (2,5 - 3%) en utilisant une chambre d&#39;induction. <ol><li> Désinfectez la zone abdominale et thoracique en utilisant largement Provo-iode, la prochaine utilisation d&#39;éthanol à 80%, répétez cette étape trois fois. </li><li> Injecter D-luciférine (Xenogen; 375 mg / kg) ip </li><li> Placez rats anesthésiés dans la chambre d&#39;imagerie IVIS (jusqu&#39;à 4 rats peuvent être analysés en même temps). </li><li> Évaluer l&#39;intensité du signal de luc + cellules à l&#39;intérieur d&#39;ISE dans les unités de photons / sec / cm 2 / steridianin la région d&#39;intérêt (ROI) au départ (120 minutes après l&#39;implantation) et aux jours 1, 2, 3, 5, 7, 10, et aux semaines 2, 3 et 4. Notez le signal de crête (apparaît environ 20 minutes après l&#39;injection D-luciférine). </li></ol> Créer un graphique (Excel) montrant l&#39;intensité du signal dans le temps. 4. Corrélation des résultats BLI et ELISPOT Le cellulaire in vivo des réponses immunitaires chez BN immunocompétents et immunodéprimés rats nus ont été étudiés après la transplantation d&#39;ISE. La rate des animaux receveurs a été récolté 5 jours après la transplantation d&#39;ISE pour isoler splénocytes destinataire. Tests ELISPOT en utilisant la mitomycine-inhibée HASTI (Sigma Aldrich, St. Louis, MO, USA) comme stimulateur et 5 splénocytes x10 bénéficiaires 6 comme cellules répondeuses ont été effectués selon le protocole du fabricant (BD Biosciences) en utilisant l&#39;IFN-γ et IL-4 revêtement plaques d&#39;enquêter TH1, TH2 et la réponse, respectivement. IFN-γ et IL-4 spots ont été significativement plus élevé dans le immunocompétents, le modèle par rapport au modèle immunodéficientes (IFN-γ: p = 0,001; IL-4: p = 0,023; t de Student-test). 5. Les résultats représentatifs: <img src="/files/ftp_upload/2605/2605fig1.jpg" alt="Figure 1" /> Figure 1. Corrélation des résultats BLI et ELISPOT a) Le cellulaire in vivo des réponses immunitaires chez immunocompétents Brown Norvège et immunodéficientes rats nus ont été étudiés après l&#39;implantation d&#39;ISE. Récipiendaire splénocytes ont été récoltés 5 jours après l&#39;implantation d&#39;ISE. IFN? et IL-4 d&#39;expression a été évaluée par tests ELISPOT en utilisant la mitomycine-inhibée ISE comme stimulateurs et 5x106 splénocytes destinataire comme cellules répondeuses. Th1 et Th2 étaient significativement plus élevés dans le modèle immunocompétentes par rapport au modèle immunodéficientes comme en témoigne IFNy et IL-4, respectivement. b) Luc HASTI + ont été transplantées dans plus de la omentus des rats BN soit immunocompétents ou immunodéprimés rats nus. La survie des cellules a été suivie par BLI longitudinalement. Le taux d&#39;animaux qui a rejeté l&#39;HASTI transplanté est présenté. Tous les rats BN rapidement rejeté les greffes de l&#39;ISE, alors que les cellules ont survécu dans des rats de cellules T déficientes.

