Los autores desean agradecer a Leica Microsystems por sus valiosos consejos sobre la imagen de las secciones de titanio y Gemini Bio-Productos para la prestación del suero porcino utilizado en este estudio. También agradecer a los NIH, por su apoyo a través de subvenciones &quot;forro autólogo EPC para mejorar la biocompatibilidad de los dispositivos de asistencia circulatoria&quot;, RC1HL099863-01. Además, estamos agradecidos por el apoyo de la Fundación Nacional de Ciencias de Postgrado de Becas de Investigación de Alexandra Jantzen. También agradecer a George rápido, Mike Lowe y Parker Ianthia por su ayuda en muchos aspectos de la intervención quirúrgica y el manejo de los animales de nuestra investigación. Steven Owen ha sido muy valioso para el mecanizado de piezas que muchos de nuestros dispositivos de siembra y corte de tubos de titanio.

<ol>
	<li>Achneck, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+of+bleeding+and+clotting+in+the+cardiac+surgery+patient:+from+vascular+endothelium+to+circulatory+assist+device+surface.">Pathophysiology of bleeding and clotting in the cardiac surgery patient: from vascular endothelium to circulatory assist device surface.</a> Circulation. 122, 2068-2077 (2010).</li><li>Achneck, H. E., Sileshi, B., Lawson, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+the+biology+of+bleeding+and+clotting+in+the+surgical+patient.">Review of the biology of bleeding and clotting in the surgical patient.</a> Vascular. 16, 6-13 (2008).</li><li>Arvidsson, S., Askendal, A., Tengvall, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+plasma+contact+activation+on+silicon,+titanium+and+aluminium.">Blood plasma contact activation on silicon, titanium and aluminium.</a> Biomaterials. 28, 1346-1354 (2007).</li><li>Achneck, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biocompatibility+of+titanium+cardiovascular+devices+seeded+with+autologous+blood-derived+endothelial+progenitor+cells:+EPC-seeded+antithrombotic+Ti+Implants.">The biocompatibility of titanium cardiovascular devices seeded with autologous blood-derived endothelial progenitor cells: EPC-seeded antithrombotic Ti Implants.</a> Biomaterials. 32, 10-18 (2011).</li><li>Kang, C., Bonneau, M., Brouland, J. P., Bal dit Sollier, C., Drouet, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+pig+models+of+venous+thrombosis+mimicking+human+disease.">In vivo pig models of venous thrombosis mimicking human disease.</a> Thromb Haemost. 89, 256-263 (2003).</li><li>Ueberrueck, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+ovine+and+porcine+animal+models+for+biocompatibility+testing+of+vascular+prostheses.">Comparison of the ovine and porcine animal models for biocompatibility testing of vascular prostheses.</a> J Surg Res. 124, 305-311 (2005).</li><li>Velik-Salchner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+values+for+thrombelastography+(ROTEM)+and+selected+coagulation+parameters+in+porcine+blood.">Normal values for thrombelastography (ROTEM) and selected coagulation parameters in porcine blood.</a> Thromb Res. 117, 597-602 (2006).</li><li>Yoder, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redefining+endothelial+progenitor+cells+via+clonal+analysis+and+hematopoietic+stem/progenitor+cell+principals.">Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals.</a> Blood. 109, 1801-1809 (2007).</li><li>Achneck, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> American Heart Association Scientific Sessions, Abstract Oral Sessions, Medical Aspects End Stage Heart Failure: Transplantation and Device Therapies. , (2010).</li><li>Achneck, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerating+titanium+ventricular+assist+device+surfaces+after+gold/palladium+coating+for+scanning+electron+microscopy.">Regenerating titanium ventricular assist device surfaces after gold/palladium coating for scanning electron microscopy.</a> Microsc Res Tech. 73, 71-76 (2010).</li><li>Ford, J. W., Welling, T. H., Stanley, J. C., Messina, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PKH26+and+125I-PKH95:+characterization+and+efficacy+as+labels+for+in+vitro+and+in+vivo+endothelial+cell+localization+and+tracking.">PKH26 and 125I-PKH95: characterization and efficacy as labels for in vitro and in vivo endothelial cell localization and tracking.</a> J Surg Res. 62, 23-28 (1996).</li></ol>

No hay conflictos de interés declarado.

