The authors acknowledge support from a Translational Breast Cancer Research Grant (14-60-26-BROC to AB) from the Breast Cancer Research Foundation and the American Association for Cancer Research.

Procedimientos que utilizan los sujetos animales han sido aprobados por el Comité de Cuidado y Uso de Animales Institucional de la Universidad de Texas en Austin. 1. Preparación preoperatoria <ol><li> Anotar el peso corporal de cada conejo. Al igual que con todos los estudios preclínicos, monitorear pesos de los animales regularmente para evaluar la toxicidad potencial. </li><li> Antes de anestesiar el conejo, llenar un tubo cónico de 50 ml con gel de ultrasonido disponibles comercialmente y centrifugado a 500 xg durante 30 s; no debe haber burbujas visibles en el gel sobre la terminación de la centrifugación. </li><li> Administrar por vía subcutánea glicopirrolato en una dosis de 0,1 mg / kg y acepromacina por vía intramuscular a una dosis de 0,75 mg / kg. Espere 15-20 minutos para que el sedante para tener efecto durante el seguimiento de los signos vitales y el comportamiento. NOTA: El glicopirrolato es un agente anticolinérgico que previene la bradicardia y reduce respiratorias y gastrointestinales secreciones. Acepromazina es un sedante que sirve como un premedicatipor la anestesia. </li><li> Administrar 35 mg / kg de ketamina y 5 mg / kg de xilazina por vía subcutánea como anestésico. Sin embargo, como el operador vuelve más experimentado y puede lograr la entrega intraductal más rápidamente, disminuir las dosis de los fármacos a 15 mg / kg de ketamina y 3 mg / kg de xilazina por vía subcutánea; esto acortará el tiempo de anestesia y el tiempo requerido para que el animal recuperarse de la anestesia. </li><li> Comprobar y documentar la frecuencia cardíaca, la SPO 2, temperatura, frecuencia respiratoria, y el color membrana mucosa cada 15 minutos. Aplicar lubricante ocular en ambos ojos. NOTA: Sólo el personal que han recibido una formación adecuada y han sido aprobados por el IACUC de su institución debe administrar o supervisar la anestesia. El uso de un monitor de anestesia veterinaria puede ayudar en la adquisición de los signos vitales y se recomienda. El monitor de anestesia veterinaria, sin embargo, no reemplaza la necesidad de comprobar manualmente el animal cada 15 minutos. Ver las instrucciones del fabricante fo el uso adecuado de un monitor de veterinaria. </li><li> Verificar el inicio de la anestesia por una pizca dedo del pie suave; el conejo debe ser no responden antes de continuar. </li><li> afeitarse con cuidado el abdomen caudal del conejo en el área alrededor de la tercera y cuarta pares de pezones inguinales. </li><li> Con la mayor parte del pelo eliminado, aplicar una crema de depilación over-the-counter de la zona afeitada. Retire la crema 10 minutos después de la aplicación usando toallas de papel húmedas mojadas con agua tibia. NOTA: piel de conejo es muy frágil / sensibles. La crema de depilación debe ser utilizado por no más de 10 min. De hecho, es más seguro para probar un &quot;punto de prueba&quot; a los 5 minutos y sólo dejar actuar durante más tiempo si punto de prueba indica un tiempo más largo se necesita tiempo. </li><li> Limpie el área con gasas empapadas en alcohol para limpiar el sitio de la inyección. </li><li> Colocar el conejo en su dorso en una cubeta en forma de V forrada con una manta de agua caliente de recirculación y una almohadilla absorbente. </li></ol> 2. Preparation del agente de contraste <ol><li> Reconstituir el reactivo no específica contraste de acuerdo con las instrucciones del fabricante. Pipetear arriba y abajo suavemente para mezclar. NOTA: El agente de contraste utilizado en este protocolo es estable a temperatura ambiente durante 4 - 6 h después de la reconstitución. Agite suavemente el vial entre cada recuperación. NOTA: El volumen de solución requerido dependerá del número de conductos a inyectar. El conejo tiene 4 aberturas de los conductos por los pezones, y 0,2 ml de solución es