Los autores agradecen al Prof. Hendrik Tevaearai y el Departamento de Cirugía Cardiovascular de apoyo financiero a la parte inicial del estudio.

NOTA: Lewis ratas machos y hembras, de 200-220 g fueron alojados en condiciones de laboratorio estándar (12 horas de luz y oscuridad ciclo, agua ad libitum y alimento, IVC jaula). Todos los animales fueron tratados de acuerdo con las recomendaciones del la Ley suiza FELASA y en materia de protección animal. 1. Cell Preparación: Aislamiento de células madre mesenquimales de la médula ósea <ol><li> Anestesiar las ratas con isoflurano al 5% y O 2 5 L / min en una cámara de inducción durante 5 min. Coloque el hocico del animal en un cono de nariz conectado al sistema de anestesia. Realizar un dedo del pie o de la cola pellizcar para confirmar la sedación. </li><li> Quitar la piel de la pierna con unas tijeras cortando la piel desde el tobillo hasta la cadera. Quitar los músculos y cortar la arteria femoral por desangramiento animal. Exponer la articulación de la cadera y luxación de la cabeza del fémur. Es importante no dañar la cabeza del fémur. Coloque los huesos en PBS estéril en un tubo de plástico de 50 ml. </li><li> Retire todos los muscles y ligamentos de los huesos sin romperlos. NOTA: Es importante que el hueso se mantiene intacta para evitar la vinculación de la solución de enjuague o contaminación con etanol. Enjuague los huesos pelados en etanol al 70% durante 5 min. </li><li> Bajo la campana de flujo laminar estéril, cortar en ambos extremos de los huesos con tijeras. Enjuague la médula ósea mediante la inyección de PBS estéril desde el extremo del hueso. Recoger ósea en un tubo de 15 ml. Centrifugar 7 min a 300 x g. </li><li> Eliminar el sobrenadante. Suspender el sedimento en 3 ml de glóbulos rojos Tampón de lisis. Incubar la suspensión durante 1 min a temperatura ambiente antes de girar de nuevo durante 7 minutos a 300 x g. En un frasco de cultivo de 150 ml, añadir medio de cultivo celular estéril (395 ml de medio IMDM, 5 ml penicilina / estreptomicina, 100 ml de FBS) y sembrar las células. </li><li> Cambie medio del segundo día para eliminar las células no adherentes. <ol><li> Realizar cambios de medio cada dos días durante 2-3 semanas, hasta que se obtiene la cantidad de células deseada. </li><li> Por otra parte, preparar el biograft por semillaing las células sobre la matriz sólida según sea necesario y descrito en otra parte 3. </li></ol></li><li> El día de la implantación cardiaca, preparar las células o cosechar el biograft justo antes de la aplicación del epicardio. <ol><li> Recoger las células por medio de solución de desprendimiento celular Accutase. Contar las células. Llenar un tubo de centrífuga de 1,5 ml con el volumen de suspensión celular calculada de obtener una cantidad de células deseado. Centrifugar durante 7 min a 300 xg y eliminar el sobrenadante. </li><li> Como alternativa, recoger el biograft del medio de cultivo celular, se lava en PBS estéril, y mantenerse en medio de cultivo fresco sin suero. </li></ol></li></ol> 2. Primera toracotomía y LAD Ligadura <ol><li> Pese la rata. Encienda la almohadilla térmica a 37 ° C. Anestesiar la rata con 5% de isoflurano y 5 L / min 100% de O 2 en una cámara de inducción de 5-7 min. Realizar un dedo del pie o de la cola pellizcar para confirmar la sedación. Coloque la rata sobre el cojín eléctrico. </li><li> Intubar al animal con un 14T catéter iv. Conectar el catéter de intubación a un ventilador de roedores programado para 2,5 L / min de oxígeno, 2,5% de isoflurano, volumen corriente de 2 ml, y una frecuencia de respiración de 90 respiraciones / min. </li><li> Preparar una solución de buprenorfina a 0,1 mg / kg. Inyectar por vía subcutánea un tercio de la solución. Afeitarse la parte izquierda del tórax. Desinfectar con una solución de Betadine 1%. </li><li> Incisión en la piel perpendicular al esternón en el cuarto espacio intercostal. Separe las 3 capas de músculo torácico (pectoral mayor, ascendiendo pectoral y pectoral oblicuo externo). Abra el cuarto espacio intercostal (entre las costillas 4 y 5). </li><li> Utilice un pequeño retractor para separar las costillas y para exponer el corazón. Abra el pericardio cuidadosamente. Busque y ligar la arteria descendente anterior coronaria (LAD) con un 7,0 sutura 4 mm por debajo del atrio. </li><li> Cerrar el espacio intercostal con dos puntos de sutura utilizando 3,0 suturas. Coloque los dos suturas proximal y distalmente desde el esternón. En primer lugar, tighten la sutura distal. Cierre la tubería de escape del ventilador durante 2 segundos con el fin de inflar el pecho y evitar cualquier neumotórax. Apriete la segunda sutura. Coloque las capas de músculos en su lugar (no se necesitan puntos de sutura). Cierre la piel con una sutura de 5,0. </li><li> Desinfectar la sutura con una solución de Betadine 1%. Inyectar el resto de la solución de buprenorfina. Apague el sistema de anestesia. Retire el catéter de intubación. </li><li> Mantenga la rata en la jaula bajo una lámpara caliente durante 1-2 horas. Mantenga un termómetro en la jaula y controlar la distancia entre la lámpara caliente y la jaula con el fin de evitar el exceso de calentamiento. Volver a la rata en la unidad IVC. Inyectar por vía subcutánea Buprenorfina 0,05-0,1 mg / kg mensaje -operatively cada 6-12 h durante 48 h. </li></ol> 3. Administración epicárdica de la Vía Tratamiento Segundo toracotomía <ol><li> Repita los pasos 2.1 a 2.3. </li><li> Incisión en la piel perpendicular al esternón en el quinto espacio intercostal. Se separa la capa 3s de los músculos torácicos (pectoral mayor, ascendiendo pectoral y pectoral oblicuo externo). Abra el quinto espacio intercostal (entre las costillas 5 y 6). </li><li> Utilice un pequeño retractor para separar las costillas y para exponer el corazón. Si hay alguna adherencia, retire con cuidado con unas pinzas finas. </li><li> Localizar el área de infarto que aparece como un área pálido por debajo de la ligadura. NOTA: Cuando sea necesario, utilice un trozo de 10 cm de sutura (7.0) que se inserta en el ápice para visualizar mejor el ventrículo izquierdo. Tire suavemente a la sutura mantenido ligeramente por una abrazadera para exponer el corazón. </li><li> Aplicar uno de los siguientes tratamientos: <ol><li> Coloque el biograft en la superficie del corazón. Utilice la jeringa precargada sellador de fibrina precalentado a temperatura ambiente durante 10 min. Aplique una gota (50-100 l) de sellador de fibrina en el biograft utilizando una aguja de 25 G Luer. Verifique que el biograft está perfectamente sellada. </li><li> Por otra parte, aplicar el sedimento de células recogido con una punta de pipeta en la superficie of el corazón en el área de pálido por debajo de la ligadura. Aplicar una gota (50-100 l) del sellador de fibrina en el sedimento celular. </li></ol></li><li> Retire el trozo de sutura desde el vértice. </li><li> Repita los pasos 2.6 a 2.8. </li></ol>

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The authors have nothing to disclose.

La ligadura permanente de LAD causa daño irreversible del miocardio. El primer modelo animal fue descrito en 1960 4. Desde entonces, se ha considerado como un modelo estándar y adecuado para MI crónica. Su estabilidad y reproducibilidad permite la evaluación experimental de las terapias para el MI 5. Mejora de los procedimientos posteriores a la descripción inicial reportaron una tasa de mortalidad operatoria del 35-13% 6. Como era de esperar, la ligadur...

Desde finales del siglo 19, la enfermedad cardiovascular sigue siendo la principal causa de muerte en serie en los países industrializados. Entre ellos, enfermedad de la arteria coronaria representa la etiología principal. Los resultados de fase aguda en el infarto de miocardio (MI) y es seguido por la remodelación de mala adaptación que se desarrolla progresivamente hacia una fase crónica e insuficiencia cardíaca severa. A pesar de los últimos avances tecnológicos y terapéuticos significativos, la morbilidad y la mortalidad debido a la progresión de la insuficiencia cardiaca sigue creciendo 1. En este contexto, la terapia celular se ha ganado un creciente interés como una nueva opción terapéutica para detener la progresión de la enfermedad hacia la insuficiencia cardíaca y para estimular la capacidad regenerativa recientemente identificado del miocardio. Las investigaciones experimentales y clínicos han proporcionado evidencia convincente de los efectos beneficiosos obtenidos después de trasplante cardíaco de diversos tipos de células. Resultados principales incluyeron la mejora de cardifunción contráctil ac, disminuyó la remodelación ventricular izquierda, redujo el tamaño del infarto, y el aumento de la densidad vascular en la zona infartada. Sin embargo, la retención de número bajo de células después de la inyección de células se mantuvo un importante inconveniente. Asociación de células con un biomatriz para mejorar la entrega de la celda 2 han contribuido a alimentar el investigador y los intereses clínicos. La ligadura de la arteria descendente anterior coronaria (LAD) es un método de referencia para la MI en modelo animal pequeño que resulta en infarto transmural y una cicatriz madura. La terapia celular aplicada en la fase crónica de MI requiere una segunda intervención quirúrgica. Una esternotomía media se lleva a cabo por lo general para permitir la inyección intramiocárdica de las células o la implantación epicárdica del biografts. Tales procedimientos quirúrgicos invasivos aumentan la tasa de mortalidad, después de la cirugía el tiempo de recuperación, dolor, y el riesgo de infección. El abordaje mínimamente invasivo que aquí se presenta no sólo evita que ese sesgo, sino también proporciona oaccesibilidad ptimal del corazón para la aplicación del tratamiento. MI e implantación epicárdica de células asociadas a un tipo de gel biomatriz se realizan en un corazón que late a través de una toracotomía intercostal izquierdo.

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			<td height="21" style="height:21px;width:216px;">SURGIPRO II 7-0 Suture</td>
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			<td style="width:119px;">381267</td>
			<td></td>
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			<td height="42" style="height:42px;width:216px;">INSPIRA ADVANCED VOLUME CONTROLLED VENTILATOR</td>
			<td style="width:104px;">HARVARD APPARATUS</td>
			<td style="width:119px;">557058</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">DUMONT #7 Forceps</td>
			<td style="width:104px;">FST Germany</td>
			<td style="width:119px;">11274-20</td>
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			<td height="21" style="height:21px;width:216px;">Fibrin sealant</td>
			<td style="width:104px;">BAXTER</td>
			<td style="width:119px;">1501441</td>
			<td>TISSEEL Glue, Frozen -20&#176;C</td>
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			<td height="42" style="height:42px;width:216px;">CASTROVIEJO EYE SPECULA</td>
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			<td>use as retractor for the ribs</td>
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			<td height="21" style="height:21px;">IMDM Glutamax</td>
			<td>Gibco</td>
			<td>31980</td>
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			<td height="21" style="height:21px;">Pen/Strep</td>
			<td>Gibco</td>
			<td>15140</td>
			<td></td>
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			<td height="21" style="height:21px;">FBS</td>
			<td>PAA Clone</td>
			<td>&#160;&#160;&#160;&#160;&#160; A15-101</td>
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		<tr height="42">
			<td height="42" style="height:42px;">bone scissors</td>
			<td style="width:104px;">Fine Science Tols</td>
			<td style="width:119px;">16044-10</td>
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			<td height="21" style="height:21px;">Red Blood Cell Lysis Solution</td>
			<td>Gentra Systems</td>
			<td>D-50k1</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Accutase cell detachment solution</td>
			<td style="width:104px;">Stem cell technology</td>
			<td style="width:119px;">7920</td>
			<td></td>
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cell-based therapy for heart failure in rat: double thoracotomy for myocardial infarction and epicardial implantation of cells and biomatrix

Terapia celular cardíaca ha ganado cada vez más interés e implantación de biomateriales asociados a células se ha convertido en un problema importante para optimizar la entrega de las células del miocardio. Modelo de roedor de infarto de miocardio (IM) que consiste en arteria descendente anterior izquierda (LAD) ligadura comúnmente se ha realizado a través de una toracotomía; una segunda operación a corazón abierto a través de una esternotomía tradicionalmente se ha realizado para la aplicación epicárdica del tratamiento. Desde la descripción del modelo de la ligadura de LAD, la tasa de mortalidad después de la cirugía se ha reducido desde 35 hasta 13%, sin embargo la segunda cirugía se ha mantenido crítico. Con el fin de mejorar la recuperación después de la cirugía y reducir el dolor y la infección, se presentan procedimientos quirúrgicos mínimamente invasivos. Se realizaron dos toracotomías, la inicial para la ligadura de LAD y la segunda para la administración epicárdica tratamiento. Biografts que consisten en células asociadas con matrices de tipo sólido o de gel se aplica sobre la ar infartadaea. LAD ligadura resultado la pérdida de la función del corazón como se confirmó por ecocardiografía realizada después de 2 y 6 semanas. Tricrómico de Goldner tinción realizó en secciones del corazón confirmó la formación de cicatriz transmural. Primera y segunda cirugías resultaron en menos que 10% de mortalidad post-operatorio.

Todos los animales se recuperaron dentro de 1 hora después de una toracotomía. La cicatrización de la herida fue rápida. No se observó ninguna infección o edema. El doble toracotomía izquierda permitió un acceso óptimo al corazón (Figura 1). El dolor y la mortalidad después de la cirugía fueron bajos. El animal se recuperó rápidamente de la cirugía y aumentaron de peso (Figura 2). Kaplan Meir porcentaje de supervivencia fue del 96% para la pri...

Watch this Scientific Journal Video about Cell-based Therapy for Heart Failure in Rat: Double Thoracotomy for Myocardial Infarction and Epicardial Implantation of Cells and Biomatrix at JoVE.com

Cell-based Therapy for Heart Failure in Rat: Double Thoracotomy for Myocardial Infarction and Epicardial Implantation of Cells and Biomatrix

Implantación de un biograft para tratar el infarto de miocardio inducido por ligadura de LAD en un modelo de roedor ha requerido convencionalmente dos cirugías a corazón abierto. Con el fin de reducir la mortalidad y proporcionar las condiciones óptimas para la fijación de biomatrices sólidos gelatinosos y asociados con las células, se han desarrollado procedimientos mínimamente invasivos.

Terapia basada en células de la insuficiencia cardiaca en rata: Doble toracotomía para infarto de miocardio y epicardio implantación de células y Biomatrix

Cardiac cell therapy has gained increasing interest and implantation of biomaterials associated with cells has become a major issue to optimize myocardial ...

cell-based-therapy-for-heart-failure-rat-double-thoracotomy-for

Research

JoVE Journal

Bioengineering

11.8K vistas.  University of Fribourg.  Implantación de un biograft para tratar el infarto de miocardio inducido por ligadura de LAD en un modelo de roedor ha requerido convencionalmente dos cirugías a corazón abierto. Con el fin de reducir la mortalidad y proporcionar las condiciones óptimas para la fijación de biomatrices sólidos gelatinosos y asociados con las células, se han desarrollado procedimientos mínimamente invasivos. 

Video: Cell-based Therapy for Heart Failure in Rat: Double Thoracotomy for Myocardial Infarction and Epicardial Implantation of Cells and Biomatrix

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or bioartificial scaffold materials, entrapment of cardiac myocytes in hydrogels such as fibrin or collagen and stacking of myocyte monolayers 1. These concepts aim at restoration of compromised cardiac function (e.g. after myocardial infarction) or as experimental models (e.g. predictive toxicology and substance screening or disease modelling). Precise monitoring of cell survival after implantation of engineered heart tissue (EHT) has now become possible using in-vivo bioluminescence imaging (BLI) techniques 2. Here we describe the generation of fibrin-based EHT from a transgenic rat strain with ubiquitous expression of firefly luciferase (ROSA/luciferase-LEW Tg; 3). Implantation is performed into the greater omentum of different rat strains to assess immune responses of the recipient organism following EHT implantation. Comparison of results generated by BLI and the Enzyme Linked Immuno Spot Technique (ELISPOT) confirm the usability of BLI for the assessment of immune responses.

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT

This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats.

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 ...

Investigación

Bioingeniería

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Trasplante De InducidaS Pluripotentes Derivadas De Células Madre Mesoangioblastos Como Progenitores Miógenos En Modelos De Ratón De La Regeneración Muscular

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. While the outer surface membrane and associated TATS membrane components appear to be continuous, there are substantial differences in lipid and protein content. In ventricular myocytes (VMs), certain TATS components are highly abundant contributing to rectilinear tubule networks and regular branching 3D architectures. It is thought that peripheral TATS components propagate action potentials from the cell surface to thousands of remote intracellular sarcoendoplasmic reticulum (SER) membrane contact domains, thereby activating intracellular Ca2+ release units (CRUs). In contrast to VMs, the organization and functional role of TATS membranes in atrial myocytes (AMs) is significantly different and much less understood. Taken together, quantitative structural characterization of TATS membrane networks in healthy and diseased myocytes is an essential prerequisite towards better understanding of functional plasticity and pathophysiological reorganization. Here, we present a strategic combination of protocols for direct quantitative analysis of TATS membrane networks in living VMs and AMs. For this, we accompany primary cell isolations of mouse VMs and/or AMs with critical quality control steps and direct membrane staining protocols for fluorescence imaging of TATS membranes. Using an optimized workflow for confocal or superresolution TATS image processing, binarized and skeletonized data are generated for quantitative analysis of the TATS network and its components. Unlike previously published indirect regional aggregate image analysis strategies, our protocols enable direct characterization of specific components and derive complex physiological properties of TATS membrane networks in living myocytes with high throughput and open access software tools. In summary, the combined protocol strategy can be readily applied for quantitative TATS network studies during physiological myocyte adaptation or disease changes, comparison of different cardiac or skeletal muscle cell types, phenotyping of transgenic models, and pharmacological or therapeutic interventions.

In cardiac myocytes, tubular membrane structures form intracellular networks. We describe optimized protocols for i) isolation of myocytes from mouse heart including quality control, ii) live cell staining for state-of-the-art fluorescence microscopy, and iii) direct image analysis to quantify the component complexity and the plasticity of intracellular membrane networks.

Análisis de Redes Tubular membrana en miocitos cardiacos de aurículas y los ventrículos

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. ...

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 ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

Evaluación de la proliferación del Keratinocyte en dos y tres dimensiones substratos de colágeno de tipo I

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...

Magnetic-, Acoustic-, and Optical-Triple-Responsive Microbubbles for Magnetic Hyperthermia and Pothotothermal Combination Cancer Therapy

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has been challenged. Here, we fabricate iron oxide nanoparticle shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform. Iron oxide nanoparticles serve as both magnetic and photothermal agents. Once intravenously injected, NSMs can be magnetically guided to the tumor site. Ultrasound triggers the release of iron oxide nanoparticles, facilitating the penetration of nanoparticles deep into the tumor due to the cavitation effect of microbubbles. Thereafter, magnetic hyperthermia and photothermal therapy can be performed on the tumor for combinational cancer therapy, a solution for cancer resistance due to the tumor heterogeneity. In this protocol, the synthesis and characterization of NSMs including structural, chemical, magnetic and acoustic properties were performed. In addition, the anti-cancer efficacy by thermal therapy was investigated using in vitro cell cultures. The proposed delivery strategy and combination therapy holds great promise in cancer treatment to improve both delivery and anticancer efficacies.

Presented here is a protocol for the fabrication of iron oxide nanoparticle-shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform for magnetic hyperthermia and photothermal combination cancer therapy.

Microburbujas magnéticas, acústicas y ópticas de triple respuesta para la hipertermia magnética y la terapia combinada de cáncer potototérmico

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has ...

Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from human induced pluripotent stem cells (hiPSCs), which are developed towards the goal of clinical use. HiPSC-derived cardiomyocytes, endothelial cells, and vascular mural cells are mixed with gel matrix and then poured into a polydimethylsiloxane (PDMS) tissue mold with rectangular internal staggered posts. By culture day 14 ECTs mature into a 1.5 cm x 1.5 cm mesh structure with 0.5 mm diameter myofiber bundles. Cardiomyocytes align to the long-axis of each bundle and spontaneously beat synchronously. This approach can be scaled up to a larger (3.0 cm x 3.0 cm) mesh ECT while preserving construct maturation and function. Thus, mesh-shaped ECTs generated from hiPSC-derived cardiac cells may be feasible for cardiac regeneration paradigms.

The present protocol generates mesh-shaped engineered cardiac tissues containing cardiovascular cells derived from human induced pluripotent stem cells to allow the investigation of cell implantation therapy for heart diseases.

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

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from ...

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering.

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

Trasplante de un parche bioimpreso 3D en un modelo murino de infarto de miocardio

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...