El método de los tubos de siembra de células-Ti que aquí se presenta permite a los médicos y científicos de forma rápida y uniforme endothelialize contacto con la sangre de las superficies de los dispositivos implantables. Ya que aislar y expandir las CPE a partir de muestras de sangre periférica, procedimiento invasivo no es importante para la cosecha de estas células. Además, el EPC son autólogas, por lo tanto, el riesgo de una reacción inmune al implante de células cabeza de serie, se elimina. Los principios que demostró en su protocolo se puede utilizar no sólo para los tubos de Ti, pero para muchos otros biomateriales, que se utilizan en la medicina cardiovascular. Los pasos críticos de este protocolo son la limpieza minuciosa de los tubos de Ti, como se comprueba que una película adherente de la adhesión celular contaminante compromisos. Además, una baja velocidad de rotación (inversamente proporcional al diámetro del tubo) es esencial durante el proceso de siembra, de manera que poco a poco EPCs pueden resolver y se adhieren a la superficie como el tubo de Ti está en movimiento. Nuestro método de siembra inmediatamente antes de la implantación evita práctico ex casos la cultura vivo, las células se adhieran de forma individual y luego formar una hoja confluente en vivo, evitando la posibilidad de la embolización como una hoja inmediatamente después de la re-establecimiento del flujo. Nuestros estudios previos muestran que una vez que el EPC ha crecido a una capa confluente, hacen una matriz extracelular a la que se adhieren firmemente, además, reducir al mínimo cualquier posible desprendimiento de una capa de endotelio. Aunque la posibilidad de desprendimiento de embólicos no se puede descartar del todo, parece ser múltiple riesgo menor que la trombosis de la superficie del dispositivo completo, el problema de que esta terapia está diseñada para prevenir. Nuestro enfoque de implantación en el corte bajo, el medio ambiente protrombótico de la vena cava inferior utiliza uno de los modelos animales más grandes de confianza para la investigación de la compatibilidad de sangre y la trombosis de los dispositivos de 5,6. Tenga en cuenta que todo el cuidado de los animales y la experimentación se llevó a cabo de acuerdo con el Instituto Nacional de Pautas de Salud para el Cuidado y Uso de Animales de Laboratorio y sólo después de la aprobación de la Universidad de Duke Cuidado de Animales institucional y el empleo. Con el fin de utilizar con éxito este método para probar la implantación de biomateriales y dispositivos, es importante esqueletizar pacientemente el segmento de vena cava inferior y ligar todos los buques de rama venosa en la preparación para la inserción del dispositivo, de forma que no presente sangrado alrededor del dispositivo se origina en una &quot;falsa luz. Otro paso importante es la adición de DPBS en el lumen del dispositivo de tal manera que las células en la superficie interior del tubo permanecer húmeda durante el cierre de la veinotomy y antes de la reperfusión se inicia. Si el dispositivo no se puede encontrar en el lugar donde se implantó, que pueden haber migrado &quot;hacia arriba&quot; en la vena cava inferior. Esto se puede prevenir mediante la colocación de una sutura (4-O Prolene) a través de la pared de la vena ya través de un 2 a 3 mm de sección de la tubería de PVC para que el tubo está firmemente anclada en su actual ubicación. En caso de que el investigador tiene dificultades para encontrar las células con fluorescencia pre-etiquetados se muestra en la Figura 8 después de la explantación en un tubo de otro modo patente, es probable que las células se han pelado como una hoja coherente. Esto se puede prevenir mediante la disección muy suave alrededor de la vena o desmontaje de las tres secciones del tubo del Ti, después de la fijación de la vena junto con el tubo. Nuestra tecnología ofrece una prueba de concepto para el dispositivo de prevención de la trombosis cardiovascular a través de EPC-siembra. Esta tecnología se puede utilizar en el desarrollo de &quot;biogénico&quot; alineado con los implantes de los pacientes propias células progenitoras endoteliales. El estudio de viabilidad de nuestro modelo animal porcino establece los primeros pasos para la traducción de esta &quot;medicina personalizada&quot; en la práctica clínica.

