Este proyecto fue financiado por la Asociación Alemana de Investigación (DFG, Excmo 4578/3-1).

A. Cartílago biopsia (Cirugía habitación; pasos 1-5 en la sala de preparación no estéril) <ol><li> Realizar un control del peso final del conejo (conejo Blanco de Nueva Zelanda, hembra, 3,5-4,0 kg de peso corporal, 6 meses de edad) con el fin de ser capaz de dosis drogas correctamente y para supervisar peso posterior a la cirugía. </li><li> Inducir la anestesia para el conejo mediante una inyección intravenosa de 10 mg / kg de propofol. </li><li> Después de la intubación, mantener la anestesia con 1,5 mg / kg / min propofol y 0,05 mg / kg de fentanilo por vía intravenosa. Supervisar la anestesia mediante capnografía, pulsioximetría y el pulso. </li><li> Afeitado de la rodilla para ser operado con una maquinilla eléctrica y el vacío de la piel. </li><li> Desinfectar la rodilla afeitado a fondo y cubrir el resto del conejo con un apósito estéril. </li><li> Se palpa la rótula y realizar una incisión en la piel en sentido medial de la rótula. </li><li> Abra la articulación de la rodilla por una artrotomía parapatelar medial en condiciones estériles. Trate de evitar el corte de cualquier pequeña dovasos sanguíneos perficial. </li><li> Desplazar la rótula lateralmente. </li><li> Inspeccione la articulación de la rodilla de las anomalías y lesiones de cartílago macroscópicos. </li><li> Retire con cuidado trozos de cartílago pequeñas (2-3 mm) fuera de la tróclea femoral ossis con un bisturí estéril (Figura 1). </li><li> Inmediatamente colocar estas biopsias de cartílago en un tubo de 50 ml con medio completo (DMEM + 10% suero de ternera fetal (FCS) + 1% de penicilina / estreptomicina (Pen / Strep)) para continuar con el cultivo in vitro en la derecha después de la operación. </li><li> Limpie los defectos y enjuague con solución salina estéril. </li><li> Vuelva a colocar la rótula en el surco troclear. </li><li> Cierre de la herida en capas con suturas solo botón (4-0 Vicryl) y una sutura cutánea continua (4-0 Monocryl). Utilice material de sutura absorbible. </li><li> Por último, sellar la herida con un apósito de pulverización permeable al vapor de agua. </li></ol> B. In vitro Cultura <ol><li> Procesar la biopsia cpiezas artilage tan pronto como sea posible después de la operación en un flujo laminar campana de cultivo de tejido estéril. </li><li> Lavar la estéril cosechada cartílago 2x en PBS con posterior centrifugación a 500 xg durante 3 minutos a TA. </li><li> Cortar cartílago en piezas de 1 mm 3, la transferencia a un Falcon 50 ml y digerir ellos en un agitador durante 30 min con 10 ml de tripsina-EDTA (0,25%) y 10 ml de PBS durante 30 min </li><li> Después de 30 min, dejar de tripsinización mediante la adición de medio completo. Entonces, agitar los trozos de cartílago para otra durante 12 horas con colagenasa A (0,21 U / mg) en medio libre de suero (DMEM + 1% Pen / Strep). </li><li> Centrifugar la solución resultante a 170 xg durante 3 minutos a TA. </li><li> Resuspender los sedimentos celulares en 5 ml de medio completo. </li><li> Seed los condrocitos aislados de hasta 25 cm 2 frascos de cultivo de tejidos en 5 ml de medio completo y manténgalo en un cultivo de tejidos humidificado incubadora a 37 ° C, 5% CO 2. </li><li> Crecer condrocitos a una densidad de 80%. </li><li> Liberar las células ffrascos