Quantification of Mouse Heart Left Ventricular Function, Myocardial Strain, and Hemodynamic Forces by Cardiovascular Magnetic Resonance Imaging

Mouse models have contributed significantly to understanding genetic and physiological factors involved in healthy cardiac function, how perturbations result in pathology, and how myocardial diseases may be treated. Cardiovascular magnetic resonance imaging (CMR) has become an indispensable tool for a comprehensive in vivo assessment of cardiac anatomy and function. This protocol shows detailed measurements of mouse heart left ventricular function, myocardial strain, and hemodynamic forces using 7-Tesla CMR. First, animal preparation and positioning in the scanner are demonstrated. Survey scans are performed for planning imaging slices in various short- and long-axis views. A series of prospective ECG-triggered short-axis (SA) movies (or CINE images) are acquired covering the heart from apex to base, capturing end-systolic and end-diastolic phases. Subsequently, single-slice, retrospectively gated CINE images are acquired in a midventricular SA view, and in 2-, 3-, and 4-chamber views, to be reconstructed into high-temporal resolution CINE images using custom-built and open-source software. CINE images are subsequently analyzed using dedicated CMR image analysis software.
Delineating endomyocardial and epicardial borders in SA end-systolic and end-diastolic CINE images allows for the calculation of end-systolic and end-diastolic volumes, ejection fraction, and cardiac output. The midventricular SA CINE images are delineated for all cardiac time frames to extract a detailed volume-time curve. Its time derivative allows for the calculation of the diastolic function as the ratio of the early filling and atrial contraction waves. Finally, left ventricular endocardial walls in the 2-, 3-, and 4-chamber views are delineated using feature-tracking, from which longitudinal myocardial strain parameters and left ventricular hemodynamic forces are calculated. In conclusion, this protocol provides detailed in vivo quantification of the mouse cardiac parameters, which can be used to study temporal alterations in cardiac function in various mouse models of heart disease.

This study describes a comprehensive cardiovascular magnetic resonance imaging (CMR) protocol to quantify the left ventricular functional parameters of the mouse heart. The protocol describes the acquisition, post-processing, and analysis of the CMR images as well as assessment of different cardiac functional parameters.

Cuantificación de la función ventricular izquierda del corazón de ratón, la distensión miocárdica y las fuerzas hemodinámicas mediante imágenes de resonancia magnética cardiovascular

Mouse models have contributed significantly to understanding genetic and physiological factors involved in healthy cardiac function, how perturbations ...

Evaluation of Cardiac Contractility Modulation Therapy in 2D Human Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used in regulatory submissions. Here, we extend their use to cardiac medical device safety or performance assessments. We developed a novel method to evaluate cardiac medical device contractile properties in robustly contracting 2D hiPSC-CMs monolayers plated on a flexible extracellular matrix (ECM)-based hydrogel substrate. This tool enables the quantification of the effects of cardiac electrophysiology device signals on human cardiac function (e.g., contractile properties) with standard laboratory equipment. The 2D hiPSC-CM monolayers were cultured for 2-4 days on a flexible hydrogel substrate in a 48-well format.
The hiPSC-CMs were exposed to standard cardiac contractility modulation (CCM) medical device electrical signals and compared to control (i.e., pacing only) hiPSC-CMs. The baseline contractile properties of the 2D hiPSC-CMs were quantified by video-based detection analysis based on pixel displacement. The CCM-stimulated 2D hiPSC-CMs plated on the flexible hydrogel substrate displayed significantly enhanced contractile properties relative to baseline (i.e., before CCM stimulation), including an increased peak contraction amplitude and accelerated contraction and relaxation kinetics. Furthermore, the utilization of the flexible hydrogel substrate enables the multiplexing of the video-based cardiac-excitation contraction coupling readouts (i.e., electrophysiology, calcium handling, and contraction) in healthy and diseased hiPSC-CMs. The accurate detection and quantification of the effects of cardiac electrophysiological signals on human cardiac contraction is vital for cardiac medical device development, optimization, and de-risking. This method enables the robust visualization and quantification of the contractile properties of the cardiac syncytium, which should be valuable for nonclinical cardiac medical device safety or effectiveness testing. This paper describes, in detail, the methodology to generate 2D hiPSC-CM hydrogel substrate monolayers.

Here, we demonstrate a non-invasive cardiac medical device contractility evaluation method using 2D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) monolayers, plated on a flexible substrate, coupled with video-based microscopy. This tool will be useful for the in vitro evaluation of the contractile properties of cardiac electrophysiology devices.

Evaluación de la terapia de modulación de la contractilidad cardíaca en cardiomiocitos derivados de células madre humanas 2D

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used ...