suficiente para llenar un árbol ductal de un adulto New Zealand White Rabbit (Oryctolaguscuniculus). Por lo tanto, un volumen total de 0,8 ml puede ser entregado a una glándula mamaria. </li></ol> 3. Entrega intraductal <ol><li> Localizar los pezones apropiados para ser inyectados; Se recomiendan los pares 3 ° y 4 ° de tetinas inguinales, ya que se visualizan fácilmente cuando el animal se coloca en su dorso. </li><li> Cargar 0,2 ml de solución salina estéril al 0,9% enuna jeringa de tuberculina de 1 ml Luer-lock con una aguja 22 G. Deseche la aguja de 22 G una vez que la solución salina es en la jeringa y reemplazarlo con una aguja de calibre 25 estéril. limpiar suavemente el área con un 85% de alcohol isopropílico en una almohadilla de gasa. </li><li> Con el bisel de la aguja y la jeringa paralela al cuerpo del animal, insertar el bisel de la aguja en el lado de la tetina y lentamente inyectar 0,1-,2 ml de solución salina; esto permitirá una mejor visualización de las aberturas de los conductos. </li><li> Cargar 0,2 ml de solución inyectable en una jeringa de tuberculina de 1 ml de cierre Luer. </li><li> Mantenga el pezón suavemente con los dedos pulgar e índice y levantarlo ligeramente para posicionarlo para la inyección intraductal; una lupa ocular portátil puede ayudar en la visualización de las aberturas de los conductos. </li><li> Mientras se mantiene la posición elevada de la tetina, cannulate cuidadosamente el conducto de interés utilizando una aguja de punta roma 25 G. </li><li> Después de la punción, gire suavemente la jeringa Luer-lock en el centro del elemento romo-taguja de infusión ip hasta que quede bloqueado en su lugar. </li><li> Levantar el pezón e inyectar la solución lentamente para minimizar el daño potencial causado por movimiento rápido del fluido dentro del conducto; En ningún momento debe haber resistencia al inyectar la solución. </li></ol> 4. imágenes por ultrasonido <ol><li> Aplicar una cantidad generosa de gel de ultrasonido se centrifugaron para la piel de la zona de interés. Asegúrese de que no haya burbujas en el gel, ya que pondrá en peligro la calidad de la imagen. </li><li> Ajuste la profundidad de imagen de 6 mm. Coloque el transductor 21 MHz en contacto con el gel y escanear el área de interés en B-mode. Observe el medio de contraste en la región explorada incluyendo la apertura ductal y en todo el conducto. NOTA: Estos ajustes se han desarrollado para su uso con una máquina de ultrasonido fotoacústica específica. Consulte la Tabla de Materiales para obtener más detalles. Puede ser necesario ajustar la potencia de transmisión y la profundidad de formación de imágenes de otros sistemas de imagen para optimizar mamariala visualización de la glándula. </li><li> Retire el gel de ultrasonido de la piel del animal con una almohadilla de gasa. </li></ol> 5. Cuidado posoperatorio <ol><li> Observe el lugar de la inyección: no debe haber signos de traumatismo en la región del pezón o en el tejido circundante, e hinchazón en el área que rodea el pezón probablemente indica una inyección almohadilla de grasa mamaria en lugar de una inyección intraductal éxito. </li><li> Colocar el conejo en una posición esternal. Si es necesario, dar 0,2 mg / kg de yohimbina vía intravenosa en la vena marginal de la oreja; esto va a invertir el efecto de la xilazina y permitir que el animal se recupere más rápidamente de la anestesia. </li><li> Monitorear el conejo cada 15 minutos durante todo el período de recuperación hasta que el animal está alerta, sensible, y mantiene una posición esternal. NOTA: Este procedimiento no debería ocasionar daño tisular o inflamación. Si enrojecimiento o inflamación se observan, administrar una dosis de meloxicam 0,1-0,2 mg / kg PO una vez que el animal está alerta y capaztomar la medicación por vía oral. Póngase en contacto con el personal veterinario de la institución para obtener más instrucciones. </li></ol>

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The authors have nothing to disclose.

