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 ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

Generación y Acrecentamiento de buques de ingeniería de tejidos en un modelo de ratón

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

Producción, Caracterización y posibles usos de un Humano esofágica mucosas Modelo ingeniería tisular 3D

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

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 ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

Ingeniería tisular tridimensional alineado astrositos redes para recapitular los mecanismos del desarrollo y facilitar la regeneración del sistema nervioso

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

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 ...