<ol>
	<li>Eschenhagen, T., Zimmermann, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+myocardial+tissue.">Engineering myocardial tissue.</a> Circ Res. 97, 1220-1231 (2005).</li><li>van der Bogt, K. E., Schrepfer, S., Yu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+transplantation+of+adipose+tissue-+and+bone+marrow-derived+mesenchymal+stem+cells+in+the+infarcted+heart.">Comparison of transplantation of adipose tissue- and bone marrow-derived mesenchymal stem cells in the infarcted heart.</a> Transplantation. 87, 642-652 (2009).</li><li>Hakamata, Y., Murakami, T., Kobayashi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Firefly+rats"+as+an+organ/cellular+source+for+long-term+in+vivo+bioluminescent+imaging.">"Firefly rats" as an organ/cellular source for long-term in vivo bioluminescent imaging.</a> Transplantation. 81, 1179-1184 (2006).</li><li>Hansen, A., Eder, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+Drug+Screening+Platform+Based+on+Engineered+Heart+Tissue.">Development of a Drug Screening Platform Based on Engineered Heart Tissue.</a> Circ Res. , (2010).</li></ol>

Avant la mise en œuvre clinique de la technologie d&#39;ISE pour la réparation cardiaque, des questions fondamentales telles que l&#39;immunogénicité de deux composants cellulaires et de la matrice ou de la survie du greffon après l&#39;implantation doivent être évalués dans des modèles expérimentaux. Imagerie par bioluminescence in vivo représente une nouvelle technique utile pour la survie des cellules de suivi après la transplantation . Nous avons réussi à généré à base de fibrine ISE conte...

<ul><li> LEW-Tg (Gt (ROSA) 26Sor-Luc) 11Jmsk rat (Rat Nombre NBPR 0299, http://www.anim.med.kyoto-u.ac.jp/NBR/) </li><li> Fibrinogène bovin (solution stock: 200 mg / ml, plus aprotinine 5 ug / ml dans le fibrinogène NaCl 0,9%, Sigma F4753) </li><li> Matrigel (BD Bioscience 356235) </li><li> DMEM (Biochrome F0415) </li><li> Sérum de cheval (Gibco 26050) </li><li> Pénicilline / streptomycine (Gibco 15140) </li><li> L&#39;insuline (Sigma-Aldrich I9278) </li><li> L&#39;aprotinine (Sigma Aldrich A1153) </li><li> D-luciférine (Biosynth L-8220) </li><li> Immuno-enzymatique plaques spot test (ELISpot) (Hassa Laborbedarf, S2EM004M99) </li></ul>

bioluminescence imaging for assessment of immune responses following implantation of engineered heart tissue (eht)

Diverses techniques d&#39;ingénierie tissulaire cardiaque ont été poursuivis dans les dernières décennies, y compris les stratégies d&#39;échafaudage en utilisant soit natif ou bioartificiel matériaux d&#39;échafaudage, piégeage des myocytes cardiaques dans les hydrogels tels que la fibrine ou de collagène et l&#39;empilement des monocouches myocytes 1. Ces concepts visent à la restauration de la fonction cardiaque compromise (par exemple après un infarctus du myocarde) ou comme modèles expérimentaux (par exemple, la toxicologie prédictive et le dépistage de substances ou de modélisation de la maladie). Un suivi précis de la survie des cellules après l&#39;implantation du tissu cardiaque d&#39;ingénierie (ISE) est désormais possible en utilisant la bioluminescence in vivo d&#39;imagerie (BLI) des techniques 2. Nous décrivons ici la génération de base de fibrine ISE partir d&#39;une souche de rats transgéniques avec expression ubiquitaire de la luciférase de luciole (ROSA / luciférase-LEW Tg; 3). L&#39;implantation est réalisée dans le grand omentum des différentes souches de rats afin d&#39;évaluer la réponse immunitaire de l&#39;organisme receveur après l&#39;implantation d&#39;ISE. Comparaison des résultats générés par BLI et l&#39;enzyme liée Technique spot Immuno (ELISPOT) confirment l&#39;utilité de BLI pour l&#39;évaluation des réponses immunitaires.

Watch this Scientific Journal Video about Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT at JoVE.com

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT

Cette vidéo montre l&#39;utilisation de<em&gt; In vivo</emL&#39;imagerie par bioluminescence&gt; pour étudier les réponses immunitaires après l&#39;implantation du tissu cardiaque d&#39;ingénierie (ISE) chez le rat.