rom de lavado en PBS 2 veces antes de exponerse al 0,05% de tripsina-EDTA durante 3 min. Una vez que los condrocitos se desprenden, dejar de tripsinización mediante la adición de 10 l de medio completo. </li><li> Se centrifuga la suspensión a 300 xg (3 min) y se resuspende el precipitado en medio completo. </li><li> Se siembran las células en matraces de cultivo tisular (75 cm 2) y divididas en una proporción de 1:03 (~ 5000 células / cm 2) cada 5 º día o al 80% de confluencia. </li><li> Determinar el número de células y la viabilidad mediante tinción con azul tripán. </li></ol> C. siembra de células de la matriz <ol><li> Antes de la implantación de lavado y las células de liberación como se describió anteriormente. Lleve a cabo el recuento de células y transferir 10 células de 5 x 4 en un tubo de microcentrífuga de 1,5 ml. A continuación, se centrifuga a 500 xg durante 5 min a TA. </li><li> Resuspender el precipitado en 15 l de medio completo para una saturación completa de la matriz. </li><li> Cortar la bicapa de colágeno andamio a las dimensiones exactas del defecto troclear por una biopsia estérilponche. </li><li> Lugar matriz en una placa de cultivo tisular de tamaño adecuado (por ejemplo, placas de 24 pocillos). </li><li> Se siembran las células sobre el lado poroso, célula-adhesivo de la matriz. Esto impide la migración de células lejos de la matriz. </li><li> Deja matrices de células sembradas sin más medio de cultivo celular en una incubadora durante 1 hora para permitir la plena adhesión de los condrocitos. </li><li> Luego, coloque célula-matriz-construcciones en recipientes de cultivo (por ejemplo, placas de 24 pocillos) con medio completo fresco. Tome les importa para lavar las células fuera de la matriz, aunque se espera una fuerte adherencia. </li><li> Ahora, célula-matriz-construcciones pueden ser llevados a la sala de cirugía de reimplantación. </li></ol> D. Matrix asistida por trasplante de condrocitos autólogos <ol><li> Realizar artrotomía parapatelar a la rodilla contralateral como se describió anteriormente (A. 1-8). </li><li> Cree dos defectos condrales aisladas en el surco troclear con una de Operación aire estérilg taladro eléctrico (3,6 mm de diámetro). </li><li> Limpie el defecto y enjuague con solución salina estéril. </li><li> Se debe prestar atención de no abrir la cavidad medular en cualquier momento. Si es necesario, sellar la parte inferior de los defectos con cauterización eléctrica frente a hemorragias. </li><li> Implante las matrices sembradas con los cultivos de condrocitos con la cara lateral hacia abajo porosa (Figura 2). A continuación, encaje a presión en los orificios de perforación y al ras de la superficie circundante. </li><li> Selle las matrices implantados con un poco de pegamento de fibrina (Tissucol Duo S) para garantizar una fijación segura. </li><li> Después de la coagulación, la ubicación de la rótula en el surco troclear y aplicar el rango de movimiento de la articulación de la rodilla un par de veces. </li><li> Desplazar la rótula lateralmente una vez más y comprobar si hay algún evento o señal de fijación inestable. Las matrices deben realizarse todavía en su lugar. </li><li> Vuelva a colocar la rótula de nuevo y terminar la operación con el cierre de la herida en capas y el aerosol de vestir como se describe anteriormente. </li></ol>

