Sonja Schrepfer, Conradi Lenard y Arne Hansen recibió fondos de la Deutsche Forschungsgemeinschaft (DFG, SCHR992/3-1, SCHR992/4-1, CO 858/1-1, HA 3423/3-1). El trabajo también fue apoyado por subvenciones de la Unión Europea a Thomas Eschenhagen (EUGeneHeart y Angioscaff).

1. EHT Generación y condiciones de cultivo Fabricación de fibrina basado en EHT se realizó como se describe en otro lugar 4. En resumen, la generación de fibrina, basado en EHT para fines de implantación, una mezcla maestra se prepara con células aisladas de neonatos ROSA / luciferasa ASE corazones de ratas transgénicas, fibrinógeno bovino y Matrigel. Agarosa moldes de fundición se prepararon utilizando separadores de teflón colocados en placas de cultivo de 6 pocillos. Después de la solidificación de la agarosa, separadores fueron retirados y bastidores de silicona después se colocaron en placas de cultivo con los mensajes de llegar en los moldes. Para generar altas tensiones, la mastermix se mezcló brevemente con una cantidad calculada de la trombina y la pipeta en el molde. Después de la polimerización del fibrinógeno, bastidores de silicona con EHTs adherido a los postes fueron removidas suavemente de los moldes de agarosa, transferidos a las nuevas 6-y placas de cultivo celular y mantiene en un 37 ° C, 7% de CO 2 de cultivo celular incubadora usando a medida medio de cultivo celular. EHT se mantuvieron bajo condiciones de cultivo celular hasta el día diez. Durante este lapso de tiempo, las células de corazón de rata neonatal comenzó a interconectar alargada, y alinear a lo largo de las líneas de fuerza en medio de mensajes de silicona. En el momento de la implantación, las contracciones espontáneas coherente se observaron, desviando los mensajes de silicona. 2. EHT implantación EHT implantación se llevó a cabo, ya sea en inmunocompetentes Brown Norway (BN) ratas, ratones o inmunodeficientes desnuda para investigar la respuesta inmune alogénico. Ratas BN y desnuda un peso de 100 - 150 g fueron adquiridos de Charles River Alemania (Sandhofer Weg 7, D-97633 Sulzfeld) y alojados en condiciones convencionales, dieta estándar comida de rata y libidum agua ad. Ratas inmunodeficientes se encuentran en las camas estéril, jaulas, agua esterilizada y se utiliza. El comité de evaluación ética alemán revisó y aprobó los procedimientos con animales. Todos los instrumentos quirúrgicos son esterilizados antes de su uso. El procedimiento de implante se realiza de la siguiente manera: Rata son anestesiados con isoflurano (2,5-3%), utilizando una cámara de inducción. La exposición humana a isoflurano se puede reducir mediante el trabajo en una mesa de corriente descendente o no de recirculación de campana. La temperatura corporal se mantiene durante el procedimiento quirúrgico. <ol><li> Afeitarse la zona abdominal y aplicar ungüento oftálmico para evitar que los ojos se sequen durante la anestesia. </li><li> Coloque el ratón sobre la espalda y colocar una máscara sobre su nariz y la boca para mantener la anestesia. </li><li> Desinfectar el área abdominal con Betadine y etanol al 80%. Hisopo de limpieza de suciedad, desde el centro del área afeitada movimiento hacia afuera, hacia la zona de pelo. </li><li> Revisar los reflejos pellizcar las patas traseras para asegurarse de que la rata es lo suficientemente anestesiado. </li><li> Coloque el animal y realizar una incisión abdominal que separa la piel y el músculo en dos pasos para abrir el abdomen. Caliente se utiliza la esterilización de cuentas entre la piel y la apertura de los músculos. </li><li> Lugar de los intestinos en una solución salina caliente guante sin polvo hidratada. Veces el guante de todo el intestino para evitar la pérdida de humedad. </li><li> Eliminar el tejido graso y exponer el bazo. </li><li> Visualizar y difundir el epiplón mayor con unas pinzas. </li><li> Corte cuidadosamente EHT de la suspensión de los mensajes de silicona y la transferencia en el epiplón mayor. </li><li> Envuelva el mayor omentus todo el EHT y fijar con dos suturas de prolene 7-0 sola (Ethicon, Norderstedt, Alemania) </li><li> Luego mueva el intestino en el abdomen. </li><li> Enjuague bien el abdomen con solución salina estéril precalentada. </li><li> Cerca de la capa muscular de la pared abdominal usando suturas de prolene 6-0 en ejecución (Ethicon, Norderstedt, Alemania). </li><li> Use 5-0 prolene (Ethicon, Norderstedt, Alemania) que se ejecuta suturas para cerrar la piel. </li><li> Mientras que la rata aún está en la anestesia, inyectar 5 mg / kg por vía subcutánea Carprofen. </li><li> Para proporcionar una analgesia suficiente para este tipo de procedimiento, un analgésico (como el metamizol) se añade al agua potable (50 mg Metamizol por 100 ml) de medicamentos para el dolor durante 3 días después del trasplante. </li></ol> 3. Bioluminiscencia de imágenes después de la implantación EHT La plataforma bioimagen IVIS (Xenogen, Alameda, CA, EE.UU.) se utiliza para obtener imágenes de bioluminiscencia no invasiva in vivo y los resultados se analizaron mediante la viva imagen IVIS (Xenogen) paquete de software. Imágenes se realiza diariamente a partir de 2 horas tras la intervención. Anestesiar la rata con isoflurano (2,5 - 3%) con una cámara de inducción. <ol><li> Desinfectar el área abdominal y torácica ampliamente mediante Provo-yodo, el próximo uso etanol al 80%, repetir este paso tres veces. </li><li> Inyectar d-luciferina (Xenogen, 375 mg / kg de peso corporal) ip </li><li> Lugar ratas anestesiadas en la cámara de imágenes IVIS (hasta 4 ratas pueden ser analizados al mismo tiempo). </li><li> Evaluar la intensidad de la señal de luc + dentro de las células EHT en unidades de fotones / s / cm 2 / steridianin la región de interés (ROI) al inicio del estudio (120 minutos después de la implantación) y en los días 1, 2, 3, 5, 7, 10, y en las semanas 2, 3 y 4. Tenga en cuenta el pico de la señal (aparece alrededor de 20 minutos después de D-luciferina de la inyección). </li></ol> Crear un gráfico (Excel) que muestra la intensidad de la señal a través del tiempo. 4. La correlación de los resultados de BLI y ELISPOT El celular in vivo la respuesta inmune en BN inmunocompetentes y inmunodeficientes ratas desnudas fueron estudiados después de EHT trasplante. El bazo de los animales receptores se cosechó 5 días después del trasplante de EHT para aislar esplenocitos destinatario. ELISPOT con mitomicina inhibe EHTs (Sigma Aldrich, St. Louis, MO, EE.UU.) como estimulador y 5 x 10 6 esplenocitos destinatario como células de respuesta se realizaron de acuerdo con el protocolo del fabricante (BD Biosciences) con IFN-γ y recubierto de IL-4 placas para investigar TH1-, y la respuesta TH2, respectivamente. IFN-γ e IL-4 puntos fueron significativamente mayores en el modelo inmunocompetentes, en comparación con el modelo de inmunodeficiencia (IFN-γ: p = 0,001; IL-4: p = 0,023; t de Student-test). 5. Los resultados representativos: <img src="/files/ftp_upload/2605/2605fig1.jpg" alt="Figura 1" /> Figura 1. Correlación de los resultados de BLI y ELISPOT a) El celular in vivo la respuesta inmune en personas inmunocompetentes Noruega Brown y inmunodeficientes ratas desnudas fueron estudiados después de la implantación EHT. Esplenocitos receptores se cosecharon cinco días después de la implantación EHT. IFN? y la expresión de IL-4 fue evaluada por ensayos de ELISPOT con mitomicina inhibe EHT como estimuladores y 5x106 esplenocitos destinatario como células de respuesta. Th1 y Th2 fueron significativamente mayores en el modelo inmunocompetentes en comparación con el modelo de inmunodeficiencia como se evidencia por IFN-y IL-4, respectivamente. b) Luc EHTs + fueron trasplantados en el mayor omentus de cualquiera de las ratas BN inmunocompetentes o inmunodeficientes ratas desnudas. Supervivencia de las células fue seguido longitudinalmente por BLI. La tasa de los animales que rechazaban el EHTs trasplantado se presenta. Todas las ratas BN rápidamente rechazó el trasplante de EHT, mientras que las células sobrevivieron en ratas de células T deficientes.

