Los autores desean agradecer Brogan invitados, Weaver Michelle, y Lippert Kristen. El financiamiento para este estudio fue proporcionado por el NIH Grant R03EB009237, así como subvenciones del NIH Formación T32EB001026-06 del Instituto Nacional de Imágenes Biomédicas y Bioingeniería y T32HL076124 05-.

1. Preparación de tejidos y Experimento de instalación <ol><li> Harvest órgano porcino inmediatamente después de la eutanasia desde una instalación de matadero o de investigación y enjuagar el exceso de sangre. Recorte el corazón del exceso de grasa y tejido, manteniendo las aurículas y la aorta intacta. Recorte la grasa para separar la arteria pulmonar de la aorta. Si hay algunos cortes en el tejido, desechar apropiadamente. </li><li> Envolver cada corazón individualmente en papel de congelador y almacenar todo el tejido en un congelador -80 ° C durante al menos 24 horas para asegurar la congelación completa. </li><li> Cuando esté listo para su uso (generalmente menos de 3 meses), descongelar un corazón intacto congelado de porcino tipo 1 en agua durante la noche sumergido en un vaso de precipitados de 4 L a 4 º C. </li><li> Después de que el corazón está completamente descongelado, acariciar el corazón seco, pesar el corazón, y registrar el peso. El corazón de un cerdo peso de mercado debe pesar aproximadamente 375-450 g. </li><li> Conecte talla 18 a la tubería Masterflex ¼ &quot;fin de un reductor de púas. Inserte el reductor de púas ytubo dentro de la aorta. Coloque 2 abrazaderas de manguera o los lazos zip seguras alrededor de la aorta, justo debajo del tronco braquiocefálico. El reductor y el tubo debe permanecer por encima de la válvula aórtica, así que las arterias coronarias pueden ser perfundidos (Figura 1). </li><li> Usar una jeringa de 30 o 60 ml para llenar el tubo con agua Tipo I. Insertar el tubo dentro del cartucho de una bomba de rodillos Masterflex en su punto medio aproximado. Sumergir la afluencia extremo de la tubería en el fondo de un vaso de precipitados de 4 L con 2,5 L de agua y asegurar la tubería. </li><li> Colocar el corazón en el vaso de precipitados lleno de agua, y cebar la bomba para eliminar burbujas de aire. Si se observan burbujas procedente de la aorta, donde se inserta el tubo, la aorta puede ser necesario reposicionar o fijarse con cintas adicionales. Un sello hermético es importante para mantener la presión adecuada durante el proceso de descelularización (Figura 2). </li><li> Coloque el vaso de precipitados que contiene 3 L 4 L de un 0,2% NaN% Trypsin/0.05 EDTA/0.05%3 solución en una placa de agitación y se calienta a 37 ° C en el proceso de preparación de la descelularización. </li></ol> 2. Tejido Enjuagues <ol><li> Ajustar la bomba a una velocidad de flujo de 400 ml / min, asegurándose de que el tamaño del tubo correcto está seleccionado. Enjuague el corazón con agua de tipo I durante 15-25 min. Como se arranca la bomba, el corazón debe hincharse y efusión de sangre de los ventrículos. Solución fresca debe ser sustituido cada 5-10 minutos, o cuando sea necesario sobre la base de la cantidad de sangre que sale del corazón. Si la sangre no está derramada desde el corazón, ajustar el tubo y las abrazaderas según sea necesario. </li><li> Detener la bomba y transferir el corazón a un vaso de precipitados separado lleno de fosfato 2X salino tamponado (PBS). Después de que el tubo se sumerge en solución, arrancar la bomba y aumentar la velocidad de flujo de 700 ml / min. El corazón debe permanecer en disolución durante 15 minutos, cambiando la solución cada 5 min. Cada cambio de solución requiere que la bomba se detiene temporalmente mientras que el tejido y el tubo se mueveal vaso de precipitados de nuevo. </li><li> Transferir el corazón de tipo I de agua durante 10 minutos y aumentar la velocidad de flujo de 750 ml / min. </li></ol> 3. Descelularización y solución de perfusión <ol><li> Transferir el corazón al vaso de precipitados que contiene 0,2% Trypsin/0.05% EDTA/0.05% NaN 3 a 37 ° C. Aumentar la velocidad de la bomba a 1.200 ml / min y arrancar la bomba. Usar una barra de agitación colocado en el fondo del vaso de precipitados para hacer circular la solución en el vaso de precipitados. El corazón