Imagerie par bioluminescence pour l&#39;évaluation des réponses immunitaires après l&#39;implantation du tissu cardiaque d&#39;ingénierie (ISE)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or ...

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Research

JoVE Journal

Bioengineering

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

9.6K vues.  University Heart Center Hamburg. Cette vidéo montre l'utilisation de In vivo

Vidéo: Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

Méthodologie élastographie par résonance magnétique pour l&#39;évaluation des tissus Conçu croissance Construct

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

Recherche

Bio-ingénierie

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of tri-leaflet polymer valve prostheses. However, identification of a protocol for initial assessment of polymer valve hydrodynamic functionality is paramount during the early stages of the design process. Traditional in vitro pulse duplicator systems are not configured to accommodate flexible tri-leaflet materials; in addition, assessment of polymer valve functionality needs to be made in a relative context to native and prosthetic heart valves under identical test conditions so that variability in measurements from different instruments can be avoided. Accordingly, we conducted hydrodynamic assessment of i) native (n = 4, mean diameter, D = 20 mm), ii) bi-leaflet mechanical (n= 2, D = 23 mm) and iii) polymer valves (n = 5, D = 22 mm) via the use of a commercially available pulse duplicator system (ViVitro Labs Inc, Victoria, BC) that was modified to accommodate tri-leaflet valve geometries. Tri-leaflet silicone valves developed at the University of Florida comprised the polymer valve group. A mixture in the ratio of 35:65 glycerin to water was used to mimic blood physical properties. Instantaneous flow rate was measured at the interface of the left ventricle and aortic units while pressure was recorded at the ventricular and aortic positions. Bi-leaflet and native valve data from the literature was used to validate flow and pressure readings. The following hydrodynamic metrics were reported: forward flow pressure drop, aortic root mean square forward flow rate, aortic closing, leakage&#160;and regurgitant volume, transaortic closing, leakage, and total energy losses. Representative results indicated that hydrodynamic metrics from the three valve groups could be successfully obtained by incorporating a custom-built assembly into a commercially available pulse duplicator system and subsequently, objectively compared to provide insights on functional aspects of polymer valve design.

There has been renewed interest in developing polymer valves. Here, the objectives are to demonstrate the feasibility of modifying a commercial pulse duplicator to accommodate tri-leaflet geometries and to define a protocol to present polymer valve hydrodynamic data in comparison to native and prosthetic valve data collected under near-identical conditions.

Protocole pour l&#39;évaluation hydrodynamique relative de Vannes polymères tri-leaflet

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

A step-by-step protocol for the inter-positional placement of Tissue Engineered Vessels (TEVs) into the carotid artery of a sheep using end-to-end anastomosis and real-time digital assessment in vivo until animal sacrifice.

Technique chirurgicale pour l&#39;implantation de l&#39;ingénierie tissulaire vasculaires greffes et suivantes<em&gt; In Vivo</em&gt; Surveillance

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

Modèle de polarisée Neural Tissue base-Silk-collagène ingénierie 3D

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate engineered heart tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 106/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human heart, indicating it to be a promising tool for cardiac drug safety screening.

Here, we show the generation of human engineered heart tissue from induced pluripotent stem cells (hiPSC)-derived cardiomyocytes. We present a method to analyze contraction force and exemplary alteration of contraction pattern by the hERG channel inhibitor E-4031. This method shows high level of robustness and suitability for cardiac drug screening.