<table border="1"><tbody><tr><td> Reactivo </td><td> Nombre de compañía </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Acepromacina </td><td> Boehringer-Ingelheim </td><td> BIC670025 </td><td> NAC # 10280002 </td></tr><tr><td> Alconox limpiador de precisión Desarrollado </td><td> Alconox </td><td> 1104 </td><td></td></tr><tr><td> Balfour quirúrgica Retractor </td><td> Adler </td><td> N / A </td><td></td></tr><tr><td> Baytril </td><td> Bayer </td><td> APVMA 46028/0705 </td><td></td></tr><tr><td> Mariposa con aguja (19G) </td><td> Terumo </td><td> SV19CLK </td><td></td></tr><tr><td> Clorhexidina </td><td> 3M </td><td> 9200 </td><td></td></tr><tr><td> Abrazaderas (45 grados) </td><td> Aesculap </td><td> FC339T </td><td></td></tr><tr><td> DPBS (-/-) </td><td> Gibco </td><td> 14190-144 </td><td></td></tr><tr><td> DPBS (+/+) </td><td> Gibco </td><td> 14040-133 </td><td></td></tr><tr><td> DuraPrep </td><td> 3M </td><td> 8635 </td><td></td></tr><tr><td> EBM-2 medianas </td><td> Lonza </td><td> CC-3156 </td><td> Base para los medio de cultivo libre de suero y completa </td></tr><tr><td> EGM-2 SingleQuots </td><td> Lonza </td><td> CC-3162 </td><td> Se utiliza con EBM-2, tanto para el medio de cultivo libre de suero y completa </td></tr><tr><td> Herramienta de electrocauterio </td><td> Valleylab </td><td> SurgII-20 </td><td></td></tr><tr><td> Materia esmeril </td><td> 3M </td><td> 60-0700-0425-8 </td><td> Grano 240 </td></tr><tr><td> Euthasol solución de la eutanasia </td><td> Virback Sanidad Animal </td><td></td><td> ANADÁ #: 200-071 </td></tr><tr><td> Fentanilo parche </td><td> Actavis </td><td></td><td> NDC 67767-120-18 # </td></tr><tr><td> Flunixina </td><td> Schering-Plough </td><td></td><td> NAC #: 10470183 </td></tr><tr><td> Sonda Foley (16F) </td><td> Bardo </td><td> 730116 </td><td></td></tr><tr><td> HBSS </td><td> Sigma </td><td> H8264-500ML </td><td></td></tr><tr><td> Pistola de aire caliente </td><td> Milwaukee </td><td> 8988-20 </td><td></td></tr><tr><td> Heparina </td><td></td><td></td><td> NDC #: 25021 </td></tr><tr><td> Histopaque-1077 </td><td> Sigma </td><td> H8889-500ML </td><td></td></tr><tr><td> Intubación Tubo </td><td> Mallinckrodt </td><td> 86113 </td><td></td></tr><tr><td> Isoflurano </td><td> MWI, Meridian </td><td></td><td> NDC # 13985-030 </td></tr><tr><td> IV catéter (18G) </td><td> Becton-Dickinson </td><td> 381547 </td><td></td></tr><tr><td> La ketamina </td><td> Fort Dodge </td><td></td><td> NDC # 0856-2013 </td></tr><tr><td> Nivel </td><td> Swanson </td><td> LLA001 </td><td></td></tr><tr><td> Luer-Lock tapón en el extremo </td><td> CML suministro </td><td> 909-001 </td><td></td></tr><tr><td> Bupivacaína </td><td> Hospira </td><td></td><td> NDC #: 0409-1560-10 </td></tr><tr><td> Metzenbaum </td><td> Adler </td><td> N / A </td><td></td></tr><tr><td> Micro-introductor (5F) </td><td> Galt </td><td> KIT 002-01 </td><td></td></tr><tr><td> Pinzas mosquito </td><td> Adler </td><td> N / A </td><td></td></tr><tr><td> Motor </td><td> Herbach y Rademan </td><td> H1-08 </td><td></td></tr><tr><td> Oximorfona </td><td> Endo Laboratorios </td><td></td><td> NDC: 63481-624-10 </td></tr><tr><td> PKH26 tinte Kit </td><td> Sigma </td><td> PKH26GL-1KT </td><td></td></tr><tr><td> Suero porcino </td><td> Gemini Bio-Productos </td><td> 100-115 </td><td> 2% de concentración en el medio de cultivo completo </td></tr><tr><td> Potts Tijeras </td><td> Adler </td><td> N / A </td><td></td></tr><tr><td> Vista la piel precisa de la grapadora </td><td> 3M </td><td> 3998 </td><td></td></tr><tr><td> Tubo de PVC </td><td> McMaster-Carr/Insultab </td><td> 7132K117 </td><td> ampliado ID 15,88 mm, recuperado ID 7,95 mm </td></tr><tr><td> Tamaño medio del ángulo derecho </td><td> Adler </td><td> N / A </td><td></td></tr><tr><td> Hoja de bisturí (# 10-15) </td><td> Bardo </td><td> 373910 </td><td></td></tr><tr><td> Tubo de silicona </td><td> McMaster-Carr </td><td> 51735K26 </td><td> 16,64 mm OD, 9,52 mm de diámetro </td></tr><tr><td> Jeringa (5 cc) </td><td> Becton-Dickinson (BD) </td><td> 309603 </td><td></td></tr><tr><td> Tegaderm </td><td> 3M </td><td> 90001 </td><td></td></tr><tr><td> Llave de tres vías </td><td> Kendall </td><td> 170060 </td><td></td></tr><tr><td> Ti tubo </td><td> Tico de titanio </td><td> N / A </td><td> Especifica como ½ &quot;OD, .065&quot; de pared, 0.370 ID &quot;, 0,1737 libras / pie </td></tr><tr><td> Tripsina </td><td> Lonza </td><td> CC-5012 </td><td></td></tr><tr><td> Neutralizar la solución de tripsina (TNS) </td><td> Lonza </td><td> CC-5002 </td><td> 0,03% </td></tr><tr><td> Vetropolycin </td><td> Pharmaderm Sanidad Animal </td><td></td><td> NAC #: 12920110 </td></tr><tr><td> Sutura Vicryl (3-0) </td><td> Ethicon </td><td> J808T </td><td></td></tr><tr><td> Agua baño de ultrasonido </td><td> Branson </td><td> B200 </td><td></td></tr></tbody></table>