Este método de suministro intraductal a la glándula mamaria de conejo se puede usar para los reactivos de contraste de ultrasonido y muchas otras soluciones acuosas, incluyendo colorantes vitales y la terapéutica. Estudios previos han demostrado la entrega intraductal de hormonas 17, 18, 19. En modelos de roedores, la entrega intraductal de los ácidos nucleicos 8, quimioterapéuticos <sup class="xref...

<html> La entrega intraductal de agentes terapéuticos se ha estudiado en modelos de roedores y en los ensayos en humanos en fase inicial 3, 4, 5, 6, 11, 12. Un reciente estudio de Fase demostró la seguridad y la viabilidad de carboplatino intraductal o intraductal doxorrubicina liposomal pegilada en las mujeres que esperan la mastectomía para el tratamiento de cáncer invasivo 2. Protocolos anteriores para la entrega intraductal se han desarrollado para ratón y de rata glándulas mamarias 6, 7, 8, 9. Para fines de investigación, inyecciones de células tumorales intraductales y la entrega de vector lentiviral de oncogenes también se han realizado en modelos de roedoresref &quot;&gt; 13, 14, 15, 16. Sin embargo, un ideal modelo in vivo del proceso de entrega intraductal debe permitir el desarrollo de nuevas clases de compuestos terapéuticos y facilitar la evaluación preclínica. Las diferencias anatómicas entre roedores y humanos han complicado la traducción de estos estudios. A diferencia de los ratones, en el que cada conducto termina en un pezón separado, el de mama humano se compone de 5 a 9 sistemas ductales independientes, cada uno con una abertura separada terminando en el pezón. Conejo glándulas mamarias albergan cuatro sistemas ductales independientes, cada uno por separado accesible a través de uno de los cuatro orificios en una sola pezón. Un modelo de conejo es más compatible con la anatomía humana y permite el estudio de la administración de fármacos intraductal en un contexto más relevante. En este caso, se utilizan dos técnicas para evaluar la entrega intraductal. La co-administración de unacolorante vital permite la visualización a través de la piel y proporciona una confirmación simple y rápida del método. Para algunas aplicaciones, puede ser preferible mayor mapeo resolución de los conductos. Presentamos aquí un protocolo para la formación de imágenes por ultrasonido de los conductos a través de la entrega intraductal de un reactivo no específica contraste.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>MicroMarker non-targeted contrast reagent</td>
			<td>VisualSonics</td>
			<td>VS-11694</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer Lock 1mL Syringes</td>
			<td>BD</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycopyrrolate 0.2mg/mL</td>
			<td>Wedgewood Compounding Pharmacy</td>
			<td>GLYCOP-INJ013VC</td>
			<td>6 month shelf life, supply may be limited.&#160;</td>
		</tr>
		<tr>
			<td>Atropine Sulfate 0.5 mg/mL</td>
			<td>Animal Health International</td>
			<td>15320764</td>
			<td>If glycopyrrolate is unavailable. Not to be combined with glycopyrrolate.</td>
		</tr>
		<tr>
			<td>Ketamine HCL 100mg/mL</td>
			<td>Animal Health International</td>
			<td>21250699</td>
			<td><a href="http://www.animalhealthinternational.com/">http://www.animalhealthinternational.com/</a></td>
		</tr>
		<tr>
			<td>Acepromazine 10mg/mL</td>
			<td>Animal Health International</td>
			<td>17640541</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine 20mg/mL</td>
			<td>Animal Health International</td>
			<td>20101547</td>
			<td></td>
		</tr>
		<tr>
			<td>Yohimbine 0.2mg/mL</td>
			<td>Animal Health International</td>
			<td>14588965</td>
			<td></td>
		</tr>
		<tr>
			<td>Hair Removing Cream</td>
			<td>Veet</td>
			<td></td>
			<td>Sensitive skin solution. Available through local retailers.</td>
		</tr>
		<tr>
			<td>Blunt tip infusion needles</td>
			<td>Sai Infusion Technology</td>
			<td>B14-50</td>
			<td><a href="http://www.sai-infusion.com/collections/blunt-needles">http://www.sai-infusion.com/collections/blunt-needles</a></td>
		</tr>
		<tr>
			<td>Veterinary Pulse Oximeter</td>
			<td>EdanUSA</td>
			<td>VE-H100B</td>
			<td><a href="http://www.edanusa.com/Product/VE-H100B-Veterinary-Pulse-Oximeter.html">http://www.edanusa.com/Product/VE-H100B-Veterinary-Pulse-Oximeter.html</a></td>
		</tr>
		<tr>
			<td>Warm Water Pump</td>
			<td>Gaymar</td>
			<td>TP700</td>
			<td></td>
		</tr>
		<tr>
			<td>Warm Water Blanket</td>
			<td>Animal Health International</td>
			<td>21232696</td>
			<td>Maxi-Therm Lite Warming Pads</td>
		</tr>
		<tr>
			<td>Ultrasound system</td>
			<td>VisualSonics</td>
			<td>Vevo 2100</td>
			<td></td>
		</tr>
	</tbody>
</table>