debe permanecer en el 0,2% Trypsin/0.05% EDTA/0.05% NaN 3 disolución a 37 ° C durante un total de tres horas. Después de 1 h, aumentar la velocidad de la bomba a 1.500 ml / min. Después de una hora adicional, aumentar la velocidad de la bomba a 1.800 ml / min. El tejido se lentamente sometido a velocidades de perfusión mayores para acondicionar el tejido y evitar la rotura de los vasos. El corazón se hinchará y casi el doble de tamaño durante este paso del protocolo. El tejido pierde su color natural, progresando desde las aurículas hasta el ápice a lo largo de thProtocolo E (Figura 3). </li><li> Después de cada perfusión solución, una de dos pasos de enjuague se realiza para eliminar los desechos celulares, residuos de productos químicos, y la lisis celular ayuda. Cada enjuague se compone de un 10 min enjuague en agua de tipo I seguido por un enjuague de 10 minutos con 2X solución de PBS a temperatura ambiente. Cada lavado consiste en la eliminación de la solución en el vaso de precipitados original, añadiendo soluciones de enjuague, y circulando el líquido de perfusión en el vaso de precipitados que contiene el corazón sumergido. Después de que el 0,2% Trypsin/0.05% EDTA/0.05% NaN 3 solución, el agua perfundir a 1.900 ml / min y, a continuación perfundir 2X PBS a 1950 ml / min. </li><li> Transferir el corazón a una solución de 3% de Triton X-100/0.05% EDTA/0.05% NaN 3 a temperatura ambiente. Aumentar la velocidad de la bomba a 2.000 ml / min y la solución de perfusión durante una hora. Quitar la solución del vaso de precipitados y reemplazar con solución fresca, aumentar la velocidad de la bomba a 2100 ml / min, y la perfusión de la solución fresca durante una hora adicional y un medio, con lo que el tiempo total en3% Triton X-100/0.05% EDTA/0.05% NaN 3 a 2,5 horas. </li><li> Enjuague el tejido en agua de tipo I en 2150 ml / min y PBS 2X a 2180 ml / min durante 10 min cada uno. </li><li> Transferir el corazón a una solución de desoxicolato sódico 4% a temperatura ambiente. Aumentar la velocidad de la bomba a 2.200 ml / min y la solución de perfusión durante 3 horas. </li><li> Enjuague el tejido en el tipo I y el agua en 2X PBS a 2.200 ml / min durante 15 min cada uno, el cambio de las soluciones después de 5-10 minutos para cada solución. Los pasos descritos perfusión se puede dividir en varios días mediante la realización de la etapa de aclarado dos veces y almacenar el corazón con una cánula adherida durante la noche a 4 ° C y se sumerge en agua de tipo I. </li><li> El día siguiente, realizar un enjuague con 5 min de tipo I de agua a 750 ml / min, seguido de un enjuague 5 min en PBS 1X a 1.500 ml / min. El protocolo a continuación, se puede continuar a la velocidad de flujo se describe en la solución apropiada. </li></ol> 4. Desinfección y procesamiento final <ol><li> Transferir el corazón a un 0,1% peracácido etic / 4% de solución de etanol y la solución de perfusión durante 1,5 horas a 2.200 ml / min. </li><li> Los enjuagues finales para el tejido se realizan todas en 2.200 ml / min. Perfundir el tejido con PBS 1X durante 15 min, seguido por dos lavados de 5 min en el tipo I de agua. Esta serie de aclarados se repite una vez más con el fin de completar el procedimiento de perfusión de solución. </li><li> Apague la bomba y retire el corazón de la solución para drenar el corazón. Cortar los lazos de la aorta, eliminar todos los tubos, y colocar el corazón en un vaso de precipitados vacío para drenar durante 1 hora. El exceso de líquido tendrá que ser drenada periódicamente. Coloque el corazón en una almohadilla absorbente para drenar totalmente el corazón (Figura 4). </li><li> Después de la mayor parte del agua se elimina, se registra el peso de la matriz extracelular cardiaca (C-ECM). El corazón se puede esperar que pierden aproximadamente 20-25% de su peso inicial durante el proceso de descelularización. </li><li> Diseccionar los ventrículos derecho e izquierdo, así como el tabique ventricular para el ADN quantificatiy en el procesamiento histológico con el fin de confirmar la descelularización completa del tejido (Figura 5). </li><li> Congelar la C-ECM a -80 º C durante al menos 2 horas antes de la liofilización. </li></ol>

