Sonja Schrepfer, Lenard Conradi und Arne Hansen erhielt finanzielle Unterstützung von der Deutschen Forschungsgemeinschaft (DFG; SCHR992/3-1, SCHR992/4-1, CO 858/1-1, HA 3423/3-1). Die Arbeit wurde auch durch Zuschüsse der Europäischen Union zu Thomas Eschenhagen (EUGeneHeart und Angioscaff) unterstützt.

1. EHT-Generation und Kulturbedingungen Herstellung von Fibrin-basierten EHT erfolgte wie an anderer Stelle beschrieben 4. In kurzen, an Fibrin-basierten EHT für die Implantation Zwecke zu generieren, wurde ein Master-Mix hergestellt, die Zellen von Neugeborenen ROSA / Luciferase-LEW transgenen Rattenherzen, Rinder-Fibrinogen und Matrigel isoliert. Agarose Gussformen wurden unter Verwendung von Teflon Abstandshalter in 6-well Kulturschalen gelegt. Nach dem Erstarren der Agarose wurden Abstandhalter entfernt und Silikon-Post-Racks wurden auf Kulturschalen mit einem Thema bis in die Formen gelegt. Zur Erzeugung von EHT, war der Mastermix kurz mit einer berechneten Menge von Thrombin gemischt und pipettiert in die Form. Nach der Polymerisation von Fibrinogen, wurden Silikon-Racks mit EHTs Einhaltung der Beiträge vorsichtig aus dem Agarose-Gießformen, die mit neuen 6-well Zellkulturschalen und in einer 37 ° C, 7% CO 2 Zellkulturbrutschrank entfernt mit maßgeschneiderten Zellkulturmedium. EHT wurden unter Zellkultur-Bedingungen bis zum zehnten Tag gehalten. Während dieser Zeitspanne begann neugeborenen Ratten Herzzellen zu langgestreckten, Interconnect und richten entlang Kraftlinien zwischen Silikon-Beiträge. Zum Zeitpunkt der Implantation wurden spontane kohärente Kontraktionen beobachtet, Ablenkung Silikon Beiträge. 2. EHT Implantation EHT-Implantation war in beiden immunkompetenten Brown Norway (BN) Ratten oder immundefizienten Nacktratten durchgeführt, um die allogene Immunantwort zu untersuchen. BN und nackte Ratten mit einem Gewicht von 100 bis 150 g wurden von Charles River Deutschland (Sandhofer Weg 7, D-97633 Sulzfeld) erworben und beherbergte unter üblichen Bedingungen, gefüttert Standard Rattenfutter und Wasser ad libidum. Immundefizienten Ratten sind in sterilen Betten, Käfige und keimfreies Wasser verwendet untergebracht. Die deutsche Ethikkommission geprüft und genehmigt das Tier Verfahren. Alle chirurgischen Instrumente sind vor dem Gebrauch sterilisiert. Die Implantation sind wie folgt durchgeführt: Rat sind mit Isofluran (2.5-3%) mit einer Induktion Kammer narkotisiert. Exposition des Menschen gegenüber Isofluran kann durch Arbeiten in einem Fallstrom Tabelle oder Nicht-Rückführung Motorhaube reduziert werden. Die Körpertemperatur ist während des chirurgischen Eingriffs beibehalten. <ol><li> Shave Bauchbereich und gelten Augensalbe auf die Augen vor dem Austrocknen während der Narkose zu verhindern. </li><li> Legen Sie die Ratte auf den Rücken und legen Sie eine Gesichtsmaske über seinen Mund und Nase zu halten die Anästhesie. </li><li> Desinfizieren Sie den Bauchbereich mit Betadine und dann 80% Ethanol. Swab von Clean bis schmutzig, von der Mitte des rasierten Bereich bewegt nach außen in Richtung der behaarten Bereich. </li><li> Überprüfen Reflexe kneifen die Hinterfüße sicher sein, dass die Ratte ausreichend betäubt ist. </li><li> Hängen Sie die Tiere und führen Sie eine Mittellinie Bauchschnitt Trennung der Haut und Muskeln in zwei Schritten auf den Bauch zu öffnen. Hot bead Sterilisation zwischen Haut und Muskel Öffnung verwendet. </li><li> Setzen Sie den Darm in einer angewärmten Kochsalzlösung befeuchtet puderfrei Handschuhe. Klappen Sie den Handschuh um den Darm, um den Verlust von Feuchtigkeit zu verhindern. </li><li> Entfernen Fettgewebe und setzen die Milz. </li><li> Visualisieren und breitete das große Netz mit einer Pinzette. </li><li> Schneiden Sie vorsichtig EHT vom Aussetzung Silikon Beiträge und Übertragung auf das große Netz. </li><li> Wickeln Sie das größere omentus rund um die EHT und fix mit zwei Einzelbetten 7-0 Prolene Fäden (Ethicon, Norderstedt, Deutschland) </li><li> Nächstes verschieben Sie den Darm wieder in den Bauch. </li><li> Spülen Sie den Bauch mit vorgewärmtem steriler Kochsalzlösung. </li><li> Schließen Sie die Muskelschicht der Bauchwand mit 6-0 Prolene fortlaufenden Nähten (Ethicon, Norderstedt, Deutschland). </li><li> Verwenden Sie 5-0 Prolene (Ethicon, Norderstedt, Deutschland) läuft Nähte auf der Haut zu schließen. </li><li> Während die Ratte immer noch in Narkose, injizieren 5 mg / kg Carprofen subkutan. </li><li> Um eine ausreichende Analgesie für diese Art von Verfahren, ein Analgetikum (z. B. Metamizol) ist mit dem Trinkwasser (50 mg Metamizol pro 100 ml) für Schmerzmittel für 3 Tage nach der Transplantation gegeben sind. </li></ol> 3. Biolumineszenz-Bildgebung des EHT nach der Implantation Die IVIS Bioimaging Plattform (Xenogen, Alameda, CA, USA) ist für nicht-invasive Biolumineszenz-Bildgebung in vivo verwendet und die Ergebnisse werden mit Hilfe der IVIS Wohnen Image (Xenogen) Software-Paket. Imaging durchgeführt wird täglich ab 2 Stunden postoperativ. Anesthetize Ratte mit Isofluran (2,5 bis 3%) mit einer Induktion Kammer. <ol><li> Desinfizieren Sie die Bauch-und Brustbereich weit über Provo-Jod, nächsten Einsatz 80% Ethanol, wiederholen Sie diesen Schritt dreimal. </li><li> Inject d-Luciferin (Xenogen; 375 mg / kg Körpergewicht) ip </li><li> Legen Sie narkotisierten Ratten in die IVIS Bildgebung Kammer (bis zu 4 Ratten können gleichzeitig analysiert werden). </li><li> Beurteilen Signalintensität luc + Zellen innerhalb EHT in Einheiten von Photonen / sec / cm 2 / steridianin der region of interest (ROI) zu Studienbeginn (120 Minuten nach der Implantation) und an den Tagen 1, 2, 3, 5, 7, 10, und in den Wochen 2, 3 und 4. Beachten Sie die Peak-Signal (erscheint ca. 20 Minuten nach dem D-Luciferin-Injektion). </li></ol> Erstellen Sie eine Grafik (Excel) zeigt die Signalstärke über die Zeit. 4. Korrelation von BLI Ergebnisse und ELISPOT Die zellulären in-vivo Immunantworten in immunkompetenten BN und immundefizienten nude Ratten wurden nach EHT Transplantation untersucht. Die Milz des Empfängers Tieren geerntet wurde 5 Tage nach EHT Transplantation Empfänger Splenozyten zu isolieren. Elispot Assays unter Verwendung von Mitomycin-gehemmte EHTs (Sigma Aldrich, St. Louis, MO, USA) als Stimulator und 5 x10 6 Empfänger Splenozyten als Responder-Zellen nach dem Protokoll des Herstellers (BD Biosciences) mit IFN-γ und IL-4 beschichtete durchgeführt wurden Platten zu untersuchen, TH1-und TH2-Antwort, bzw.. IFN-γ-und IL-4-Spots waren signifikant höher in der immunkompetenten, Modell im Vergleich zu den immundefizienten Modell (IFN-γ: p = 0,001; IL-4: p = 0,023; Student-t-Test). 5. Repräsentative Ergebnisse: <img src="/files/ftp_upload/2605/2605fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Korrelation von BLI Ergebnisse und ELISPOT a) Die zellulären in vivo Immunantworten in immunkompetenten Brown Norwegen und immundefizienten nude Ratten wurden nach EHT-Implantation untersucht. Empfänger Splenozyten wurden 5 Tage nach der Implantation EHT geerntet. IFN? und IL-4-Expression wurde durch ELISPOT-Assays mit Mitomycin-gehemmte EHT als Stimulatoren und 5x106 Empfänger Splenozyten als Responder-Zellen ausgewertet. Th1 und Th2-Antworten waren signifikant höher in der immunkompetenten Modell im Vergleich zu den immundefizienten Modell als durch IFNy-und IL-4 Produktion bzw. belegt. b) Luc + EHTs wurden in den größeren omentus entweder immunkompetenten BN Ratten oder immundefizienten nude Ratten transplantiert. Überleben der Zellen wurde in Längsrichtung durch BLI gefolgt. Die Rate der Tiere, dass die transplantierten EHTs abgelehnt wird vorgestellt. Alle BN Ratten schnell wies die EHT-Transplantationen, während die Zellen überlebten in T-Zell-defizienten Ratten.