intraductal delivery to the rabbit mammary gland

Tratamientos intraductales localizado para el cáncer de mama ofrecen ventajas potenciales, incluyendo la entrega eficiente con el tumor y la reducción de la toxicidad sistémica y los efectos adversos 1, 2, 3, 4, 5, 6, 7. Sin embargo, persisten algunos desafíos antes de que estos tratamientos pueden ser aplicados más ampliamente. El desarrollo y la validación de la terapéutica intraductales en un modelo animal apropiado facilitar el desarrollo de estrategias terapéuticas para los pacientes intraductales. Mientras que la glándula mamaria de ratón ha sido ampliamente utilizado como un sistema modelo de desarrollo mamario y la tumorigénesis, la anatomía es distinta de la glándula humano. Un modelo animal más grande, como el conejo, puede servir como un modelo mejor para la estructura de la glándula mamaria y desarrollo terapéutico intraductal. Cªontraste a ratones, en la que diez árboles ductales están distribuidas espacialmente a lo largo del eje del cuerpo, cada uno que termina en una tetina separada, la glándula mamaria de conejo se asemeja más a la glándula humana, con múltiples sistemas ductales se solapan y que salen a través de aberturas separadas en un pezón. A continuación, presentamos los métodos mínimamente invasivos para el suministro de reactivos directamente en el conducto mamario de conejo y para la visualización de la entrega de sí mismo con imágenes de ultrasonido de alta resolución.

Aquí, se muestra que la entrega intraductal de reactivos de contraste a los conductos mamarios de un conejo puede lograrse sin trauma al tejido (Figura 2). En conejos, cuatro sistemas ductales separadas convergen en un pezón y de este modo se puede acceder de forma individual y la imagen utilizando este método. aberturas ductales individuales se visualizaron fácilmente; en cuenta el marcado de una segunda abertura ductal adyacente al conducto canulado en la F...

Watch this Scientific Journal Video about Intraductal Delivery to the Rabbit Mammary Gland at JoVE.com

Intraductal Delivery to the Rabbit Mammary Gland

Here, we describe a technique for the localized delivery of reagents to the rabbit mammary gland via an intraductal injection. In addition, we describe a protocol for visualization and the confirmation of delivery by high-resolution ultrasound imaging of contrast agents.

Intraductal La entrega al conejo Glándula Mamaria

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and ...

intraductal-delivery-to-the-rabbit-mammary-gland

Research

JoVE Journal

Bioengineering

9.2K vistas.  The University of Texas at Austin. Here, we describe a technique for the localized delivery of reagents to the rabbit mammary gland via an intraductal injection. In addition, we describe a protocol for visualization and the confirmation of delivery by high-resolution ultrasound imaging of contrast agents. 