autologous endothelial progenitor cell-seeding technology and biocompatibility testing for cardiovascular devices in large animal model

Dispositivos implantables cardiovasculares son fabricadas con materiales artificiales (por ejemplo, el titanio (Ti), politetrafluoroetileno expandido), que plantean el riesgo de formación de tromboembolias 1,2,3. Hemos desarrollado un método para la línea de la superficie interior de los tubos de Ti con autólogos derivados de la sangre las células progenitoras humanas o porcinas endoteliales (CPE) 4. Mediante la implantación de tubos de Ti contiene una capa confluente de EPCs porcina en la vena cava inferior (VCI) de los cerdos, hemos probado la biocompatibilidad mejorada de la superficie de las células sembradas en el medio ambiente protrombótico de un modelo animal de gran tamaño y lo comparó con modificar las superficies de metal desnudo 5,6,7 (Figura 1). Este método puede ser usado para endothelialize dispositivos en cuestión de minutos de la implantación y prueba de su función antitrombótica in vivo. Sangre periférica se obtuvo a partir de 50 kg Yorkshire porcina y su fracción de células mononucleares de cultivo para aislar CPE 4,8. Ti tubos (9,4 mm de diámetro) se pre-cortado en tres secciones 4,5 cm longitudinal y vuelto a montar con bomba de calor tubo retráctil. Un dispositivo de siembra fue construido, que permite la rotación lenta de las trompas de Ti. Se realizó una laparotomía en los cerdos y exterioriza el intestino y la vejiga urinaria. Disección cortante y contundente fue utilizado para esqueletizar el IVC desde su bifurcación distal de la arteria renal derecha proximal. Los tubos de Ti se rellenaron con fluorescencia marcado autólogo suspensión del CPE y gira a 10 RPH x 30 min para conseguir células de revestimiento uniforme 9. Después de la administración de 100 USP / kg de heparina, ambos extremos de la vena cava inferior y la vena lumbar se sujetaron. A veinotomy de 4 cm se realizó y se inserta el dispositivo y lleno de tampón fosfato salino. A medida que el veinotomy se cerró con una sutura de Prolene 4-0 en funcionamiento, una pinza fue trasladado a al aire de la VCI. Al final del procedimiento, la fascia se aproximó con 0-PDS (sutura de polidioxanona), el espacio subcutáneo cierra con Vicryl 2-0 y la piel con grapas cerrado. Después de 3 a 21 días, los cerdos fueron sacrificados, el dispositivo explantado en bloque y fija. Los tubos de Ti fueron desmontados y las superficies internas de imágenes con un microscopio de fluorescencia. Hemos encontrado que el metal desnudo tubos Ti completamente oculto, mientras que los tubos de EPC cabeza de serie, quedó patente. Además, hemos sido capaces de demostrar una capa confluente de EPC en el interior de la superficie de contacto con la sangre. En conclusión, nuestra tecnología se puede utilizar para endothelialize tubos Ti minutos después de la implantación de EPC autólogos para prevenir la trombosis del dispositivo. Nuestro método quirúrgico permite probar la biocompatibilidad mejorada de estos dispositivos modificados con mínima pérdida de sangre y EPC cabeza de serie, la interrupción de la superficie.