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Los autores no tienen nada que revelar.

El protocolo presentado proporciona una técnica establecida 9,12,13 y fácilmente reproducible para aislar condrocitos autólogos para la posterior proliferación y re-implante en defectos del cartílago creados artificialmente en las rodillas de conejo. El uso de condrocitos autólogos para la remodelación y reparación de lesiones del cartílago articular ya está en uso clínico proporcionar resultados satisfactorios a largo plazo 6. Los principales problemas como ...

<table border="1">
	<tbody>
		<tr>
			<td>Name of reagent/equipment</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Biochrom AG</td>
			<td>F 0415</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase A</td>
			<td>Roche</td>
			<td>10 103 586 001</td>
			<td>0.21 U/mg</td>
		</tr>
		<tr>
			<td>Fetal calf serum (FCS)</td>
			<td>PAN Biotech GmbH</td>
			<td>3702-P103009</td>
			<td></td>
		</tr>
		<tr>
			<td>Propofol</td>
			<td>Fresenius Kabi</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Biochrom AG</td>
			<td>A 2213</td>
			<td>10,000 U/ml/10,000 &mu;g/ml</td>
		</tr>
		<tr>
			<td>PBS Dulbecco (1X)</td>
			<td>Biochrom AG</td>
			<td>L1815</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (70%)</td>
			<td>Merck KgaA</td>
			<td>410230</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.25 %/0.02 %</td>
			<td>Biochrom AG</td>
			<td>L2163</td>
			<td>in PBS w/o Ca2+, Mg2+</td>
		</tr>
		<tr>
			<td>Fentanyl</td>
			<td>Delta Select GmBH</td>
			<td>1819340</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl solution (0.9%)</td>
			<td>Bbraun</td>
			<td>8333A193</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture dishes 100 mm/150 mm</td>
			<td>TPP AG</td>
			<td>93100/93150</td>
			<td>Growth area 60.1 mm2/147.8 mm2</td>
		</tr>
		<tr>
			<td>Tissue culture flasks 25/75 mm2</td>
			<td>TPP AG</td>
			<td>90025/90075</td>
			<td>25 mm2, 75 mm2</td>
		</tr>
		<tr>
			<td>Centrifuge Tubes (50 ml)</td>
			<td>TPP AG</td>
			<td>91050</td>
			<td>Gamma-sterilized</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Brand GmbH+Co KG</td>
			<td>717810</td>
			<td>Neubauer</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution 0.4%</td>
			<td>Sigma-Aldrich</td>
			<td>L8154</td>
			<td></td>
		</tr>
		<tr>
			<td>Spray dressing (OpSite)</td>
			<td>Smith&amp;Nephew</td>
			<td>66004978</td>
			<td>Permeable for water vapor</td>
		</tr>
		<tr>
			<td>Chondro-Gide&Ograve;</td>
			<td>Geistlich Pharma AG</td>
			<td>30915.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy Punch</td>
			<td>pfm medical ag</td>
			<td>48351</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissucol Duo S</td>
			<td>Baxter</td>
			<td>3419627</td>
			<td>0.5 ml</td>
		</tr>
	</tbody>
</table>

matrix-assisted autologous chondrocyte transplantation for remodeling and repair of chondral defects in a rabbit model

Defectos del cartílago articular se considera un problema de salud importante porque el cartílago articular tiene una capacidad limitada para la auto-regeneración 1. Lesiones del cartílago no tratadas conducen a dolor en curso, afectar negativamente a la calidad de vida y predisponer para la osteoartritis. Durante las últimas décadas, varias técnicas quirúrgicas se han desarrollado para el tratamiento de estas lesiones. Sin embargo, hasta ahora no era posible lograr una reparación completa en términos de cubrir el defecto de cartílago hialino articular o de proporcionar satisfactoria recuperación a largo plazo 2-4. Por lo tanto, las lesiones del cartílago articular siguen siendo un objetivo prioritario para las técnicas regenerativas como la ingeniería de tejidos. En contraste con otras técnicas quirúrgicas, que a menudo conducen a la formación de tejido fibroso o fibrocartilaginoso, Ingeniería de Tejidos apunta a restaurar completamente la compleja estructura y las propiedades del cartílago articular original utilizando el potencial condrogénico de las células trasplantadas. Rn los últimos desarrollos abren posibilidades prometedoras para las terapias de regeneración del cartílago. El primer enfoque basado en células para el tratamiento de cartílago de grosor completo o lesiones osteocondrales se llevó a cabo en 1994 por Lars Peterson y Mats Brittberg que fue pionero en la implantación de condrocitos autólogos clínica (ACI) 5. Hoy en día, la técnica está bien establecido clínicamente para el tratamiento de grandes defectos del cartílago hialino de la rodilla, el mantenimiento de buenos resultados clínicos incluso 10 a 20 años después de la implantación 6. En los últimos años, la implantación de condrocitos autólogos se sometió a una rápida progresión. El uso de un tridimensional de colágeno-matriz artificial en el que las células se vuelven a plantar posteriormente se hizo más y más popular 7-9. MACT comprende de dos procedimientos quirúrgicos: En primer lugar, con el fin de recoger los condrocitos, una biopsia de cartílago necesita ser realizada a partir de una zona de cartílago no soporte de peso de tél articulación de la rodilla. Entonces, los condrocitos se extraen, se purificaron y se expandieron a un número suficiente de células in vitro. Los condrocitos se sembraron a continuación sobre una matriz tridimensional y pueden ser posteriormente reimplantados. Cuando se prepara un implante de tejido modificado, la tasa de proliferación y la capacidad de diferenciación son cruciales para una exitosa regeneración de los tejidos 10. Se cree que el uso de una matriz tridimensional como un portador de células para apoyar estas características celulares 11. El siguiente protocolo se resumir y demostrar una técnica para el aislamiento de condrocitos a partir de biopsias de cartílago, su proliferación in vitro y su siembra en una matriz en 3D (Condro-Gide, Biomateriales Geistlich, Wollhusen, Suiza). Finalmente, se describirá la implantación de las célula-matriz-constructos en defectos condrales creadas artificialmente de articulación de la rodilla de un conejo. Esta técnica se puede utilizar como un entorno experimentalpara experimentos adicionales de la reparación del cartílago.

La técnica quirúrgica descrita permite un éxito en el aislamiento y la implantación de condrocitos autólogos en un defecto condral artificial. La configuración experimental dio como resultado una integración con éxito del implante en el cartílago circundante. Después de 12 semanas in vivo, el defecto condral se llenó por tejido de reparación con una superficie homogénea e intacto, lo que reduce el estrés de cizallamiento y el daño al implante (Figura 4).</stro...

Watch this Scientific Journal Video about Matrix-assisted Autologous Chondrocyte Transplantation for Remodeling and Repair of Chondral Defects in a Rabbit Model at JoVE.com

Matrix-assisted Autologous Chondrocyte Transplantation for Remodeling and Repair of Chondral Defects in a Rabbit Model

Se describe una técnica experimental para el tratamiento de defectos condrales en la articulación de la rodilla del conejo. La implantación de condrocitos autólogos sembrados en una matriz es un método bien aceptado para la remodelación y reparación de lesiones del cartílago articular que proporcionan resultados satisfactorios a largo plazo. Matrix asistida por el trasplante de condrocitos autólogos (MACT) ofrece un método de implantación estandarizada y clínicamente establecida.

Matrix asistida por trasplante de condrocitos autólogos para la remodelación y reparación de defectos condrales en un modelo de conejo

Articular cartilage defects are considered a major health problem because articular cartilage has a limited capacity for self-regeneration 1. Untreated ...

matrix-assisted-autologous-chondrocyte-transplantation-for-remodeling

Research

JoVE Journal

Medicine

14.3K vistas.  Klinikum rechts der Isar der Technischen Universität München. Se describe una técnica experimental para el tratamiento de defectos condrales en la articulación de la rodilla del conejo. La implantación de condrocitos autólogos sembrados en una matriz es un método bien aceptado para la remodelación y reparación de lesiones del cartílago articular que proporcionan resultados satisfactorios a largo plazo. Matrix asistida por el trasplante de condrocitos autólogos (MACT) ofrece un método de implantación estandarizada y clíni...

Video: Matrix-assisted Autologous Chondrocyte Transplantation for Remodeling and Repair of Chondral Defects in a Rabbit Model

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

Un modelo de ratón En el útero Trasplante

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

Investigación

Medicina

Development of Obliterative Bronchiolitis in a Murine Model of Orthotopic Lung Transplantation

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and standardized not only for the study of allo-rejection but also between syngeneic strains for examining mechanisms of ischemia-reperfusion injury after lung transplantation. Although the application of the rat and other large animal model 2 contributed significantly to the elucidation of these studies, the scope of those investigations is limited by the scarcity of knockout and transgenic rats. Due to no effective therapies for obliterative bronchiolitis, the leading cause of death in lung transplant patients, there has been an intensive search for pre-clinical models that replicate obliterative bronchiolitis. The tracheal allograft model is the most widely used and may reproduce some of the histopathologic features of obliterative bronchiolitis 3. However, the lack of an intact vasculature with no connection to the recipient's conducting airways, and incomplete pathologic features of obliterative bronchiolitis limit the utility of this model 4. Unlike transplantation of other solid organs, vascularized mouse lung transplants have only recently been reported by Okazaki and colleagues for the first time in 2007 5. Applying the basic principles of the rat lung transplant, our lab initiated the obliterative bronchiolitis model using minor histoincompatible antigen murine orthotopic single-left lung transplants which allows the further study of obliterative bronchiolitis immunopathogenesis6.