Video: Intraductal Delivery to the Rabbit Mammary Gland

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. 
The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.
Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

Multiparamétrico óptico Cartografía del corazón de conejo Langendorff perfundidos

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

Investigación

Bioingeniería

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

Diseño de un biorreactor de presión cíclica de la Ex Vivo Estudio de las válvulas aórtica del corazón

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively for tissue regeneration, a number of variables must be taken into account. These variables include: the total volume and surface area of the implantation site, the mechanical properties of the tissue and the tissue microenvironment, which includes the amount of vascularization and the components of the extracellular matrix. Therefore, the materials being used to deliver these cells must be biocompatible with a defined chemical composition while maintaining a mechanical strength that mimics the host tissue. These materials must also be permeable to oxygen and nutrients to provide a favorable microenvironment for cells to attach and proliferate. Chitosan, a cationic polysaccharide with excellent biocompatibility, can be easily chemically modified and has a high affinity to bind with in vivo macromolecules4-5. Chitosan mimics the glycosaminoglycan portion of the extracellular matrix, enabling it to function as a substrate for cell adhesion, migration and proliferation. In this study we utilize chitosan in the form of microspheres to deliver adipose-derived stem cells (ASC) into a collagen based three-dimensional scaffold6. An ideal cell-to-microsphere ratio was determined with respect to incubation time and cell density to achieve maximum number of cells that could be loaded. Once ASC are seeded onto the chitosan microspheres (CSM), they are embedded in a collagen scaffold and can be maintained in culture for extended periods. In summary, this study provides a method to precisely deliver stem cells within a three dimensional biomaterial scaffold.

A major hurdle in current stem cell therapies is determining the most effective method to deliver these cells to host tissues. Here, we describe a chitosan-based delivery method that is efficient and simple in approach, while allowing adipose-derived stem cells to maintain their multipotency.

La construcción de un hidrogel de colágeno para la entrega de la madre microesferas cargadas con células quitosano

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively ...

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant exogenous molecular probes into cells. This work reports a microfluidic electroporation platform capable of performing multiple molecule delivery to mammalian cells with precise and molecular-dependent parameter control. The system&#8217;s ability to isolate cells with uniform size distribution allows for less variation in electroporation efficiency per given electric field strength; hence enhanced sample viability. Moreover, its process visualization feature allows for observation of the fluorescent molecular uptake process in real-time, which permits prompt molecular delivery parameter adjustments in situ for efficiency enhancement. To show the vast capabilities of the reported platform, macromolecules with different sizes and electrical charges (e.g., Dextran with MW of 3,000 and 70,000 Da) were delivered to metastatic breast cancer cells with high delivery efficiencies (&#62;70%) for all tested molecules. The developed platform has proven its potential for use in the expansion of research fields where on-chip electroporation techniques can be beneficial.

A microfluidic vortex assisted electroporation platform was developed for sequential delivery of multiple molecules into identical cell populations with precise and independent dosage control. The system&#8217;s size based target cell purification step preceding electroporation aided to enhance molecular delivery efficiency and processed cell viability.

Vortex asistida Microscale Electroporador para entrega secuencial Molecular

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

Adaptación del Proceso Electrohilado para proporcionar tres ambientes únicos para un Tri-capa<em&gt; In Vitro</em&gt; Modelo de la pared de la vía aérea

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is limited to cell proliferation and gene expression endpoints. However, in the organism, mammary morphogenesis occurs in a 3D environment. 3D culture systems help bridge the gap between monolayer cell culture (2D) and the complexity of the organism. Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action. It uses the commercially available hormone-responsive human breast epithelial cell line, T47D, and rat tail collagen type 1 as a matrix. This 3D culture model responds to the main mammotropic hormones: estradiol, progestins and prolactin. The influence of these hormones on epithelial morphogenesis can be observed after 1- or 2-week treatment according to the endpoint. The 3D cultures can be harvested for analysis of epithelial morphogenesis, cell proliferation and gene expression.

We describe a 3D culture model of the human breast epithelium that is suitable to study hormone action.

Un 3D Cultura Modelo sensibles a hormonas de la glándula mamaria humana epitelio

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is ...