Watch this Scientific Journal Video about Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model at JoVE.com

Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model

Un método para la siembra de titanio contacto con la sangre de biomateriales con células autólogas y las pruebas de biocompatibilidad se describe. Este método utiliza células progenitoras endoteliales y los tubos de titanio, cabeza de serie en cuestión de minutos de implantación quirúrgica en porcino venas cavas. Esta técnica se puede adaptar a muchos otros dispositivos biomédicos implantables.

Autólogo de células progenitoras endoteliales-Siembra Tecnología y las pruebas de biocompatibilidad para dispositivos cardiovasculares en el modelo animal de gran

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk ...

autologous-endothelial-progenitor-cell-seeding-technology

Research

JoVE Journal

Bioengineering

20.3K vistas.  Duke University. Un método para la siembra de titanio contacto con la sangre de biomateriales con células autólogas y las pruebas de biocompatibilidad se describe. Este método utiliza células progenitoras endoteliales y los tubos de titanio, cabeza de serie en cuestión de minutos de implantación quirúrgica en porcino venas cavas. Esta técnica se puede adaptar a muchos otros dispositivos biomédicos implantables.

Video: Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model

Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation, Barrier Function, and Motility

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. To this end, cells are grown in special culture chambers on top of opposing, circular gold electrodes. A constant small alternating current is applied between the electrodes and the potential across is measured. The insulating properties of the cell membrane create a resistance towards the electrical current flow resulting in an increased electrical potential between the electrodes. Measuring cellular impedance in this manner allows the automated study of cell attachment, growth, morphology, function, and motility. Although the ECIS measurement itself is straightforward and easy to learn, the underlying theory is complex and selection of the right settings and correct analysis and interpretation of the data is not self-evident. Yet, a clear protocol describing the individual steps from the experimental design to preparation, realization, and analysis of the experiment is not available. In this article the basic measurement principle as well as possible applications, experimental considerations, advantages and limitations of the ECIS system are discussed. A guide is provided for the study of cell attachment, spreading and proliferation; quantification of cell behavior in a confluent layer, with regard to barrier function, cell motility, quality of cell-cell and cell-substrate adhesions; and quantification of wound healing and cellular responses to vasoactive stimuli. Representative results are discussed based on human microvascular (MVEC) and human umbilical vein endothelial cells (HUVEC), but are applicable to all adherent growing cells.

This protocol reviews Electric Cell-substrate Impedance Sensing, a method to record and analyze the impedance spectrum of adherent cells for the quantification of cell attachment, proliferation, motility, and cellular responses to pharmacological and toxic stimuli. Detection of endothelial permeability and assessment of cell-cell and cell-substrate contacts are emphasized.

Eléctrico de la célula-sustrato Impedancia de detección para la cuantificación de la proliferación endotelial, la función de barrera, y la motilidad

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. ...

Investigación

Bioingeniería

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to model well differentiated human airway epithelium and heart muscle cells. This could be an invaluable tool to study the deleterious effects of toxic inhaled chemicals, such as chlorine, that can normally interact with the cell surfaces, and form various byproducts upon reacting with water, and limiting their effects in submerged cultures. Our model using well differentiated human airway epithelial cell cultures at air-liqiuid interface circumvents this limitation as well as provides an opportunity to evaluate critical mechanisms of toxicity of potential poisonous inhaled chemicals. We describe enhanced loss of membrane integrity, caspase release and death upon toxic inhaled chemical such as chlorine exposure. In this article, we propose methods to model chlorine exposure in mammalian heart and airway epithelial cells in culture and simple tests to evaluate its effect on these cell types.

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

This protocol is designed to demonstrate exposure method of cell cultures to inhaled toxic chemicals. Exposure of differentiated air-liquid&#160;interface (ALI) cultures of airway epithelial cells provides a unique model of airway exposure to toxic gases such as chlorine. In this manuscript we describe effect of chlorine exposure on air-liquid&#160;interface cultures of epithelial cells and submerged culture of cardiomyocytes. In vitro exposure systems allow important mechanistic studies to evaluate pathways that could then be utilized to develop novel therapeutic agents.

In vitro modelo de cultivo celular para Toxic inhalado Prueba química

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

Capa por capa deposición de colágeno en dispositivos de microfluidos para microtejidos Estabilización

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

Ferromagnetic Bare Metal Stent for Endothelial Cell Capture and Retention

Rapid endothelialization of cardiovascular stents is needed to reduce stent thrombosis and to avoid anti-platelet therapy which can reduce bleeding risk. The feasibility of using magnetic forces to capture and retain endothelial outgrowth cells (EOC) labeled with super paramagnetic iron oxide nanoparticles (SPION) has been shown previously. But this technique requires the development of a mechanically functional stent from a magnetic and biocompatible material followed by in-vitro and in-vivo testing to prove rapid endothelialization. We developed a weakly ferromagnetic stent from 2205 duplex stainless steel using computer aided design (CAD) and its design was further refined using finite element analysis (FEA). The final design of the stent exhibited a principal strain below the fracture limit of the material during mechanical crimping and expansion. One hundred stents were manufactured and a subset of them was used for mechanical testing, retained magnetic field measurements, in-vitro cell capture studies, and in-vivo implantation studies. Ten stents were tested for deployment to verify if they sustained crimping and expansion cycle without failure. Another 10 stents were magnetized using a strong neodymium magnet and their retained magnetic field was measured. The stents showed that the retained magnetism was sufficient to capture SPION-labeled EOC in our in-vitro studies. SPION-labeled EOC capture and retention was verified in large animal models by implanting 1 magnetized stent and 1 non-magnetized control stent in each of 4 pigs. The stented arteries were explanted after 7 days and analyzed histologically. The weakly magnetic stents developed in this study were capable of attracting and retaining SPION-labeled endothelial cells which can promote rapid healing.

Our goals were to design, manufacture and test ferromagnetic stents for endothelial cell capture. Ten stents were tested for fracture and 10 more stents were tested for retained magnetism. Finally, 10 stents were tested in-vitro and 8 more stents were implanted in 4 pigs to show cell capture and retention.

Ferromagnético Bare Metal stent para la captura de la célula endotelial y Retención

Rapid endothelialization of cardiovascular stents is needed to reduce stent thrombosis and to avoid anti-platelet therapy which can reduce bleeding risk. ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

Fabricación de pozos de agarosa personalizado para la siembra de células y tejido anillo autoensamblaje con moldes impresa en 3D

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

Dispositivos eléctricos y de campo magnético para la estimulación de tejidos biológicos

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...

A Large Animal Model for Pulmonary Hypertension and Right Ventricular Failure: Left Pulmonary Artery Ligation and Progressive Main Pulmonary Artery Banding in Sheep

Decompensated right ventricular failure (RVF) in pulmonary hypertension (PH) is fatal, with limited medical treatment options. Developing and testing novel therapeutics for PH requires a clinically relevant large animal model of increased pulmonary vascular resistance and RVF. This manuscript discusses the latest development of the previously published ovine PH-RVF model that utilizes left pulmonary artery (PA) ligation and main PA occlusion. This model of PH-RVF is a versatile platform to control not only the disease severity but also the RV&#39;s phenotypic response.
Adult sheep (60-80 kg) underwent left PA (LPA) ligation, placement of main PA cuff, and insertion of RV pressure monitor. PA cuff and RV pressure monitor were connected to subcutaneous ports. Subjects underwent progressive PA banding twice per week for 9 weeks with sequential measures of RV pressure, PA cuff pressures, and mixed venous blood gas (SvO2). At the initiation and endpoint of this model, ventricular function and dimensions were assessed using echocardiography. In a representative group of 12 animal subjects, RV mean and systolic pressure increased from 28 &#177; 5 and 57 &#177; 7 mmHg at week 1, respectively, to 44 &#177; 7 and 93 &#177; 18 mmHg (mean &#177; standard deviation) by week 9. Echocardiography demonstrated characteristic findings of PH-RVF, notably RV dilation, increased wall thickness, and septal bowing. The longitudinal trend of SvO2 and PA cuff pressure demonstrates that the rate of PA banding can be titrated to elicit varying RV phenotypes. A faster PA banding strategy led to a precipitous decline in SvO2 &#60; 65%, indicating RV decompensation, whereas a slower, more paced strategy led to the maintenance of physiologic SvO2 at 70%-80%. One animal that experienced the accelerated strategy developed several liters of pleural effusion and ascites by week 9. This chronic PH-RVF model provides a valuable tool for studying molecular mechanisms, developing diagnostic biomarkers, and enabling therapeutic innovation to manage RV adaptation and maladaptation from PH.

This manuscript describes the surgical technique and experimental approach to develop severe right ventricular pressure overload to model their adaptive and maladaptive phenotypes.

Un modelo animal grande para la hipertensión pulmonar y la insuficiencia ventricular derecha: ligadura de la arteria pulmonar izquierda y bandas progresivas de la arteria pulmonar principal en ovejas

Decompensated right ventricular failure (RVF) in pulmonary hypertension (PH) is fatal, with limited medical treatment options. Developing and testing ...

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.

Producción automatizada a gran escala de esferoides de células madre derivadas de tejido adiposo para bioimpresión 3D

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue ...

In Silico Clinical Trials for Cardiovascular Disease

The SILICOFCM project mainly aims to develop a computational platform for in silico clinical trials of familial cardiomyopathies (FCMs). The unique characteristic of the platform is the integration of patient-specific biological, genetic, and clinical imaging data. The platform allows the testing and optimization of medical treatment to maximize positive therapeutic outcomes. Thus, adverse effects and drug interactions can be avoided, sudden cardiac death can be prevented, and the time between the commencement of drug treatment and the desired result can be shortened. This article presents a parametric model of the left ventricle automatically generated from patient-specific ultrasound images by applying an electromechanical model of the heart. Drug effects were prescribed through specific boundary conditions for inlet and outlet flow, ECG measurements, and calcium function for heart muscle properties. Genetic data from patients were incorporated through the material property of the ventricle wall. Apical view analysis involves segmenting the left ventricle using a previously trained U-net framework and calculating the bordering rectangle based on the length of the left ventricle in the diastolic and systolic cycle. M-mode view analysis includes bordering of the characteristic areas of the left ventricle in the M-mode view. After extracting the dimensions of the left ventricle, a finite elements mesh was generated based on mesh options, and a finite element analysis simulation was run with user-provided inlet and outlet velocities. Users can directly visualize on the platform various simulation results such as pressure-volume, pressure-strain, and myocardial work-time diagrams, as well as animations of different fields such as displacements, pressures, velocity, and shear stresses.

In Silico Clinical Trials for Cardiovascular Disease

This protocol demonstrates the workflow of the SILICOFCM platform for automatically generating a parametric model of the left ventricle from patient-specific ultrasound images by applying a multi-scale electromechanical model of the heart. This platform enables in silico clinical trials intended to reduce real clinical trials and maximize positive therapeutic outcomes.

In Silico Ensayos clínicos para enfermedades cardiovasculares

The SILICOFCM project mainly aims to develop a computational platform for in silico clinical trials of familial cardiomyopathies (FCMs). The unique ...

Evaluación de las propiedades hidrodinámicas en válvulas aórticas porcinas montadas

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

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

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

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

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

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