Automatisé Contraction L&#39;analyse des tissus humains Engineered cardiaque pour le dépistage de la sécurité cardiaque médicaments

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

Custom Engineered Tissue Culture Molds from Laser-etched Masters

As the field of tissue engineering has continued to mature, there has been increased interest in a wide range of tissue parameters, including tissue shape. Manipulating tissue shape on the micrometer to centimeter scale can direct cell alignment, alter effective mechanical properties, and address limitations related to nutrient diffusion. In addition, the vessel in which a tissue is prepared can impart mechanical constraints on the tissue, resulting in stress fields that can further influence both the cell and matrix structure. Shaped tissues with highly reproducible dimensions also have utility for in vitro assays in which sample dimensions are critical, such as whole tissue mechanical analysis.
This manuscript describes an alternative fabrication method utilizing negative master molds prepared from laser etched acrylic: these molds perform well with polydimethylsiloxane (PDMS), permit designs with dimensions on the centimeter scale and feature sizes smaller than 25 &#181;m, and can be rapidly designed and fabricated at a low cost and with minimal expertise. The minimal time and cost requirements allow for laser etched molds to be rapidly iterated upon until an optimal design is determined, and to be easily adapted to suit any assay of interest, including those beyond the field of tissue engineering.

Herein we present a rapid, facile, and low-cost method for fabricating custom polydimethylsiloxane molds that can be used for producing hydrogel-based engineered tissues with complex geometries. We additionally describe results from mechanical and histological assessments conducted on engineered cardiac tissues produced using this technique.

Coutume machinée moules de vitroplants de maîtres gravé au Laser

As the field of tissue engineering has continued to mature, there has been increased interest in a wide range of tissue parameters, including tissue ...

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation.

Ensemencement et Implantation d’un greffon biosynthétique de trachéale tissulaire dans un modèle murin

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant technique with nutritional support. Recently, there have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in early epithelization in the special environment of peristalsis. Here, we developed an artificial esophagus that can improve the regeneration of the esophageal mucosa and muscle layers through a two-layered tubular scaffold, a mesenchymal stem cell-based bioreactor system, and a bypass feeding technique with modified gastrostomy. The scaffold is made of polyurethane (PU) nanofibers in a cylindrical shape with a three-dimensional (3D) printed polycaprolactone strand wrapped around the outer wall. Prior to transplantation, human-derived mesenchymal stem cells were seeded into the lumen of the scaffold, and bioreactor cultivation was performed to enhance cellular reactivity. We improved the graft survival rate by applying surgical anastomosis and covering the implanted prosthesis with a thyroid gland flap, followed by temporary nonoral gastrostomy feeding. These grafts were able to recapitulate the findings of initial epithelialization and muscle regeneration around the implanted sites, as demonstrated by histological analysis. In addition, increased elastin fibers and neovascularization were observed in the periphery of the graft. Therefore, this model presents a potential new technique for circumferential esophageal reconstruction.

Esophageal reconstruction is a challenging procedure, and development of a tissue-engineered esophagus that enables regeneration of esophageal mucosa and muscle and that can be implanted as an artificial graft is necessary. Here, we present our protocol to generate an artificial esophagus, including scaffold manufacturing, bioreactor cultivation, and various surgical techniques.

Greffe tissulaire pour la reconstruction oesophagielle circonférence chez les rats

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant ...

Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from human induced pluripotent stem cells (hiPSCs), which are developed towards the goal of clinical use. HiPSC-derived cardiomyocytes, endothelial cells, and vascular mural cells are mixed with gel matrix and then poured into a polydimethylsiloxane (PDMS) tissue mold with rectangular internal staggered posts. By culture day 14 ECTs mature into a 1.5 cm x 1.5 cm mesh structure with 0.5 mm diameter myofiber bundles. Cardiomyocytes align to the long-axis of each bundle and spontaneously beat synchronously. This approach can be scaled up to a larger (3.0 cm x 3.0 cm) mesh ECT while preserving construct maturation and function. Thus, mesh-shaped ECTs generated from hiPSC-derived cardiac cells may be feasible for cardiac regeneration paradigms.

The present protocol generates mesh-shaped engineered cardiac tissues containing cardiovascular cells derived from human induced pluripotent stem cells to allow the investigation of cell implantation therapy for heart diseases.

Préparation de tissus cardiaques en forme de maille dérivés de cellules iPS humaines pour la réparation myocardique in vivo

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from ...

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.