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes. The emergence of prion diseases in wildlife populations and their increasing threat to human health has led to increased efforts to find a treatment for these diseases. Recent studies have found numerous anti-prion compounds that can either inhibit the infectious PrPRes isomer or down regulate the normal cellular prion protein. However, most of these compounds do not cross the blood brain barrier to effectively inhibit PrPRes formation in brain tissue, do not specifically target neuronal PrPC, and are often too toxic to use in animal or human subjects. 
We investigated whether siRNA delivered intravascularly and targeted towards neuronal PrPC is a safer and more effective anti-prion compound. This report outlines a protocol to produce two siRNA liposomal delivery vehicles, and to package and deliver PrP siRNA to neuronal cells. The two liposomal delivery vehicles are 1) complexed-siRNA liposome formulation using cationic liposomes (LSPCs), and 2) encapsulated-siRNA liposome formulation using cationic or anionic liposomes (PALETS). For the LSPCs, negatively charged siRNA is electrostatically bound to the cationic liposome. A positively charged peptide (RVG-9r [rabies virus glycoprotein]) is added to the complex, which specifically targets the liposome-siRNA-peptide complexes (LSPCs) across the blood brain barrier (BBB) to acetylcholine expressing neurons in the central nervous system (CNS). For the PALETS (peptide addressed liposome encapsulated therapeutic siRNA), the cationic and anionic lipids were rehydrated by the PrP siRNA. This procedure results in encapsulation of the siRNA within the cationic or anionic liposomes. Again, the RVG-9r neuropeptide was bound to the liposomes to target the siRNA/liposome complexes to the CNS. Using these formulations, we have successfully delivered PrP siRNA to AchR-expressing neurons, and decreased the PrPC expression of neurons in the CNS.

The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases.

Entrega de Therapeutic siRNA al SNC Usando catiónicos y aniónicos Los liposomas

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  The ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

La fabricación de espumas y microportadores derivados de la matriz extracelular como específica de tejido de cultivo celular y las plataformas de entrega

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Retroductal Nanoparticle Injection to the Murine Submandibular Gland

Two common goals of salivary gland therapeutics are prevention and cure of tissue dysfunction following either autoimmune or radiation injury. By locally delivering bioactive compounds to the salivary glands, greater tissue concentrations can be safely achieved versus systemic administration. Furthermore, off target tissue effects from extra-glandular accumulation of material can be dramatically reduced. In this regard, retroductal injection is a widely used method for investigating both salivary gland biology and pathophysiology. Retroductal administration of growth factors, primary cells, adenoviral vectors, and small molecule drugs has been shown to support gland function in the setting of injury. We have previously shown the efficacy of a retroductally injected nanoparticle-siRNA strategy to maintain gland function following irradiation. Here, a highly effective and reproducible method to administer nanomaterials to the murine submandibular gland through Wharton's duct is detailed (Figure 1). We describe accessing the oral cavity and outline the steps necessary to cannulate Wharton's duct, with further observations serving as quality checks throughout the procedure.

Local drug delivery to the submandibular glands is of interest in understanding salivary gland biology and for the development of novel therapeutics. We present an updated and detailed retroductal injection protocol, designed to improve delivery accuracy and experimental reproducibility. The application presented herein is the delivery of polymeric nanoparticles.

Inyección de nanopartículas de Retroductal a la glándula submaxilar de ratón

Two common goals of salivary gland therapeutics are prevention and cure of tissue dysfunction following either autoimmune or radiation injury. By locally ...

Growth and Characterization of Irradiated Organoids from Mammary Glands

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell monolayers. Although they cannot completely model in vivo complexity, they retain some functionality of the original organ. In cancer models, organoids are commonly used to study tumor cell invasion. This protocol aims to develop and characterize organoids from the normal and irradiated mouse mammary gland tissue to evaluate the radiation response in normal tissues. These organoids can be applied to future in vitro cancer studies to evaluate tumor cell interactions with irradiated organoids. Mammary glands were resected, irradiated to 20 Gy and digested in a collagenase VIII solution. Epithelial organoids were separated via centrifugal differentiation, and 3D organoids were developed in 96-well low-adhesion microplates. Organoids expressed the characteristic epithelial marker cytokeratin 14. Macrophage interaction with the organoids was observed in co-culture experiments. This model may be useful for studying tumor-stromal interactions, infiltration of immune cells, and macrophage polarization within an irradiated microenvironment.

Organoids developed from mouse mammary glands were irradiated and characterized to assess epithelial traits and interactions with immune cells. Irradiated organoids can be used to better evaluate cell-cell interactions that may lead to tumor cell recruitment in irradiated normal tissue.

Crecimiento y caracterización de organoides irradiados de glándulas mamarias

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell ...