This work was supported by National Institutes of Health Grant to A.S.M. (EY021784).

El siguiente protocolo cumple con las directrices de Animales Institucional de Cuidado y Uso Comité Thomas Jefferson University y con la Declaración de ARVO para el uso de animales en Vision Research. 1. Instalación y Preparación de lentes para la Cultura ex vivo de la herida <ol><li> Coloque tres platos de 100 mm de Petri en una campana de flujo laminar estéril. Llenar dos de los platos de Petri a medio camino con tampón Tris / Dextrosa (tampón TD; NaCl 140 mM, 5 mM KCl, 0,7 mM Na 2 PO 4, 5 mM D-glucosa, 8,25 mM Tris Base, pH a 7,4 con HCl) a TA , dejando la tercera vacío. Medios de cultivo Pre-caliente (Media suplementado con 199, 1% de L-glutamina y 1% de penicilina / estreptomicina) a 37 o C. Nota: El estándar de las heridas de curación medios de cultivo es libre de suero, como ocurre in vivo; sin embargo, los cultivos de reparación de la herida se pueden cultivar con éxito en condiciones de medios definidos que incluyen suero u otros factores. </li><li> Retire fértil d embrionariaay 15 White Leghorn huevos de pollo de la incubadora (celebrada en 37,7 ° C con suave balanceo) </li><li> Coloca el huevo seleccionado en la campana de flujo laminar y fuera de la cáscara limpia con etanol al 70% a partir de una botella de lavado. Llevar a cabo todos los procedimientos siguientes en condiciones asépticas en la campana de flujo laminar, el uso de soluciones e instrumentos estériles. </li><li> Grieta de huevo y colocar contenido en el vacío de 100 mm placa de Petri. Decapitar embrión utilizando pinzas estándar y tijeras finas. Coloque la cabeza del embrión de pollo en una placa de Petri que contiene tampón TD y disponer adecuadamente del resto del embrión. Opcionalmente mantener las cabezas de embrión de pollo en tampón TD por un corto período de tiempo, no más de 15 min. </li><li> Coloque la cabeza del embrión de pollo en una tapa de placa de Petri. Con unas pinzas de alta precisión, retire la lente junto con su humor vítreo adjunta del ojo en la siguiente secuencia. Apriete la parte posterior del ojo con las pinzas para crear una pequeña abertura en la parte posterior del ojo. </li><li> Luego, sujete el humor vítreo wITH pinzas y tirar suavemente en el vítreo con un movimiento de balanceo, el vítreo con el objetivo puesto serán desalojados del ojo. Coloque la lente / vítreo en la placa de Petri restante contiene tampón TD. Permita que los lentes se mantengan en tampón TD durante no más de 30 min. </li><li> Mover la lente a una nueva tapa de placa de petri bajo un microscopio de disección. Desde este punto de realizar todos los pasos bajo un microscopio de disección. Con unas pinzas de alta precisión cepillarse cuidadosamente cualquier cuerpo ciliar (células pigmentadas) que fueron desalojados con la lente usando el borde de la pinza, teniendo la precaución de no dañar el tejido objetivo. Nota: Extracción del cuerpo ciliar se asegura de que los tipos de células que no son endógenas a la lente no se incluyen en la cultura reparación de la herida. </li><li> Separar la lente del humor vítreo con pinzas de alta precisión pellizcando fuera del cuerpo vítreo de su asociación con la cápsula posterior del cristalino. </li><li> Uso de alta precisión fórceps transferir la lente en una pequeña gotade tampón TD (aproximadamente 200 l) en una placa de cultivo tisular de 35 mm. </li></ol> 2. Realización de Cirugía de Catarata Mock <ol><li> Oriente la lente en la gota de tampón TD en el plato 35 mm con el aspecto anterior de la lente hacia arriba. Nota: El anterior de la lente se identifica fácilmente por la presencia de un anillo denso en el tejido que toma nota de la frontera entre la parte anterior y la región ecuatorial del epitelio del cristalino. En contraste, hay una ausencia de marcas en la parte posterior de la cápsula de la lente a la que están unidos los células de fibra óptica. </li><li> El uso de dos pinzas de alta precisión hacer una pequeña incisión (aproximadamente 850μm) en el centro de la cápsula anterior de la lente, la membrana basal gruesa que rodea el tejido del cristalino, y su epitelio anterior del