<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>

Bevor klinische Umsetzung der EHT-Technologie für die Herz-Reparatur, müssen grundlegende Fragen wie die Immunogenität der beiden Mobilfunk-und Matrix-Komponenten oder Transplantat-Überlebens nach der Implantation in experimentellen Modellen bewertet werden. In-vivo-Biolumineszenz-Bildgebung stellt eine nützliche neue Technik für die Überwachung der das Überleben der Zelle nach der Transplantation . Wir haben erfolgreich Fibrin-basierten EHT generiert mit Luciferase positive (luc +) Herzzellen durch Anp...

<ul><li> LEW-Tg (Gt (ROSA) 26Sor-luc) 11Jmsk Ratten (NBPR Ratte Nummer 0299, http://www.anim.med.kyoto-u.ac.jp/NBR/) </li><li> Bovine Fibrinogen (Stammlösung: 200 mg / ml plus Aprotinin 5 ug / ml Fibrinogen in NaCl 0,9%, Sigma F4753) </li><li> Matrigel (BD Bioscience 356235) </li><li> DMEM (Biochrome F0415) </li><li> Pferdeserum (Gibco 26050) </li><li> Penicillin / Streptomycin (Gibco 15140) </li><li> Insulin (Sigma-Aldrich I9278) </li><li> Aprotinin (Sigma Aldrich A1153) </li><li> d-Luciferin (Biosynth L-8220) </li><li> Enzyme-Linked Immuno Spot (ELISPOT) Assay-Platten (Hassa Laborbedarf, S2EM004M99) </li></ul>

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

Verschiedene Techniken der kardialen Tissue Engineering haben in den vergangenen Jahrzehnten auch Gerüste Strategien entweder nativ oder bioartifiziellen Gerüstmaterial, Einschluss von Herzmuskelzellen in Hydrogele wie Fibrin oder Kollagen und Stapeln von Myozyten Monoschichten dem 1. verfolgt. Diese Konzepte zielen auf die Wiederherstellung der Herzfunktion (z. B. nach Myokardinfarkt) oder als experimentelle Modelle (zB zur prädiktiven Toxikologie und Wirkstoff-Screening oder Krankheit Modellierung). Präzise Überwachung der das Überleben der Zelle nach Implantation von Engineered Heart Tissue (EHT) ist inzwischen möglich, mit in-vivo Biolumineszenz-Bildgebung (BLI) Techniken 2. Hier beschreiben wir die Erzeugung von Fibrin-basierten EHT aus einem transgenen Ratten-Stamm mit ubiquitäre Expression von Luciferase (ROSA / Luciferase-LEW Tg, 3). Die Implantation ist in das große Netz verschiedener Zuchtlinien, um Immunreaktionen des Empfängerorganismus folgenden EHT Implantation beurteilen durchgeführt. Ein Vergleich der Ergebnisse von BLI und die Enzyme Linked Immuno Spot Technique (ELISPOT) bestätigen die Verwendbarkeit von BLI für die Beurteilung der Immunantwort generiert.

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

Dieses Video demonstriert die Verwendung von<em&gt; In vivo</em&gt; Biolumineszenz-Bildgebung zu Immunreaktionen nach Implantation von Engineered Heart Tissue (EHT) in Ratten zu untersuchen.

Biolumineszenz-Bildgebung für die Beurteilung von Immunantworten nach der Implantation von Engineered Heart Tissue (EHT)

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

bioluminescence-imaging-for-assessment-immune-responses-following

Research

JoVE Journal

Bioengineering

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

9.6K Aufrufe.  University Heart Center Hamburg. Dieses Video demonstriert die Verwendung von In vivo Biolumineszenz-Bildgebung zu Immunreaktionen nach Implantation von Engineered Heart Tissue (EHT) in Ratten zu untersuchen.

Serie: 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).

Magnet-Resonanz-Elastographie Methodik für die Beurteilung von Tissue Engineering gewonnenen Konstrukts Wachstum

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

Forschung

Biotechnik

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.

Protokoll für Relative Hydrodynamische Beurteilung der Tri-Polymer Merkblatt Ventile

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.

Operationstechnik zur Implantation von Tissue Engineered Gefäßprothesen und Folge<em&gt; In-vivo-</em&gt; Überwachung

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.

Engineered 3D Silk-Kollagen-basierten Modell von polarisiertem 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 ...

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.

Automatische Kontraktions Analyse menschlicher Engineered Herzgewebe für Herz Drug Safety Screening

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.

Custom-Engineered Tissue Culture Formen von gelasert meistern

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.

Aussaat und Implantation von einem biosynthetischen Tissue-Engineering trachealen Transplantat in einem Mausmodell

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.

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.

Vorbereitung von Mesh-Shaped Engineered Cardiac Tissues derived from Human iPS Cells for In Vivo Myokard repair

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.