Obliterative bronchiolitis is the key impediment to the long-term survival of lung transplant recipients and the lack of a robust preclinical model precludes examining obliterative bronchiolitis immunopathogenesis. Unlike other solid organ transplants, vascularized mouse lung transplantation has only recently been developed. Here we show our independently developed obliterative bronchiolitis model after murine orthotopic single-lung transplantation.

El desarrollo de bronquiolitis obliterante en un modelo murino de trasplante de pulmón ortotópico

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and ...

A Mouse Fetal Skin Model of Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

Un ratón fetal Modelo de la piel de la reparación de heridas sin cicatrices

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

A Rat Carotid Balloon Injury Model to Test Anti-vascular Remodeling Therapeutics

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is also a valuable model system to test, and to evaluate therapeutics and drugs that negate maladaptive remodeling in the vessel. The injury, or barotrauma, in the vessel lumen caused by an inflated balloon via an inserted catheter induces subsequent neointimal growth, often leading to hyperplasia or thickening of the vessel wall that narrows, or obstructs the lumen. The method described here is sufficiently sensitive, and the results can be obtained in relatively short time (2 weeks after the surgery). The efficacy of the drug or therapeutic against the induced-remodeling can be evaluated either by the post-mortem pathological and histomorphological analysis, or by ultrasound sonography in live animals. In addition, this model system has also been used to determine the therapeutic window or the time course of the administered drug. These studies can leadto the development of a better administrative strategy and a better therapeutic outcome. The procedure described here provides a tool for translational studies that bring drug and therapeutic candidates from bench research to clinical applications.

The rat carotid balloon injury model described below allows researchers to evaluate drugs or therapeutics that negate injury-induced arterial hyperplasia. Detailed pre-surgical preparation, surgical procedure, and post-surgical cares of the animal are described.

Una rata carótida lesión con el balón modelo para probar Terapéutica Remodelación Anti-vasculares

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is ...

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the treatment of many hematopoietic diseases in humans, as their use offers stable transgene expression due to the vector&#39;s ability to integrate into the host genome. This method demonstrates the application of ex vivo gene therapy of hepatocytes to a large animal model of hereditary tyrosinemia type I. This process consists of 1) isolation of primary hepatocytes from the autologous donor/recipient animal, 2) ex vivo gene delivery via hepatocyte transduction with a lentiviral vector, and 3) autologous transplant of corrected hepatocytes via portal vein injection. Success of the method generally relies upon efficient and sterile removal of the liver resection, careful handling of the excised specimen for isolation of viable hepatocytes sufficient for re-engrafting, high-percentage transduction of the isolated cells, and aseptic surgical procedures throughout to prevent infection. Technical failure at any of these steps will result in low yield of viable transduced hepatocytes for autologous transplant or infection of the donor/recipient animal. The pig model of human type 1 hereditary tyrosinemia (HT-1) chosen for this approach is uniquely amenable to such a method, as even a small percentage of engraftment of corrected cells will lead to repopulation of the liver with healthy cells based on a powerful selective advantage over native-diseased hepatocytes. Although this growth selection will not be true for all indications, this approach is a foundation for expansion into other indications and allows for manipulation of this environment to address additional diseases, both within the liver and beyond, while controlling for exposure to viral vector and opportunity for off-target toxicity and tumorigenicity.

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

This protocol is intended to describe porcine hepatocyte isolation and ex vivo gene delivery to cure models of metabolic diseases via autologous cell transplantation. Although this particular model enjoys unique advantages that favor successful therapy, the application is a relevant foundation to address additional diseases and indications.

Terapia del Gene mediada por vectores lentivirales de hepatocitos Ex Vivo para trasplante autólogo en cerdos

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the ...

Transthoracic Echocardiographic Examination in the Rabbit Model

Large animal models such as the rabbit are valuable for translational preclinical research. Rabbits have a similar cardiac electrophysiology compared to that of humans and that of other large animal models such as dogs and pigs. However, the rabbit model has the additional advantage of lower maintenance costs compared to other large animal models. The longitudinal evaluation of cardiac function using echocardiography, when appropriately implemented, is a useful methodology for preclinical assessment of novel therapies for heart failure with reduced ejection fraction (e.g. cardiac regeneration). The correct use of this non-invasive tool requires the implementation of a standardized examination protocol following international guidelines. Here we describe, step by step, a detailed protocol supervised by veterinary cardiologists for performing echocardiography in the rabbit model, and demonstrate how to correctly obtain the different echocardiographic views and imaging planes, as well as the different imaging modes available in a clinical echocardiography system routinely used in human and veterinary patients.

Here we describe, step by step, a detailed protocol for performing echocardiography in the rabbit model. We show how to correctly obtain the different echocardiographic views and imaging planes, as well as the different imaging modes available in a clinical echocardiography system routinely used in human and veterinary patients.

Examen Ecocardiográfico Transtorácico en el Modelo Conejo

Large animal models such as the rabbit are valuable for translational preclinical research. Rabbits have a similar cardiac electrophysiology compared to ...

Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These conditions include retinitis pigmentosa (RP) and advanced forms of age-related macular degeneration (AMD), such as geographic atrophy (GA). Together, these disorders represent a significant proportion of currently untreatable blindness globally. These unmet medical needs have generated heightened academic interest in developing methods of RPE replacement. Among the animal models commonly utilized for preclinical testing of therapeutics, the non-human primate (NHP) is the only animal model that has a macula. As it shares this anatomical similarity with the human eye, the NHP eye is an important and appropriate preclinical animal model for the development of advanced therapy medicinal products (ATMPs) such as RPE cell therapy.
This manuscript describes a method for the submacular transplantation of an RPE monolayer, cultured on a polyethylene terephthalate (PET) cell carrier, underneath the macula onto a surgically created RPE wound in immunosuppressed NHPs. The fovea-the central avascular portion of the macula-is the site of the greatest mechanical weakness during the transplantation. Foveal trauma will occur if the initial subretinal fluid injection generates an excessive force on the retina. Hence, slow injection under perfluorocarbon liquid (PFCL) vitreous tamponade is recommended with a dual-bore subretinal injection cannula at low intraocular pressure (IOP) settings to create a retinal bleb. 
Pretreatment with an intravitreal plasminogen injection to release parafoveal RPE-photoreceptor adhesions is also advised. These combined strategies can reduce the likelihood of foveal tears when compared to conventional techniques. The NHP is a key animal model in the preclinical phase of RPE cell therapy development. This protocol addresses the technical challenges associated with the delivery of RPE cellular therapy in the NHP eye.

The non-human primate (NHP) is an ideal model for studying human retinal cellular therapeutics due to the anatomical and genetic similarities. This manuscript describes a method for submacular transplantation of retinal pigment epithelial cells in the NHP eye and strategies to prevent intraoperative complications associated with macular manipulation.

Trasplante de epitelio pigmentario retiniano en un modelo de primate no humano para enfermedades degenerativas de la retina

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These ...

A Simplified Model for Heterotopic Heart Valve Transplantation in Rodents

There is an urgent clinical need for heart valve replacements that can grow in children. Heart valve transplantation is proposed as a new type of transplant with the potential to deliver durable heart valves capable of somatic growth with no requirement for anticoagulation. However, the immunobiology of heart valve transplants remains unexplored, highlighting the need for animal models to study this new type of transplant. Previous rat models for heterotopic aortic valve transplantation into the abdominal aorta have been described, though they are technically challenging and costly. For addressing this challenge, a renal subcapsular transplant model was developed in rodents as a practical and more straightforward method for studying heart valve transplant immunobiology. In this model, a single aortic valve leaflet is harvested and inserted into the renal subcapsular space. The kidney is easily accessible, and the transplanted tissue is securely contained in a subcapsular space that is well vascularized and can accommodate a variety of tissue sizes. Furthermore, because a single rat can provide three donor aortic leaflets and a single kidney can provide multiple sites for transplanted tissue, fewer rats are required for a given study. Here, the transplantation technique is described, providing a significant step forward in studying the transplant immunology of heart valve transplantation.