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+tissue+and+organ+decellularization.">Strategies for tissue and organ decellularization.</a> Journal of cellular biochemistry. , (2012).</li><li>Gilbert, T. W., Sellaro, T. L., Badylak, S. 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Dr. Gilbert estaba en el Consejo Asesor Científico de ACELL, Inc., mientras que el estudio se está haciendo, y recientemente se convirtió en el vicepresidente de Investigación y Desarrollo. ACELL, Inc. vende matriz vejiga urinaria y no tiene interés comercial en el presente estudio.

El actual estudio se describe la metodología para la descelularización coherente y eficaz de un corazón porcino. El protocolo era una modificación de un informe publicado anteriormente 1, e incluyó una exposición más larga al caudal y la presión aumentada, que proporcionan resultados más reproducibles. El tejido resultante descelularizado cumplido con todos los criterios publicados por descelularización éxito de tejido 2. Los cambios frecuentes de solución se realiza para limitar la rei...

<table border="1"><tbody><tr><td> Nombre del reactivo / material </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Tripsina </td><td> Gibco </td><td> 15090 </td><td></td></tr><tr><td> EDTA </td><td> Pescador </td><td> BP120-500 </td><td></td></tr><tr><td> NaN 3 </td><td> Sigma </td><td> S2002-500G </td><td></td></tr><tr><td> Triton X-100 </td><td> Sigma </td><td> X100-1L </td><td></td></tr><tr><td> 10X PBS </td><td> Pescador </td><td> BP399-20 </td><td></td></tr><tr><td> Desoxicolato de sodio </td><td> Sigma </td><td> D6750-500G </td><td></td></tr><tr><td> El ácido peracético </td><td> Pfaltz y Bauer </td><td> P05020 </td><td> 35% CAS # 79-21-0 </td></tr><tr><td> Etanol </td><td> Pharmco </td><td> 111000200 </td><td></td&gt; </tr><tr><td> Masterflex Drive Pump </td><td> Cole Parmer </td><td> SI-07.524-50 </td><td></td></tr><tr><td> Tubería Masterflex </td><td> Cole Parmer </td><td> 96400-18 </td><td> Tamaño 18 </td></tr><tr><td> Reductor de púas </td><td> Cole Parmer </td><td> EW-30612-20 </td><td></td></tr><tr><td> Beaker 4L </td><td> Fisher Scientific </td><td> 02-540T </td><td></td></tr></tbody></table>

procedure for decellularization of porcine heart by retrograde coronary perfusion