cristalino asociada, agarrando el tejido con uno fórceps en cada mano y tirando suavemente en direcciones opuestas. </li><li> Retire la masa celular de fibra, lo que constituye la mayor parte del tejido de la lente, dislodging desde sus archivos adjuntos al epitelio de la lente y que rodea la cápsula del cristalino por hidro-elución (un enfoque clásico utilizado en la cirugía de cataratas, modelada en la Figura 1A). </li><li> Llene una jeringa de 1 ml con una punta de la aguja 27.5 G con 300 l de tampón TD. Inserte la punta de la aguja en la incisión hecha en la cápsula anterior del cristalino, y aproximadamente a mitad de camino en la lente. </li><li> Presione suavemente la jeringa inyectar el tampón TD en la masa celular de fibra lente. Inyectar entre 50 y 200 TD l, y nunca más de 300 l. Observe la masa celular de fibra aflojando en sí de la cápsula epitelio y lente. </li><li> Con unas pinzas de alta precisión, retire la masa celular de fibra aflojado desde la lente a través del sitio de la incisión anterior. Nota: Este procedimiento deja la cápsula posterior del cristalino membrana basal a la cual las células de fibra se habían unido despojada de las células, y un epitelio del cristalino lesionado justo al lado de este sitio. </li></ol> 3. preparing la lente Herido de Ex Vivo Cultura <ol><li> Acoplar la bolsa capsular de la lente que resulta de la cirugía de cataratas se ha descrito anteriormente en la placa de cultivo, con el lado de células hasta, al hacer cinco cortes en la cara anterior de la bolsa capsular. </li><li> Cortar perpendicular a la zona de la incisión original a través del ecuador de la lente. Acoplar los resultantes cinco &quot;flaps&quot; de cápsula del cristalino con el epitelio adjunta en el lado de la cápsula placa de cultivo abajo, célula hacia arriba. Nota de la lente heridos ex vivo ahora debe asumir una estrella o la forma del flowerlike (véase la Figura 1B). </li><li> Para asegurar la cápsula para el plato, presione suavemente hacia abajo con las pinzas en cada punto de la estrella. Esto hará una pequeña hendidura en los cinco consejos más exteriores del explante y el resultado en apego sostenido al plato. Nota: Es posible dañar la cápsula durante este procedimiento y por lo que es importante para asegurar la cápsula al plato tan cerca de la punta de la flaps como sea posible, así como para hacer la menor cantidad de puntos de sujeción como sea posible (generalmente dos, máximo de tres por FLAP). </li><li> Retire el tampón TD desde el plato de 35 mm y reemplazarlo con 1,5 ml de medio de pre-calentado. Cubra el plato de 35 mm con su tapa y colocar en la incubadora (37 ° C, 5% de CO 2). </li></ol> 4. Separación de la Zona Central de Migración (CMZ), donde Re-epitelización de la zona de la herida de la Cápsula Posterior produce, desde el archivo adjunto original Zone (OAZ) de las células epiteliales de la lente, para el análisis cuantitativo. Nota: Las células comienzan a moverse en la región CMZ inmediatamente en respuesta a una lesión. Por día uno en la cultura suficientes células están migrando a través de la CMZ para el análisis molecular y bioquímico, después de la separación de la CMZ y la OAZ por micro-disección 23. Este protocolo consiste en la extracción de una solapa (OAZ) a la vez desde la zona de la herida de la cápsula. <ol><li> Observar un Demarcatión, claramente visible bajo el microscopio de disección, entre el OAZ y CMZ (véase la figura 2A). El uso de dos pinzas de alta precisión, sujete con ambas pinzas en el borde de la línea de OAZ / CMZ, uno justo al lado de la otra, a cada lado de la línea (ver la Figura 2A, B, flecha). </li><li> Con una mano / un fórceps en el lado CMZ siguen aferrarse a la cultura heridos, mientras que con la otra mano / pinzas, tire suavemente de la OAZ largo de la línea OAZ / CMZ. La CMZ separa fácilmente del OAZ largo de esta línea. Continuar por esta línea alrededor de toda la cultura hasta que las dos regiones están completamente separadas. </li><li> Estudiar las Oaz y CMZ fracciones separadas para el análisis molecular, tales como 24 o bioquímica análisis de ARN-Seq como western blot o co-inmunoprecipitación 23,25. </li></ol>

