Sonja Schrepfer, Lenard Conradi e Arne Hansen recebeu financiamento da Deutsche Forschungsgemeinschaft (DFG; SCHR992/3-1, SCHR992/4-1, CO 858/1-1, HA 3423/3-1). O trabalho também foi suportado por concessões da União Européia para Thomas Eschenhagen (EUGeneHeart e Angioscaff).

1. EHT Geração e Cultura Condições Fabricação de fibrina baseado EHT foi realizada conforme já descrito 4. Em resumo, para gerar fibrina baseado EHT para fins de implantação, um master mix foi preparado contendo células isoladas de ROSA neonatal / luciferase-LEW corações de ratos transgênicos, fibrinogênio bovino e Matrigel. Moldes de fundição de agarose foram preparadas utilizando espaçadores Teflon colocado em pratos bem 6-cultura. Após a solidificação da agarose, espaçadores foram retirados e racks pós silicone foram colocados em placas de cultura com posts atingindo os moldes. Para gerar EHT, a Mastermix foi brevemente misturado com um montante calculado de trombina e pipetado no molde. Após a polimerização do fibrinogênio, racks de silicone com EHTs aderir aos postos foram delicadamente retirados dos moldes de fundição agarose, transferidos para novas 6-bem os pratos de cultura de células e mantidas em 37 ° C, 7% CO 2 de cultura de células incubadora usando custom-made meio de cultura celular. EHT foram mantidos em condições de cultura de células até o dia dez. Durante este período, as células do coração de ratos neonatal começou a interligar, de forma alongada e alinham ao longo linhas de força entre posts silicone. No momento da implantação, as contrações espontâneas coerente foram observados, desviando posts silicone. 2. Implantação EHT EHT implantação foi realizado em ambos os imunocompetentes Brown Norway (BN) ratos ou ratos imunodeficientes nua para investigar a resposta imunológica alogênica. BN e nu ratos pesando 100-150 g foram adquiridos da Charles River Alemanha (Sandhofer Weg 7, D-97633 Sulzfeld) e alojados em condições convencionais, alimentados padrão ração e água ad libidum. Ratos imunodeficientes estão alojados em cama estéril, gaiolas, e água esterilizada é utilizada. O comitê de revisão ética alemã analisou e aprovou os procedimentos animal. Todos os instrumentos cirúrgicos são esterilizados antes da utilização. O procedimento de implante são realizadas da seguinte forma: Ratos são anestesiados com isoflurano (2,5-3%) utilizando uma câmara de indução. A exposição humana à isoflurano pode ser reduzida através do trabalho em uma tabela downdraft ou capuz não recirculação. Temperatura corporal é mantida durante o procedimento cirúrgico. <ol><li> Shave área abdominal e aplicar pomada para evitar os olhos de secar durante a anestesia. </li><li> Coloque o rato sobre as suas costas e colocar uma máscara sobre seu nariz e boca para manter a anestesia. </li><li> Desinfetar a área abdominal com Betadine e, em seguida, o etanol 80%. Cotonete limpo para sujo, do centro da área raspada se movendo para fora em direção à área de cabelos. </li><li> Verifique reflexos beliscar as patas traseiras para ter a certeza que o rato é suficientemente anestesiados. </li><li> Drape do animal e realizar uma incisão mediana abdominal separando a pele eo músculo em duas etapas para abrir o abdômen. Esterilização talão quente é usado entre a pele e abertura do músculo. </li><li> Coloque o intestino em uma luva de pó aquecido salina hidratada livre. Dobre a luva ao redor do intestino para evitar a perda de umidade. </li><li> Remover tecido adiposo e expor o baço. </li><li> Visualize e se espalhou omento maior com uma pinça. </li><li> Corte cuidadosamente EHT de suspender posts silicone e transferir para o omento maior. </li><li> Enrole a omentus maior em torno da EHT e corrigir usando dois única prolene 7-0 (Ethicon, Norderstedt, Alemanha) </li><li> Em seguida mova o intestino volta para o abdômen. </li><li> Lave o abdome com solução salina estéril pré-aquecido. </li><li> Feche a camada muscular da parede abdominal com prolene 6-0 sutura (Ethicon, Norderstedt, Alemanha). </li><li> Use 5-0 prolene (Ethicon, Norderstedt, Alemanha), sutura contínua para fechar a pele. </li><li> Enquanto o rato ainda está em anestesia, injectar 5 carprofeno mg / kg por via subcutânea. </li><li> Para oferecer analgesia suficiente para este tipo de procedimento, um analgésico (como metamizol) é adicionado à água potável (50 mg por 100 ml metamizol) para dor medicação por 3 dias após o transplante. </li></ol> 3. Imagem de bioluminescência de Implantação Após EHT A plataforma bioimaging IVIS (Xenogen, Alameda, CA, EUA) é usado para não-invasivos de imagem de bioluminescência in-vivo e os resultados são analisados ​​utilizando a imagem viva IVIS (Xenogen) pacote de software. Imagem é realizada diariamente a partir 2 horas pós-operatório. Anestesiar ratos com isoflurano (2,5 - 3%), utilizando uma câmara de indução. <ol><li> Desinfectar a zona abdominal e torácica amplamente utilizando Iodo-Provo, use próxima de etanol 80%, repita este passo três vezes. </li><li> Injetar d-luciferina (Xenogen; 375 peso corporal mg / kg) ip </li><li> Lugar ratos anestesiados para a imagem IVIS câmara (até 4 ratos podem ser analisados ​​ao mesmo tempo). </li><li> Avaliar a intensidade do sinal luc + células dentro EHT em unidades de fótons / seg / cm 2 / steridianin a região de interesse (ROI) no início do estudo (120 minutos após o implante) e nos dias 1, 2, 3, 5, 7, 10, e nas semanas 2, 3 e 4. Observe o sinal de pico (aparece cerca de 20 minutos após o d-Luciferina injecção). </li></ol> Criar um gráfico (Excel), mostrando a intensidade do sinal ao longo do tempo. 4. Correlação de Resultados BLI e ELISPOT O celular in vivo respostas imunes em imunocompetentes e imunodeficientes BN ratos nus foram estudados após EHT transplante. O baço dos animais receptores foi colhida 5 dias após o transplante EHT para isolar esplenócitos destinatário. Ensaios de ELISPOT com mitomicina-inibida EHTs (Sigma Aldrich, St. Louis, MO, EUA) como estimulador e 5 x10 6 esplenócitos destinatário como células de resposta foram realizados de acordo com protocolo do fabricante (BD Biosciences) IFN-γ usando e IL-4 revestido placas para investigar TH1, TH2 e resposta, respectivamente. IFN-γ e IL-4 spots foram significativamente maiores no modelo, imunocompetentes em relação ao modelo imunodeficientes (IFN-γ: p = 0,001; IL-4: p = 0,023; Student t-test). 5. Resultados representativos: <img src="/files/ftp_upload/2605/2605fig1.jpg" alt="Figura 1" /> Figura 1. Correlação de resultados BLI e ELISPOT a) O celular in vivo respostas imunes em imunocompetentes Brown Noruega e imunodeficientes ratos nus foram estudados após o implante EHT. Esplenócitos destinatário foram colhidas cinco dias após o implante EHT. IFN? e IL-4 expressão foi avaliada por ensaios ELISpot com mitomicina-inibida EHT como estimuladores e 5x106 esplenócitos destinatário como células responder. Th1 e Th2 foram significativamente maiores no modelo imunocompetentes em relação ao modelo imunodeficientes como evidenciado pelo IFNγ e IL-4, respectivamente. b) EHTs Luc + foram transplantadas para o omentus maior de ambos os ratos BN imunocompetentes ou imunodeficientes ratos nude. Sobrevivência celular foi seguido por longitudinalmente BLI. A taxa de animais que rejeitou a EHTs transplantado é apresentado. Todos os ratos BN rapidamente rejeitou a transplantes EHT, enquanto as células sobreviveram em ratos células T deficiente.

<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>

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Antes da implementação clínica da tecnologia EHT para reparação cardíaca, questões fundamentais, como a imunogenicidade de ambos os componentes celulares e da matriz ou sobrevida do enxerto após o implante devem ser avaliados em modelos experimentais. In-vivo de imagem de bioluminescência representa uma nova técnica útil para a sobrevivência da célula de acompanhamento após o transplante . Nós gerado com sucesso fibrina baseado EHT contendo luciferase positivo (luc +) células do coração pela a...

<ul><li> LEW-Tg (Gt (ROSA) 26Sor-luc) 11Jmsk ratos (NBPR número de ratos 0299, http://www.anim.med.kyoto-u.ac.jp/NBR/) </li><li> Fibrinogênio bovino (solução estoque: 200 mg / ml, mais aprotinina 5 mg / ml em NaCl fibrinogênio 0,9%, Sigma F4753) </li><li> Matrigel (BD Bioscience 356235) </li><li> DMEM (Biochrome F0415) </li><li> Soro de cavalo (Gibco 26050) </li><li> Penicilina estreptomicina / (Gibco 15140) </li><li> Insulina (Sigma-Aldrich I9278) </li><li> Aprotinina (Sigma Aldrich A1153) </li><li> d-luciferina (Biosynth L-8220) </li><li> Imunoenzimático spot (ELISpot) placas de ensaio (Hassa Laborbedarf, S2EM004M99) </li></ul>

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

Várias técnicas de engenharia de tecido cardíaco foram perseguidos nas últimas décadas, incluindo estratégias de andaimes utilizando materiais nativa ou bioartificial andaime, aprisionamento de miócitos cardíacos em hidrogéis, tais como fibrina ou colágeno e empilhamento de monocamadas miócitos 1. Esses conceitos visem a restauração de comprometimento da função cardíaca (por exemplo, após infarto do miocárdio) ou como modelos experimentais (por exemplo, toxicologia preditiva e triagem substância ou modelagem de doença). Monitoramento preciso de sobrevivência das células após o implante do tecido do coração engenharia (EHT) tornou-se possível usando in-vivo de imagem de bioluminescência (BLI) técnicas de 2. Aqui, descrevemos a geração de fibrina baseado EHT de uma linhagem de ratos transgênicos com expressão ubíqua de firefly luciferase (ROSA / luciferase-LEW Tg; 3). Implantação é realizada no omento maior de linhagens de ratos diferentes para avaliar a resposta imune do organismo receptor após a implantação EHT. Comparação dos resultados gerados pelo BLI e Enzyme Linked Immuno Technique spot (ELISPOT) confirmam a usabilidade de BLI para a avaliação das respostas imunes.

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

Este vídeo demonstra o uso de<em&gt; In vivo</em&gt; Imagem de bioluminescência para estudar as respostas imune após o implante do tecido do coração Engineered (EHT) em ratos.

Imagem bioluminescência para avaliação das respostas imune após o implante do tecido do coração Engineered (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 visualizações.  University Heart Center Hamburg. Este vídeo demonstra o uso de In vivo Imagem de bioluminescência para estudar as respostas imune após o implante do tecido do coração Engineered (EHT) em ratos.

Vídeo: 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 por Ressonância Magnética para a Avaliação de engenharia de tecidos Crescimento 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 ...

Bioengenharia

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.

Protocolo Relativo Avaliação hidrodinâmica de Tri-folheto polímero Válvulas

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.

Técnica Cirúrgica para a Implantação de engenharia de tecidos enxertos vasculares e Subsequente<em&gt; In Vivo</em&gt; 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 ...

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.

Baseada em Silk-colágeno Engineered 3D Model of Tissue polarizada Neural

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 Análise contração do Coração Humano engenharia de tecidos para Screening Cardiac 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.

Custom Engineered moldes de cultura de tecidos de Masters gravados a 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.

A propagação e a implantação de um enxerto traqueal biossintética engenharia de tecido em um modelo do rato

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.

Enxerto projetado por tecido para reconstrução circunferencial do Esôfago em Ratos

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.

Preparação de tecidos cardíacos projetados em forma de malha derivados de células iPS humanas para reparo de miocárdio 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.