<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>

Antes de la aplicación clínica de la tecnología EHT para la reparación cardiaca, las preguntas fundamentales tales como la inmunogenicidad de los componentes celulares y la matriz o la supervivencia del injerto después de la implantación deben ser evaluados en modelos experimentales. In vivo imágenes de bioluminiscencia representa una técnica nueva y útil para la supervivencia de la célula de seguimiento tras el trasplante . Hemos logrado generar fibrina basado en EHT contiene luciferasa positivo (luc...

<ul><li> LEW-Tg (Gt (ROSA) 26Sor-luc) 11Jmsk ratas (número NBPR rata 0299, http://www.anim.med.kyoto-u.ac.jp/NBR/) </li><li> Fibrinógeno bovino (solución madre: 200 mg / ml aprotinina más 5 mg / ml de fibrinógeno en NaCl 0,9%, Sigma F4753) </li><li> Matrigel (BD Bioscience 356235) </li><li> DMEM (Biochrome F0415) </li><li> Suero de caballo (Gibco 26050) </li><li> Penicilina / estreptomicina (Gibco 15140) </li><li> La insulina (Sigma-Aldrich I9278) </li><li> Aprotinina (Sigma Aldrich A1153) </li><li> d-luciferina (Biosynth L-8220) </li><li> Inmunoenzimático lugar (ELISPOT) placas de ensayo (Hassa Laborbedarf, S2EM004M99) </li></ul>

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

Diversas técnicas de la ingeniería de tejidos cardíacos han llevado a cabo en las últimas décadas, incluidas las estrategias de andamios utilizando materiales de andamiaje o nativo o bioartificiales, el atrapamiento de los miocitos cardíacos en los hidrogeles, como la fibrina o el colágeno y el apilamiento de las monocapas miocitos 1. Estos conceptos tienen por objeto la restauración de compromiso de la función cardíaca (por ejemplo después de un infarto de miocardio) o como modelos experimentales (por ejemplo, toxicología predictiva y la detección de sustancias o modelos de enfermedad). Un control preciso de la supervivencia celular después de la implantación del tejido del corazón de ingeniería (EHT) se ha convertido en posible con in-vivo de imágenes de bioluminiscencia (BLI) técnicas 2. A continuación se describe la generación de fibrina, basado en EHT a partir de una cepa de rata transgénicos con expresión ubicua de la luciferasa de luciérnaga (ROSA / Tg luciferasa-ASE, 3). La implantación se realiza en el epiplón mayor de cepas de ratas diferentes para evaluar la respuesta inmune del organismo receptor después de la implantación EHT. Comparación de los resultados generados por BLI y la enzima técnica vinculada al contado Inmuno (ELISPOT) confirman la utilidad de BLI para la evaluación de la respuesta inmune.

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 video muestra el uso del<em&gt; In vivo</em&gt; Imágenes de bioluminiscencia para estudiar la respuesta inmune después de la implantación de tejido cardiaco de Ingeniería (EHT) en ratas.

Bioluminiscencia de imágenes para la evaluación de la respuesta inmune después de la implantación de tejido cardiaco de Ingeniería (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 vistas.  University Heart Center Hamburg. Este video muestra el uso del In vivo Imágenes de bioluminiscencia para estudiar la respuesta inmune después de la implantación de tejido cardiaco de Ingeniería (EHT) en ratas.

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).

Metodología elastografía por resonancia magnética para la Evaluación de los tejidos Diseñado crecimiento 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 ...

Investigación

Bioingeniería

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 para la evaluación relativa hidrodinámico de Tri-lámina polimérica 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 quirúrgica para la implantación de la ingeniería tisular injertos vasculares y posterior<em&gt; En Vivo</em&gt; Monitoreo

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.

Modelo de polarizado Neural tisular basada Silk-colágeno-Engineered 3D

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

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

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

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

Análisis automatizado contracción de ingeniería tisular para la Detección del Corazón Humano cardíaca 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 diseñado moldes de cultivo de tejidos de maestros grabado a láser

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.

Siembra e implantación de un injerto traqueal tejido-dirigida biosintético en un modelo 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.

Injerto con ingeniería de tejido para la reconstrucción esofágica circunferencial en ratas

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.

Preparación de tejidos cardíacos de ingeniería en forma de malla derivados de células iPS humanas para la reparación del miocardio 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.