<ol>
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The authors declare that they have no competing financial interests.

Here is described a technique for preparing a culture model of wound repair that involves performing an ex vivo cataract surgery on chick embryo lenses after their removal from the eye. The lens epithelium responds to this clinically relevant wounding with a repair process that closely mimics that which occurs in vivo, and shares features with wound repair in other epithelial tissues2,4. While the protocol is straightforward and simple to follow, performing mock cataract surgery with embryoni...

El, simulacro de cirugía de catarata clínicamente relevante, ex vivo modelo de curación de heridas epiteliales aquí descrito fue desarrollado para proporcionar una herramienta para la investigación de los mecanismos que regulan la reparación de los tejidos epiteliales en respuesta a una lesión. Las principales características que fueron destinadas para la creación de este modelo incluyen 1) proporcionar condiciones que replican de cerca la respuesta in vivo para herir en un entorno cultural, 2) la facilidad de la modulación de los elementos reguladores de la reparación, y 3) capacidad de la imagen del proceso de reparación, en su totalidad, en tiempo real. El reto, por lo tanto, era crear un modelo de cultivo en el que era posible estudiar y manipular, reparación de la herida epitelial en microambiente nativa de las células. La disponibilidad de este modelo de herida de reparación de abre nuevas posibilidades para la identificación de las señales de señalización endógenos de la matriz de proteínas, citocinas y quimiocinas que regulan el proceso de reparación. Además, el modelo es ideal para examinar cómo unn epitelio es capaz de moverse como una lámina colectiva para volver a epitelizar el área de la herida 2,3, y para determinar el linaje de células mesenquimales líder en el borde de la herida que funciona en la dirección de la migración colectiva del epitelio lesionado 4. Este modelo también ofrece una plataforma con la que identificar terapias que podrían promover la cicatrización efectiva y prevenir la reparación de heridas aberrante 5. Ya hay una serie de modelos de heridas de reparación disponibles, tanto en la cultura como in vivo, que han proporcionado la mayor parte de lo que se conoce sobre el proceso de reparación de heridas hoy. En modelos de lesión de origen animal, tales como la córnea y la piel 6-12 13-17, hay la oportunidad de estudiar la respuesta del tejido a heridas en el contexto de todos los mediadores de reparación que podrían estar involucrados en el proceso, incluyendo las contribuciones de la vasculatura y el sistema nervioso. Sin embargo, hay limitaciones a la manipulación de la expericondiciones mentales en vivo, y aún no es posible llevar a cabo estudios de imagen de la respuesta de reparación in vivo, de forma continua en el tiempo. En contraste, la mayoría de los modelos de cultivo in vitro de reparación de la herida, tales como la herida de cero, pueden ser fácilmente manipulados y siguieron a lo largo del tiempo, pero carecen el contexto ambiental de estudiar la cicatrización de heridas en el tejido in vivo. Mientras que los modelos ex vivo ofrecen la ventaja de estudiar el proceso de reparación de la lesión continua en el tiempo en el contexto de microambiente de las células junto con la capacidad de modular los reguladores moleculares de reparación en cualquier punto en el proceso de tiempo, hay pocos modelos que se ajustan a estos parámetros. Aquí se describe un procedimiento para generar altamente reproducible ex vivo epitelial herida culturas que reproducen la respuesta de un tejido epitelial a una herida fisiológico de curación. Uso de la lente de embrión de pollo como una fuente de tejido, un ex vivo mock se lleva a cabo la cirugía de catarata. La lente es un tejido ideal para utilizar para estos estudios ya que es auto-contenida dentro de una cápsula gruesa membrana basal, avascular, no inervado, y libre de cualquier estroma asociado 18,19. En la enfermedad humana, la cirugía de catarata se ocupa de la pérdida de la visión debido a la opacificación de la lente, y consiste en la extirpación de la masa celular de fibra de lente, que comprende el grueso de la lente. Después de la cirugía de cataratas visión se restaura mediante la inserción de una lente intraocular artificial. El procedimiento de la cirugía de cataratas, a través de la eliminación de las células de fibra, induce una respuesta lesión en el epitelio