Perfusión basada descelularización órgano completo recientemente ha ganado interés en el campo de la ingeniería de tejidos como un medio para crear un sitio determinado andamios de matriz extracelular, mientras que en gran medida se mantiene la arquitectura nativa del andamio. Hasta la fecha, este enfoque se ha utilizado en una variedad de sistemas de órganos, incluyendo el corazón, pulmón, hígado y 1-5. Los métodos anteriores para descelularización tejidos sin una red vascular fácilmente accesible se han basado en la exposición prolongada del tejido a soluciones de detergentes, ácidos, o tratamientos enzimáticos como un medio para eliminar los componentes celulares y nucleares del medio ambiente circundante extracelular 6-8. Sin embargo, la eficacia de estos métodos con bisagras en la capacidad de las soluciones para impregnar el tejido por medio de difusión. En contraste, la perfusión de órganos a través del sistema vascular natural, reduce eficazmente la distancia de difusión y transporte facilitado de decellularizatien agentes en el tejido y los componentes celulares de los tejidos. En este documento, se describe un método para decellularize completamente un corazón porcino intacta a través de perfusión retrógrada coronaria. El protocolo produjo un completamente descelularizado cardíaco matriz extracelular (ECM-c) andamio con la estructura tridimensional del corazón intacto. Nuestro método utiliza una serie de enzimas, detergentes y ácidos junto con enjuagues hipertónicas e hipotónicas para ayudar en la lisis y la eliminación de las células. El protocolo utilizado una solución de tripsina para separar las células de la matriz seguido por soluciones de Triton X-100 desoxicolato de sodio y para ayudar en la eliminación de material celular. El protocolo descrito también utiliza una velocidad de perfusión de más de 2 L / min durante largos períodos de tiempo. La alta velocidad de flujo, junto con los cambios solución permitió el transporte de agentes para el tejido sin contaminación de restos celulares y se aseguró eficaz enjuague del tejido. El método descrito eliminado todo el material nuclear de natiVE tejido cardíaco porcino, la creación de un sitio específico cardíaco ECM andamio que se puede utilizar para una variedad de aplicaciones.

El efecto de la descelularización sobre corazones porcinos enteros, naturalmente, varía debido a las diferencias de tamaño, las presiones y disposición de los vasos. Por lo tanto, la composición exacta de los medios de andamiaje de la matriz extracelular derivados no será el mismo de corazón a corazón. La realización del protocolo descrito producirá un corazón que aparece blanco o translúcido, lo que indica la pérdida de material celular. Sin embargo, está ampliamente aceptado que un tejido puede ser consi...

Watch this Scientific Journal Video about Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion at JoVE.com

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

Un método para eliminar rápida y completamente los componentes celulares de un corazón porcino intacta a través de perfusión retrógrada se describe. Este método rinde un sitio específico cardíaco extracelular andamio matriz que tiene el potencial para su uso en diversas aplicaciones clínicas.

Procedimiento para descelularización de corazón porcino por perfusión coronaria retrógrada

Perfusion-based  whole organ decellularization has recently gained interest in the field of  tissue engineering as a means to create site-specific ...

procedure-for-decellularization-porcine-heart-retrograde-coronary

Research

JoVE Journal

Bioengineering

23.7K vistas.  McGowan Institute for Regenerative Medicine. Un método para eliminar rápida y completamente los componentes celulares de un corazón porcino intacta a través de perfusión retrógrada se describe. Este método rinde un sitio específico cardíaco extracelular andamio matriz que tiene el potencial para su uso en diversas aplicaciones clínicas.

Video: Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

Decellularization and Recellularization of Whole Livers

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to recreate or mimic the liver microstructure with grounds up approach using tissue engineering and microfabrication techniques have not been successful so far due to this design challenge. In addition, synthetic biomaterials used to create scaffolds for liver tissue engineering applications have been limited in inducing tissue regeneration and repair in large part due to the lack of specific cell binding motifs that would induce the proper cell functions2. Decellularized native tissues such blood vessels3and skin4on the other hand have found many applications in tissue engineering, and have provided a practical solution to some of the challenges. The advantage of decellularized native matrix is that it retains, to an extent, the original composition, and the microstructure, hence enhancing cell attachment and reorganization5.
In this work we describe the methods to perform perfusion-decellularization of the liver, such that an intact liver bioscaffold that retains the structure of major blood vessels is obtained. Further, we describe methods to recellularize these bioscaffolds with adult primary hepatocytes, creating a liver graft that is functional in vitro, and has the vessel access necessary for transplantation in vivo.

Perfusion decellularization is a novel technique to produce whole liver scaffolds that retains the organ's extracellular matrix composition and microarchitecture. Herein, the method of preparing whole organ scaffolds using perfusion decellularization and subsequent repopulation with hepatocytes is described. Functional and transplantable liver grafts can be generated using this technique.

Descelularización y recelularización de hígados enteros

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to ...

Investigación

Bioingeniería

Procedure for Decellularization of Rat Livers in an Oscillating-pressure Perfusion Device

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent liver decellularization have already been published. We aimed to improve the decellularization process by construction of a proprietary perfusion device enabling selective perfusion via the portal vein and/or the hepatic artery. Furthermore, we sought to perform perfusion under oscillating surrounding pressure conditions to improve the homogeneity of decellularization. The homogeneity of perfusion decellularization has been an underestimated factor to date. During decellularization, areas within the organ that are poorly perfused may still contain cells, whereas the extracellular matrix (ECM) in well-perfused areas may already be affected by alkaline detergents. Oscillating pressure changes can mimic the intraabdominal pressure changes that occur during respiration to optimize microperfusion inside the liver. In the study presented here, decellularized rat liver matrices were analyzed by histological staining, DNA content analysis and corrosion casting. Perfusion via the hepatic artery showed more homogenous results than portal venous perfusion did. The application of oscillating pressure conditions improved the effectiveness of perfusion decellularization. Livers perfused via the hepatic artery and under oscillating pressure conditions showed the best results. The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

Procedimiento para Descelularización de rata hígados en un dispositivo de perfusión oscilante presión

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent ...

Decellularization of Whole Human Heart Inside a Pressurized Pouch in an Inverted Orientation

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ultimately rejection can occur. Creating a functional, autologous bio-artificial heart could solve these challenges. Biofabrication of a heart comprised of scaffold and cells is one option. A natural scaffold with tissue-specific composition as well as micro- and macro-architecture can be obtained by decellularizing hearts from humans or large animals such as pigs. Decellularization involves washing out cellular debris while preserving 3D extracellular matrix and vasculature and allowing &#34;cellularization&#34; at a later timepoint. Capitalizing on our novel finding that perfusion decellularization of complex organs is possible, we developed a more &#34;physiological&#34; method to decellularize non-transplantable human hearts by placing them inside a pressurized pouch, in an inverted orientation, under controlled pressure. The purpose of using a pressurized pouch is to create pressure gradients across the aortic valve to keep it closed and improve myocardial perfusion. Simultaneous assessment of flow dynamics and cellular debris removal during decellularization allowed us to monitor both fluid inflow and debris outflow, thereby generating a scaffold that can be used either for simple cardiac repair (e.g. as a patch or valve scaffold) or as a whole-organ scaffold.

This method enables decellularization of a complex solid organ using a simple protocol based on osmotic shock and perfusion of ionic detergent with minimal organ matrix disruption. It comprises a novel decellularization technique for human hearts inside a pressurized pouch with real-time monitoring of flow dynamics and cellular debris outflow.

Descelularización de todo humano corazón dentro de una bolsa presurizada en una orientación invertida

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ...

Isolation and Decellularization of a Whole Porcine Pancreas

Tissue engineering of the whole pancreas can improve current treatments for diabetes mellitus. The ultimate goal is to tissue engineer pancreas from an allogeneic or xenogeneic source with human cells. A demonstration of methods for the efficient dissection, decellularization, and recellularization of porcine pancreas might benefit the field. Akin to human pancreases, porcine pancreases have a special anatomical arrangement with three lobes (splenic, duodenal, and connection) rounded by the duodenum and small intestine. The duodenal lobe of the pancreas connects to the duodenum by several small blood vessels. Tissue engineering of the pancreas is complicated because of its exocrine and endocrine nature. In this paper, we show a detailed protocol to dissect the whole porcine pancreas and decellularize it with detergents while saving its structure and some extracellular matrix components. To achieve complete perfusion, the aorta is chosen as inlet and the portal vein as outlet. The other blood vessels (hepatic artery, splenic vein, splenic artery,&#160;mesenteric artery and vein tree) and bile duct are ligated. To prevent the formation of thrombus, the pig is heparinized and, immediately after dissection, the organ is flushed with cold heparin. To inhibit the action of exocrine enzymes, the pancreas decellularization is set at 4 &#176;C. The decellularization is performed by perfusion of Triton X-100, sodium deoxycholate, and deoxyribonuclease, with an intermittent and final extensive washing. With a successful decellularization, the pancreas appears white, and a histological evaluation with hematoxylin and eosin shows an absence of nuclei with a preserved extracellular matrix structure. Thus, the proposed method can be used to successfully dissect and decellularize whole porcine pancreas.