This protocol describes a simple and efficient method for the transplantation of aortic valve leaflets under the renal capsule to allow for the study of alloreactivity of heart valves.

Un modelo simplificado para el trasplante heterotópico de válvulas cardíacas en roedores

There is an urgent clinical need for heart valve replacements that can grow in children. Heart valve transplantation is proposed as a new type of ...

Left Lung Orthotopic Transplantation in a Juvenile Porcine Model for ESLP

Lung transplantation is the gold-standard treatment for end-stage lung disease, with over 4,600 lung transplantations performed worldwide annually. However, lung transplantation is limited by a shortage of available donor organs. As such, there is high waitlist mortality. Ex situ lung perfusion (ESLP) has increased donor lung utilization rates in some centers by 15%-20%. ESLP has been applied as a method to assess and recondition marginal donor lungs and has demonstrated acceptable short- and long-term outcomes following transplantation of extended criteria donor (ECD) lungs. Large animal (in vivo) transplantation models are required to validate ongoing in vitro research findings. Anatomic and physiologic differences between humans and pigs pose significant technical and anesthetic challenges. An easily reproducible transplant model would permit the&#160;in vivo&#160;validation of current ESLP strategies and the preclinical evaluation of various interventions designed to improve donor lung function. This protocol describes a porcine model of orthotopic left lung allotransplantation. This includes anesthetic and surgical techniques, a customized surgical checklist, troubleshooting, modifications, and the benefits and limitations of the approach.

This protocol describes a juvenile porcine model of orthotopic left lung allotransplantation designed for use with ESLP research. Focus is made on anesthetic and surgical techniques, as well as critical steps and troubleshooting.

Trasplante ortotópico de pulmón izquierdo en un modelo porcino juvenil para LESP

Lung transplantation is the gold-standard treatment for end-stage lung disease, with over 4,600 lung transplantations performed worldwide annually. ...

Transcatheter Pulmonary Valve Replacement from Autologous Pericardium with a Self-Expandable Nitinol Stent in an Adult Sheep Model

Transcatheter pulmonary valve replacement has been established as a viable alternative approach for patients suffering from right ventricular outflow tract or bioprosthetic valve dysfunction, with excellent early and late clinical outcomes. However, clinical challenges such as stented heart valve deterioration, coronary occlusion, endocarditis, and other complications must be addressed for lifetime application, particularly in pediatric patients. To facilitate the development of a lifelong solution for patients, transcatheter autologous pulmonary valve replacement was performed in an adult sheep model. The autologous pericardium was harvested from the sheep via left anterolateral minithoracotomy under general anesthesia with ventilation. The pericardium was placed on a 3D shaping heart valve model for non-toxic cross-linking for 2 days and 21 h. Intracardiac echocardiography (ICE) and angiography were performed to assess the position, morphology, function, and dimensions of the native pulmonary valve (NPV). After trimming, the crosslinked pericardium was sewn onto a self-expandable Nitinol stent and crimped into a self-designed delivery system. The autologous pulmonary valve (APV) was implanted at the NPV position via left jugular vein catheterization. ICE and angiography were repeated to evaluate the position, morphology, function, and dimensions of the APV. An APV was successfully implanted in&#160;sheep J. In this paper, sheep J was selected to obtain representative results. A 30 mm APV with a Nitinol stent was accurately implanted at the NPV position without any significant hemodynamic change. There was no paravalvular leak, no new pulmonary valve insufficiency, or stented pulmonary valve migration. This study demonstrated the feasibility and safety, in a long-time follow-up, of developing an APV for implantation at the NPV position with a self-expandable Nitinol stent via jugular vein catheterization in an adult sheep model.

This study demonstrates the feasibility and safety of developing an autologous pulmonary valve for implantation at the native pulmonary valve position by using a self-expandable Nitinol stent in an adult sheep model. This is a step toward developing transcatheter pulmonary valve replacement for patients with right ventricular outflow tract dysfunction.

Reemplazo de la válvula pulmonar transcatéter del pericardio autólogo con un stent de nitinol autoexpandible en un modelo de oveja adulta

Transcatheter pulmonary valve replacement has been established as a viable alternative approach for patients suffering from right ventricular outflow ...