de la lente adyacente, que responde por la reepitelización de la zona posterior de la cápsula del cristalino que había sido ocupada por las células de fibra. En la cirugía de cataratas, como en la mayoría de las respuestas de reparación de la herida, hay a veces se produce un resultado fibrótica aberrante a la respuesta de cicatrización de heridas, asociado con la aparición de miofibroblastos, que en la lente que se conoce como Posterior Capsule opacificación 20-22. Para generar el modelo de cicatrización de heridas cirugía de cataratas, un procedimiento de cirugía de cataratas es imitado en lentes retira del ojo de embrión de pollo para producir una lesión fisiológica. Remoción microquirúrgica de las células resultados de fibra de la lente en un área de la herida circular muy consistente rodeado por las células epiteliales de la lente. Esta población de células permanece firmemente unido a la cápsula de la membrana basal de la lente y se lesiona por el procedimiento quirúrgico. Las células epiteliales migran a la zona denudada de la membrana basal endógena para sanar la herida, dirigido por una población de células mesenquimales vimentina ricos conocidos en el proceso de reparación como células líder 1. Con este modelo de la respuesta de un epitelio a la lesión puede ser visualizado y siguió con el tiempo en el contexto de microambiente de las células fácilmente. Las células son fácilmente accesibles a las modificaciones de la expresión o activación de moléculas espera que juegue un papel en la reparación de heridas. Una característica poderosa del XXes el modelo es la capacidad de aislar y estudiar los cambios a la migración específica en el marco de la cicatrización de heridas. La capacidad de preparar un gran número de cultivos in vivo de edad emparejados ex cicatrización de heridas para los estudios es otra ventaja de este modelo. De este modo, este modelo de sistema ofrece una oportunidad única de separar los mecanismos de reparación de la herida y la terapéutica de las pruebas para determinar su efecto sobre el proceso de cicatrización de la herida. Se espera que el modelo de la cirugía de cataratas ex vivo simulacro de tener una amplia aplicación, proporcionando un recurso crítico para el estudio de los mecanismos de reparación del daño.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Fisher Scientific</td>
			<td>S271-3</td>
			<td>Use at 140mM in TD Buffer</td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Fisher Scientific</td>
			<td>P217-500</td>
			<td>Use at 5mM in TD Buffer</td>
		</tr>
		<tr>
			<td>Sodium Phosphate (Na2HPO4)</td>
			<td>Sigma</td>
			<td>S0876</td>
			<td>Use at .7mM in TD Buffer</td>
		</tr>
		<tr>
			<td>D-glucose (Dextrose)</td>
			<td>Fisher Scientific</td>
			<td>D16-500</td>
			<td>Use at 0.5mM in TD Buffer</td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Fisher Scientific</td>
			<td>BP152-1</td>
			<td>Use at 8.25mM in TD Buffer</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Fisher Scientific</td>
			<td>A144-500</td>
			<td>Use to pH TD buffer to 7.4</td>
		</tr>
		<tr>
			<td>Media 199</td>
			<td>GIBCO</td>
			<td>11150-059</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Corning/CellGro</td>
			<td>25-005-CI</td>
			<td>Use at 1% in Media199</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Corning/CellGro</td>
			<td>30-002-CI</td>
			<td>Use at 1% in Media199</td>
		</tr>
		<tr>
			<td>100mm petri dishes</td>
			<td>Fisher Scientific</td>
			<td>FB0875711Z</td>
			<td></td>
		</tr>
		<tr>
			<td>Stericup Filter Unit</td>
			<td>Millipore</td>
			<td>SCGPU01RE</td>
			<td>Use to filter sterilize Media</td>
		</tr>
		<tr>
			<td>Dumont #5 forceps (need 2)</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>35mm Cell Culture Dish</td>
			<td>Corning</td>
			<td>430165</td>
			<td></td>
		</tr>
		<tr>
			<td>27 Gauge 1mL SlipTip with precision glide needle</td>
			<td>BD</td>
			<td>309623</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors</td>
			<td>Fine Science Tools</td>
			<td>14058-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Forceps</td>
			<td>Fine Science Tools</td>
			<td>91100-12</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Other Items Needed: General dissection instruments,&#160; fertile white leghorn chicken eggs,&#160;</td>
		</tr>
		<tr>
			<td colspan="4">check egg incubator (humidified, 37.7&#176;C), laminar flow hood, binocular stereovision dissecting&#160;</td>
		</tr>
		<tr>
			<td>microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

establishment of a clinically relevant ex vivo mock cataract surgery model for investigating epithelial wound repair in a native microenvironment

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to follow and manipulate the wound response ex vivo in an environment that closely mimics that of epithelial tissue injury in vivo. This issue was addressed by creating a clinically relevant epithelial ex vivo injury-repair model based on cataract surgery. In this culture model, the response of the lens epithelium to wounding can be followed live in the cells&#8217; native microenvironment, and the molecular mediators of wound repair easily manipulated during the repair process. To prepare the cultures, lenses are removed from the eye and a small incision is made in the anterior of the lens from which the inner mass of lens fiber cells is removed. This procedure creates a circular wound on the posterior lens capsule, the thick basement membrane that surrounds the lens. This wound area where the fiber cells were attached is located just adjacent to a continuous monolayer of lens epithelial cells that remains linked to the lens capsule during the surgical procedure. The wounded epithelium, the cell type from which fiber cells are derived during development, responds to the injury of fiber cell removal by moving collectively across the wound area, led by a population of vimentin-rich repair cells whose mesenchymal progenitors are endogenous to the lens1. These properties are typical of a normal epithelial wound healing response. In this model, as in vivo, wound repair is dependent on signals supplied by the endogenous environment that is uniquely maintained in this ex vivo culture system, providing an ideal opportunity for discovery of the mechanisms that regulate repair of an epithelium following wounding.

Creado ex vivo modelo para estudiar el proceso de cicatrización de heridas en microambiente nativa de las células Para investigar los mecanismos implicados en la regulación de la cicatrización de heridas de un epitelio dentro microambiente nativa de las células, se ha creado un modelo de la cirugía de catarata clínicamente relevante ex vivo simulacro. Este modelo se crea a partir de tejido de lente que ofrece muchas ventajas, debido a sus propiedades intrínsecas: 1)...

Watch this Scientific Journal Video about Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment at JoVE.com

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment.

Establecimiento de un punto de vista clínico relevante<em&gt; Ex Vivo</em&gt; Catarata Mock Modelo de Cirugía en Investigando epitelial reparación de heridas en un microambiente Nativo

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to ...

establishment-clinically-relevant-ex-vivo-mock-cataract-surgery-model

Research

JoVE Journal

Developmental Biology

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

6.5K vistas.  Thomas Jefferson University. Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment. 

Video: Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

La captura de Reparación de Tejidos en pez cebra larvas con Time-lapse Brightfield Estereomicroscopía

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

Investigación

Biología del Desarrollo

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing cardiovascular progenitors during development and disease. While a number of factors have been implicated in epicardium-derived cell (EPDC) mobilization, the mechanisms governing their subsequent migration and differentiation are poorly understood. Here, we present in vitro and ex vivo strategies to study EPDC motility and differentiation. First, we describe a method of obtaining primary epicardial cells by outgrowth culture from the embryonic mouse heart. We also introduce a detailed protocol to assess three-dimensional migration of labeled EPDC in an organ culture system. We provide evidence using these techniques that genetic deletion of myocardin-related transcription factors in the epicardium attenuates EPDC migration. This approach serves as a platform to evaluate candidate modifiers of EPDC biology and could be used to develop genetic or chemical screens to identify novel regulators of EPDC mobilization that might be useful for cardiac repair.

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

Here, we describe methods for isolating primary mouse epicardial cells by an outgrowth culture assay and assessing the functional migration of epicardial-derived cells (EPDC) using an ex vivo heart culture system. These protocols are suitable for identifying genetic and chemical modulators of epicardial epithelial-to-mesenchymal transition (EMT) and motility.

Consecuencia del epicardio y Ensayo de Cultivo<em&gt; Ex Vivo</em&gt; Evaluación de la migración de células derivadas del epicardio

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing ...

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

An example of adenoviral gene therapy in the human diabetic organ-cultured corneas is presented towards the normalization of delayed wound healing and markedly reduced epithelial stem cell marker expression in these corneas. It also describes the optimization of this process in stem cell-enriched limbal epithelial cultures.

Terapia génica adenoviral para Diabetic Queratopatía: Efectos en la curación de heridas y Stem Cell expresión del marcador en córneas de órganos cultivados humanos y células epiteliales Limbal

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene ...

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of morphogenetic events, such as gastrulation2, neurulation3-5, somitogenesis6, heart bending7,8, and brain formation9-13, during early embryogenesis. Key to understanding morphogenetic processes is to follow them dynamically by time-lapse imaging. The acquisition of time-lapse movies of chick embryogenesis ex ovo has been limited either to short time windows or to the need for an incubator to control temperature and humidity around the embryo14. Here, we present a new technique to culture chick embryos ex ovo for high-resolution time-lapse imaging using transmitted light microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique (EC-culture)1 and allows for the culturing of chick embryos without the need for a climate chamber. The embryo is sandwiched between two identical filter paper carriers and is kept fully submerged in a simple, temperature-controlled medium covered by a layer of light mineral oil. Starting from the primitive streak stage (Hamburger-Hamilton stage 5, HH5)15 up to at least the 28-somite stage (HH16)15, embryos can be cultured with either their ventral or dorsal side up. This allows the acquisition of time-lapse movies covering about 30 hr of embryonic development. Representative time-lapse frames and movies are shown. Embryos are compared morphologically to an embryo cultured in the standard EC-culture.
The submerged filter paper sandwich provides a stable environment to study early dorsal and ventral morphogenetic processes. It also allows for live fluorescence imaging and micromanipulations, such as microsurgery, bead implantation, microinjection, gene silencing, and electroporation, and has a strong potential to be combined with immersion objectives for laser-based imaging (including light-sheet microscopy).

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Here, we present a new technique to culture chick embryos ex ovo for time-lapse imaging using transmitted light and fluorescence microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique1 that allows for the culturing of chick embryos fully-submerged in a liquid culture medium.

Un filtro de papel sumergido Sandwich para el largo plazo<em&gt; Ex Ovo</em&gt; Time-lapse de pollo temprano embriones

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of ...

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka and zebrafish larvae are widely used to analyze the behavior of bone cells during bone formation, degeneration, and repair. Their optical clarity allows the visualization of fluorescently labeled bone cells and fluorescent dyes bound to the mineralized skeletal matrix. Our lab has generated transgenic medaka fish that express the osteoclast-inducing factor Receptor Activator of Nuclear-factor &#954;B Ligand (RANKL) under the control of a heat&#160;shock-inducible promoter. Ectopic expression of RANKL results in the excess formation of activated osteoclasts, which can be visualized in reporter lines with nlGFP expression under the control of the cathepsin K (ctsk) promoter. RANKL induction and ectopic osteoclast formation leads to severe osteoporosis-like phenotypes. Compound transgenic medaka lines that express ctsk:nlGFP in osteoclasts, as well as mCherry under the control of the osterix (osx) promoter in premature osteoblasts, can be used to study the interaction of both cell types. This facilitates the in vivo observation of cellular behavior under conditions of bone degeneration and repair. Here, we describe the use of this system to test a drug commonly used in human osteoporosis therapy and describe a protocol for live imaging. The medaka model complements studies in cell culture and mice, and offers a novel system for the in vivo analysis of drug action in the skeletal system.

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Small laboratory fish have become popular models for bone research on the mechanisms underlying human bone disorders and for the screening of bone-modulating drugs. In this report, we describe a protocol to assess the effect of alendronate on bone cells in medaka larvae with osteoporotic lesions.

El tratamiento de drogas y<em&gt; En Vivo</em&gt; Imagen de osteoblasto-osteoclastos Interacciones en un modelo Medaka pescado osteoporosis

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka ...

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own body. The ability to grow bone de novo using a patient's own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient's own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.

Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

El aislamiento rápido de BMPR-IB + derivadas de tejido adiposo células estromales de uso en un Defecto La curación Modelo de calota

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own ...

A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor metastasis. In this assay, cells are grown to form a confluent monolayer and a mechanical wound is created by scratching with a device. Then the migration rate of the cells towards the denuded area can be monitored by imaging. Our 8-channel mechanical wounder is designed to tackle most of the problems associated with the cell migration assay. Firstly, our wounder can be easily sterilized by autoclaving or with common disinfectants. Secondly, the individual adjustable pins allow even contact with the cell culture plate so that sharp and reproducible wounds can be created. Thirdly, the guiding bars on both sides of the wounder ensure consistent wounding position in each well. The use of disposable plastic pipette tips for wounding can further provide better handling of the wounder as well as to minimize cross-contamination. In conclusion, our cell wounder can provide researchers with a user friendly and reproducible device for performing the cell migration assay using the standard 96-well culture plate.

Here, we present a protocol to perform a semi-high throughput cell migration assay on a 96-well cell culture plate. This protocol is a fast, simple and economical method to create consistent scratch wounds on a cell monolayer.

Un dispositivo para realizar la migración de células / Curación de Heridas en una placa de 96 pocillos

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor ...

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding and invasion. Current animal models for the study of T. cruzi allow limited observation of parasites in vivo, representing a challenge for understanding parasite behavior during the initial stages of infection in humans. This protozoan has a flagellar stage in both vector and mammalian hosts, but there are no studies describing its motility in vivo.The objective of this project was to establish a live vertebrate zebrafish model to evaluate T. cruzi motility in the vascular system. Transparent zebrafish larvae were injected with fluorescently labeled trypomastigotes and observed using light sheet fluorescence microscopy (LSFM), a noninvasive method to visualize live organisms with high optical resolution. The parasites could be visualized for extended periods of time due to this technique&#39;s relatively low risk of photodamage compared to confocal or epifluorescence microscopy. T. cruzi parasites were observed traveling in the circulatory system of live zebrafish in different-sized blood vessels and the yolk. They could also be seen attached to the yolk sac wall and to the atrioventricular valve despite the strong forces associated with heart contractions. LSFM of T. cruzi-inoculated zebrafish larvae is a valuable method that can be used to visualize circulating parasites and evaluate their tropism, migration patterns, and motility in the dynamic environment of the cardiovascular system of a live animal.