Tissue engineering of the whole pancreas is a challenge because of its exocrine and endocrine functions. We show a method for the dissection of an intact porcine pancreas and the process of successful decellularization by perfusion of detergents Triton X-100, sodium deoxycholate, and deoxyribonuclease.

Aislamiento y la descelularización de un páncreas todo porcino

Tissue engineering of the whole pancreas can improve current treatments for diabetes mellitus. The ultimate goal is to tissue engineer pancreas from an ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

Detección de endotoxinas en las formulaciones Nano mediante ensayos de Limulus Amoebocyte Lysate (LAL)

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart

The ability to measure biomarkers in vivo relevant to the assessment of disease progression is of great interest to the scientific and medical communities. The resolution of results obtained from current methods of measuring certain biomarkers can take several days or weeks to obtain, as they can be limited in resolution both spatially and temporally (e.g., fluid compartment microdialysis of interstitial fluid analyzed by enzyme-linked immunosorbent assay [ELISA], high-performance liquid chromatography [HPLC], or mass spectrometry); thus, their guidance of timely diagnosis and treatment is disrupted. In the present study, a unique technique for detecting and measuring peptide transmitters in vivo through the use of a capacitive immunoprobe biosensor (CI probe) is reported. The fabrication protocol and in vitro characterization of these probes are described. Measurements of sympathetic stimulation-evoked neuropeptide Y (NPY) release in vivo are provided. NPY release is correlated to the sympathetic release of norepinephrine for reference. The data demonstrate an approach for the fast and localized measurement of neuropeptides in vivo. Future applications include intraoperative real-time assessment of disease progression and minimally invasive catheter-based deployment of these probes.

Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart

Established immunochemical methods to measure peptide transmitters in vivo rely on microdialysis or bulk fluid draw to obtain the sample for offline analysis. However, these suffer from spatiotemporal limitations. The present protocol describes the fabrication and application of a capacitive immunoprobe biosensor that overcomes the limitations of the existing techniques.

Medición in vivo resuelta en el tiempo de la dinámica de neuropéptidos mediante inmunosonda capacitiva en corazón porcino

The ability to measure biomarkers in vivo relevant to the assessment of disease progression is of great interest to the scientific and medical ...

Procurement and Perfusion-Decellularization of Porcine Vascularized Flaps in a Customized Perfusion Bioreactor

Large volume soft tissue defects lead to functional deficits and can greatly impact the patient&#39;s quality of life. Although surgical reconstruction can be performed using autologous free flap transfer or vascularized composite allotransplantation (VCA), such methods also have disadvantages. Issues such as donor site morbidity and tissue availability limit autologous free flap transfer, while immunosuppression is a significant limitation of VCA. Engineered tissues in reconstructive surgery using decellularization/recellularization methods represent a possible solution. Decellularized tissues are generated using methods that remove native cellular material while preserving the underlying extracellular matrix (ECM) microarchitecture. These acellular scaffolds can then be subsequently recellularized with recipient-specific cells.
This protocol details the procurement and decellularization methods used to achieve acellular scaffolds in a pig model. In addition, it also provides a description of the perfusion bioreactor design and setup. The flaps include the porcine omentum, tensor fascia lata, and the radial forearm. Decellularization is performed via ex vivo perfusion of low concentration sodium dodecyl sulfate (SDS) detergent followed by DNase enzyme treatment and peracetic acid sterilization in a customized perfusion bioreactor.
Successful tissue decellularization is characterized by a white-opaque appearance of flaps macroscopically. Acellular flaps show the absence of nuclei on histological staining and a significant reduction in DNA content. This protocol can be used efficiently to generate decellularized soft tissue scaffolds with preserved ECM and vascular microarchitecture. Such scaffolds can be used in subsequent recellularization studies and have the potential for clinical translation in reconstructive surgery.

The protocol describes the surgical procurement and subsequent decellularization of vascularized porcine flaps by the perfusion of sodium dodecyl sulfate detergent through the flap vasculature in a customized perfusion bioreactor.

Obtención y perfusión-descelularización de colgajos vascularizados porcinos en un biorreactor de perfusión personalizado

Large volume soft tissue defects lead to functional deficits and can greatly impact the patient&#39;s quality of life. Although surgical reconstruction ...

Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an evolving reconstructive avenue for transferring multiple tissues as a composite subunit. Despite the promising nature of VCA, the long-term immunosuppressive requirements are a significant limitation due to the increased risk of malignancies, end-organ toxicity, and opportunistic infections. Tissue engineering of acellular composite scaffolds is a potential alternative in reducing the need for immunosuppression. Herein, the procurement of a rat hindlimb and its subsequent decellularization using sodium dodecyl sulfate (SDS) is described. The procurement strategy presented is based upon the common femoral artery. A machine perfusion-based bioreactor system was constructed and used for ex vivo decellularization of the hindlimb. Successful perfusion decellularization was performed, resulting in a white translucent-like appearance of the hindlimb. An intact, perfusable, vascular network throughout the hindlimb was observed. Histological analyses showed the removal of nuclear contents and the preservation of tissue architecture across all tissue compartments.

Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

We describe the surgical technique and decellularization process for composite rat hindlimbs. Decellularization is conducted using low-concentration sodium dodecyl sulfate through an ex vivo machine perfusion system.

Obtención y descelularización de extremidades posteriores de ratas utilizando un biorreactor basado en perfusión ex vivo para alotrasplante compuesto vascularizado

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an ...

Evaluación de las propiedades hidrodinámicas en válvulas aórticas porcinas montadas

An Ex Vivo Porcine Model for Hydrodynamic Testing of Experimental Aortic Valve Procedures and Novel Medical Devices

The options for testing new cardiac procedures and investigative medical devices prior to use in an animal model are limited. In this study, we present a method for mounting a porcine aortic valve in a pulse duplicator to evaluate its hydrodynamic properties. These properties can then be evaluated before and after the procedure under investigation is performed and/or the investigative medical device is applied. Securing the inflow segment presents some difficulty owing to the lack of circumferential myocardium in the left ventricular outflow tract. This method addresses that issue by securing the inflow segment using the anterior leaflet of the mitral valve and then suturing the left ventricular free wall around the inflow fixture. The outflow segment is secured simply by inserting the fixture into an incision in the superior aspect of the aortic arch. We found that specimens had significantly different hydrodynamic properties before and after tissue fixation. This finding induced us to use fresh specimens in our testing and should be considered when using this method. In our work, we used this method to test novel intracardiac patch materials for use in the valvular position by performing an aortic valve neocuspidization procedure (Ozaki procedure) on the mounted porcine aortic valves. These valves were tested before and after the procedure to assess the change in hydrodynamic properties in comparison to the native valve. Herein, we report a platform for hydrodynamic testing of experimental aortic valve procedures that enables comparison with the native valve and between different devices and techniques used for the procedure under investigation.

Autor destacado: Avance de las pruebas de procedimientos cardíacos antes de embarcarse en estudios con animales grandes 

We present a method for mounting a porcine aortic valve on a pulse duplicator to test its hydrodynamic properties. This method can be used to determine the change in hydrodynamics after the application of an experimental procedure or novel medical device prior to use in a large animal model.

Un modelo porcino ex vivo para pruebas hidrodinámicas de procedimientos experimentales de válvulas aórticas y nuevos dispositivos médicos

The options for testing new cardiac procedures and investigative medical devices prior to use in an animal model are limited. In this study, we present a ...