Sonja Schrepfer, Lenard Conradi e Arne Hansen ha ricevuto un finanziamento dalla Deutsche Forschungsgemeinschaft (DFG, SCHR992/3-1, SCHR992/4-1, CO 858/1-1, HA 3423/3-1). Il lavoro è stato sostenuto anche da finanziamenti dell&#39;Unione europea di Thomas Eschenhagen (EUGeneHeart e Angioscaff).

1. EHT Generation e la cultura Condizioni Fabbricazione di prodotti a base di fibrina EHT è stata eseguita come descritto altrove 4. In breve, per generare fibrina basato EHT per scopi impianto, una master mix è stato preparato contenente cellule isolate da neonatale ROSA / luciferasi-LEW cuori di ratto transgenico, fibrinogeno bovina e Matrigel. Agarosio stampi sono stati preparati con distanziali in teflon posto in ben 6 piatti della cultura. Dopo la solidificazione di agarosio, distanziatori sono stati rimossi e rastrelliere messaggio silicone sono stati messi sui piatti della cultura con messaggi raggiungendo negli stampi. Per generare EHT, il Mastermix fu brevemente mescolato con un importo calcolato della trombina e pipettati nello stampo. Dopo la polimerizzazione del fibrinogeno, rack in silicone con EHTs aderendo alla posti sono stati delicatamente rimossi dalla stampi l&#39;agarosio, trasferito al nuovo cambio a 6 e piatti di coltura cellulare e mantenuto a 37 ° C, 7% di CO 2 colture cellulari incubatore usando su misura terreno di coltura delle cellule. EHT sono stati tenuti in condizioni di coltura delle cellule fino a dieci giorni. Durante questo lasso di tempo, neonatale cellule del cuore di ratto cominciò a forma allungata, di interconnessione e allineare lungo linee di forza tra i messaggi di silicone. Al momento dell&#39;impianto, spontanea coerente contrazioni sono state osservate, deviando i messaggi di silicone. 2. EHT impianto EHT impianto è stato eseguito in entrambi i immunocompetenti Brown Norway (BN), ratti o topi immunodeficienti nudo per studiare la risposta immunitaria allogenico. Ratti BN e nudo peso di 100 - 150 g sono stati acquistati dalla Charles River Germania (Sandhofer Weg 7, D-97633 Sulzfeld) e alloggiati in condizioni convenzionali, alimentati standard di ratto chow e libidum ad acqua. Topi immunodeficienti sono ospitati, in camera sterile, gabbie, e viene utilizzata acqua sterilizzata. Il comitato etico tedesco ha esaminato e approvato le procedure animali. Tutti gli strumenti chirurgici vengono sterilizzati prima dell&#39;uso. La procedura di impianto vengono effettuate come segue: Rat sono anesthesized con isoflurano (2,5-3%) con una camera di induzione. L&#39;esposizione umana a isoflurano può essere ridotto di lavoro in una tabella rovesciati o non ricircolo cappuccio. La temperatura corporea è mantenuta durante la procedura chirurgica. <ol><li> Shave zona addominale e applicare una pomata oculare per evitare che gli occhi si secchi durante l&#39;anestesia. </li><li> Posizionare il topo sul dorso e posto una maschera sul suo naso e la bocca per mantenere l&#39;anestesia. </li><li> Disinfettare la zona addominale con Betadine e poi a 80% di etanolo. Tampone pulito da sporcare, dal centro dell&#39;area rasata in movimento verso l&#39;esterno verso l&#39;area dai capelli. </li><li> Controllare riflessi pizzicare i piedi posteriori per essere sicuri che il topo è sufficientemente anestetizzati. </li><li> Drape l&#39;animale ed effettuare una incisione addominale mediana che separa la pelle ei muscoli in due fasi per aprire l&#39;addome. Sterilizzazione a caldo tallone viene utilizzato tra la pelle e l&#39;apertura del muscolo. </li><li> Posto l&#39;intestino in un guanto salina riscaldata idratata in polvere libera. Piegare il guanto intorno l&#39;intestino per prevenire la perdita di umidità. </li><li> Rimuovere il tessuto adiposo ed esporre la milza. </li><li> Visualizzare e maggiore diffusione omento con una pinza. </li><li> Accuratamente tagliato EHT di sospendere i messaggi in silicone e di trasferimento nel omento maggiore. </li><li> Avvolgere maggiore è la omentus intorno al EHT e fissare con due singoli 7-0 suture prolene (Ethicon, Norderstedt) </li><li> Avanti spostare l&#39;intestino nuovamente dentro l&#39;addome. </li><li> Lavare l&#39;addome preriscaldata con soluzione salina sterile. </li><li> Chiudere lo strato muscolare della parete addominale con suture 6-0 prolene esecuzione (Ethicon, Norderstedt, Germania). </li><li> Utilizzare 5-0 prolene (Ethicon, Norderstedt) in esecuzione suture per chiudere la pelle. </li><li> Mentre il topo è ancora in anestesia, iniettare 5 mg / kg per via sottocutanea Carprofen. </li><li> Per fornire l&#39;analgesia sufficiente per questo tipo di procedura, un analgesico (come Metamizol) viene aggiunto all&#39;acqua potabile (50 mg Metamizol per 100 ml) per farmaci antidolorifici per 3 giorni post-trapianto. </li></ol> 3. Imaging bioluminescenza di EHT impianto seguito La piattaforma di bioimmagini IVIS (Xenogen, Alameda, CA, USA) è usato per imaging non invasiva bioluminescenza in vivo ei risultati sono analizzati utilizzando l&#39;immagine vivente IVIS (Xenogen), pacchetto software. Imaging viene effettuata ogni giorno dalle 2 ore dopo l&#39;intervento. Anestetizzare ratto con isoflurano (2,5 - 3%) con una camera di induzione. <ol><li> Disinfettare la zona addominale e toracica utilizzando ampiamente Provo-Iodio, utilizzare prossimi 80% di etanolo, ripetere questa operazione tre volte. </li><li> Iniettare d-Luciferin (Xenogen; 375 mg / kg di peso corporeo) ip </li><li> Luogo in topi anestetizzati l&#39;imaging IVIS camera (fino a 4 ratti possono essere analizzati allo stesso tempo). </li><li> Valutare l&#39;intensità del segnale di luc + cellule all&#39;interno EHT in unità di fotoni / sec / cm 2 / steridianin la regione di interesse (ROI) al basale (120 minuti dopo l&#39;impianto) e nei giorni 1, 2, 3, 5, 7, 10, e alle settimane 2, 3 e 4. Si noti il ​​picco del segnale (sembra circa 20 minuti dopo il d-Luciferin iniezione). </li></ol> Creare un grafico (Excel) che mostra l&#39;intensità del segnale nel tempo. 4. Correlazione dei risultati BLI e ELISPOT Le risposte immunitarie cellulari in-vivo in BN immunocompetenti e immunodeficienti topi nudi sono stati studiati dopo EHT trapianto. La milza di animali destinatario è stato raccolto 5 giorni dopo il trapianto EHT di isolare splenociti destinatario. ELISPOT con mitomicina-inibito EHTs (Sigma Aldrich, St. Louis, MO, USA) come stimolatore e 5 x10 6 splenociti destinatario come cellule responder sono stati eseguiti secondo il protocollo del produttore (BD Biosciences) con IFN-γ e IL-4 rivestiti piastre di indagare TH1-, e la risposta TH2, rispettivamente. IFN-γ e IL-4 punti erano significativamente più alti nel immunocompetenti, modello rispetto al modello immunodeficienti (IFN-γ: p = 0,001; IL-4: p = 0,023; Student t-test). 5. Rappresentante dei risultati: <img src="/files/ftp_upload/2605/2605fig1.jpg" alt="Figura 1" /> Figura 1. Correlazione dei risultati BLI e ELISPOT a) Il cellulare in vivo delle risposte immunitarie in immunocompetenti Brown Norvegia e topi immunodeficienti nudi sono stati studiati dopo l&#39;impianto EHT. Splenociti destinatario sono state raccolte 5 giorni dopo l&#39;impianto EHT. IFN? e IL-4 l&#39;espressione è stata valutata mediante ELISPOT mitomicina inibito EHT come stimolatori e 5x106 splenociti destinatario come cellule responder. Risposte Th1 e Th2 sono risultati significativamente più elevati nel modello immunocompetenti rispetto al modello immunodeficienti come evidenziato da IFNγ e IL-4 produzione rispettivamente. b) Luc EHTs + sono state trapiantate in maggiore è la omentus delle due ratti BN immunocompetenti o immunodeficienti topi nudi. Sopravvivenza delle cellule in senso longitudinale è stata seguita da BLI. La percentuale di animali che ha respinto il EHTs trapiantato è presentato. Tutti i ratti BN rapidamente respinto l&#39;trapianti EHT, mentre le cellule sopravvissute nei topi cellule T carente.

<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>

Nessun conflitto di interessi dichiarati.

Prima applicazione clinica della tecnologia EHT per la riparazione cardiaca, questioni fondamentali come l&#39;immunogenicità di entrambi i componenti cellulari e la matrice o la sopravvivenza del trapianto dopo l&#39;impianto devono essere valutati in modelli sperimentali. In-vivo di imaging bioluminescenza rappresenta una tecnica nuova e utile per la sopravvivenza delle cellule di monitoraggio dopo il trapianto . Siamo riusciti a base di fibrina generato EHT contenente luciferasi positivo (luc +) le cellule ...

<ul><li> LEW-Tg (Gt (ROSA) 26Sor-luc) 11Jmsk ratti (numero ratto NBPR 0299, http://www.anim.med.kyoto-u.ac.jp/NBR/) </li><li> Fibrinogeno bovina (soluzione madre: 200 mg / ml aprotinina, più 5 mcg / ml fibrinogeno in NaCl 0,9%, Sigma F4753) </li><li> Matrigel (BD Bioscience 356235) </li><li> DMEM (Biochrome F0415) </li><li> Siero di cavallo (Gibco 26050) </li><li> Penicillina / streptomicina (Gibco 15140) </li><li> Insulina (Sigma-Aldrich I9278) </li><li> Aprotinina (Sigma Aldrich A1153) </li><li> d-Luciferin (Biosynth L-8220) </li><li> Enzyme-linked immunosorbent spot (ELISpot) piastre di test (Hassa Laborbedarf, S2EM004M99) </li></ul>

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

Varie tecniche di ingegneria tissutale cardiaco sono stati perseguiti negli ultimi decenni anche le strategie di impalcature utilizzando nativi o bioartificiale materiali patibolo, intrappolamento dei miociti cardiaci in idrogel come la fibrina o collagene e di sovrapposizione dei monostrati miociti 1. Questi concetti mirano al recupero della compromissione della funzione cardiaca (ad esempio dopo infarto miocardico) o come modelli sperimentali (es. tossicologia predittiva e screening sostanza o modelli di malattia). Monitoraggio preciso della sopravvivenza cellulare dopo l&#39;impianto del tessuto cardiaco ingegnerizzati (EHT) è ora diventato possibile usare in vivo bioluminescenza imaging (BLI) Tecniche 2. Qui si descrive la generazione di fibrina a base di EHT da un ceppo di topo transgenico con espressione ubiquitaria luciferasi di lucciola (ROSA / luciferasi-LEW Tg, 3). L&#39;impianto viene eseguito nel omento maggiore di ceppi di topo diversi per valutare le risposte immunitarie dell&#39;organismo ricevente dopo l&#39;impianto EHT. Confronto dei risultati generati dal BLI e la Enzyme Linked Immuno Tecnica Spot (ELISPOT) confermano l&#39;utilizzabilità del BLI per la valutazione delle risposte immunitarie.

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

Questo video dimostra l&#39;uso di<em&gt; In vivo</em&gt; Immagini bioluminescenza per studiare le risposte immunitarie dopo l&#39;impianto di tessuto cardiaco Composti (EHT) nei ratti.

Imaging bioluminescenza per la valutazione delle risposte immunitarie A seguito di impianto di tessuto cardiaco Composti (EHT)

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

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Bioengineering

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

9.6K Views.  University Heart Center Hamburg. Questo video dimostra l'uso di In vivo Immagini bioluminescenza per studiare le risposte immunitarie dopo l'impianto di tessuto cardiaco Composti (EHT) nei ratti.

Video: 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).

Metodologia Elastografia risonanza magnetica per la valutazione dei Tissue Engineered Construct crescita

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

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Bioingegneria

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.

Protocollo per la relativa valutazione idrodinamica del Tri-volantino Polymer Valvole

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.

Tecnica chirurgica per l&#39;impianto di ingegneria tessutale vascolari Innesti e successive<em&gt; In Vivo</em&gt; Monitoraggio

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.

A base di seta-collagene Engineered Modello 3D del tessuto neurale polarizzata

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.

Automated Analysis Contrazione di ingegneria dei tessuti umani Cuore per lo screening cardiaco Drug Safety

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.

Abitudine costruita stampi di coltura del tessuto da maestri inciso al 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.

Semina e impianto di un innesto trachea tessutale biosintetico in un modello murino

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.

Innesto ingegnerizzato per la ricostruzione esofagea circonferenza nei ratti

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.

Preparazione di tessuti cardiaci ingegnerizzati a forma di mesh derivati da cellule iPS umane per la riparazione miocardia 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.