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

In this protocol, fluorescently labeled T. cruzi&#160;were injected into transparent zebrafish larvae, and parasite motility was observed in vivo using light sheet fluorescence microscopy.

Establecimiento de pez cebra Larval como modelo Animal para investigar Trypanosoma cruzi motilidad En Vivo

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding ...

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological questions in cell and developmental biology. In addition, organoids together with recent technical advances in gene editing and viral gene delivery promises to advance medical research and development of new drugs for treatment of diseases. Organoids grown in vitro in basement matrix provide powerful model systems for studying the behavior and function of various proteins and are well suited for live-imaging of fluorescent-tagged proteins. However, establishing the expression and localization of the endogenous proteins in ex vivo tissue and in in vitro organoids is important to verify the behavior of the tagged proteins. To this end we have developed and modified tissue isolation, fixation, and immuno-labeling protocols for localization of microtubules, centrosomal, and associated proteins in ex vivo intestinal tissue and in in vitro intestinal organoids. The aim was for the fixative to preserve the 3D architecture of the organoids/tissue while also preserving antibody antigenicity and enabling good penetration and clearance of fixative and antibodies. Exposure to cold depolymerizes all but stable microtubules and this was a key factor when modifying the various protocols. We found that increasing the ethylenediaminetetraacetic acid (EDTA) concentration from 3 mM to 30 mM gave efficient detachment of villi and crypts in the small intestine while 3 mM EDTA was sufficient for colonic crypts. The developed formaldehyde/methanol fixation protocol gave very good structural preservation while also preserving antigenicity for effective labeling of microtubules, actin, and the end-binding (EB) proteins. It also worked for the centrosomal protein ninein although the methanol protocol worked more consistently. We further established that fixation and immuno-labeling of microtubules and associated proteins could be achieved with organoids isolated from or remaining within the basement matrix.

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

We present protocols for isolation of intestinal 3D structures from in vivo tissue and in vitro basement matrix embedded organoids, and detail different fixation and staining protocols optimized for immuno-labeling of microtubule, centrosomal, and junctional proteins as well as cell markers including the stem cell protein Lgr5.

Inmuno-fluorescente etiquetado de microtúbulos y proteínas centrosómicas en tejido Intestinal Ex Vivo y 3D In Vitro Intestinal organitas

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological ...

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic age-related macular degeneration (AMD) which represents 90% of the cases. Therapies are needed to slow or retard the development of geographic atrophy (GA), and understanding Bruch&#39;s membrane pathology is part of this process. Alterations in human Bruch&#39;s membrane precede the progression of AMD by contributing to the damage of retinal pigment epithelial (RPE) cells. Given the lack of sufficient animal models to study AMD, ex vivo models of aged human Bruch&#39;s membrane serve as a useful tool to study the behavior of RPE cells from immortalized and primary cell lines as well as RPE lines derived from induced pluripotent stem cells (iPSCs). Here, we present a detailed method that allows one to determine the effects of RPE cell behavior seeded on harvested human Bruch&#39;s membrane explants from human donors, including attachment, apoptosis and proliferation, ability to phagocytize photoreceptor outer segments, establishment of polarity, and gene expression. This assay provides an ex vivo model of aged Bruch&#39;s membrane to assess the functional characteristics of RPE cells when seeded on aged/compromised extracellular matrix.

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch&#039;s Membrane

The goal of this protocol is to demonstrate the culturing of retinal pigment epithelial (RPE) cells on aged and/or diseased human Bruch&#39;s membrane. This method is suitable to study RPE cell behavior on a compromised extracellular matrix.

Cultivo de células epiteliales del pigmento retiniano en un modelo Ex Vivo de la membrana de Bruch humano envejecido

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic ...