Tracy C. Grikscheit, Erik R. Barthel, y Frédéric G. Sala son apoyados por el Instituto de Medicina Regenerativa de California (CIRM), números de concesión RN2-00946-1 (TCG) y TG2-01168 (ERB, FGS). Allison L. Speer es una Sociedad de Cirujanos de la Universidad de Ethicon erudito. Yashuhiro Torashima es financiado por un Hospital de Niños de Los Angeles Fellowship Saban Research Institute de Desarrollo Profesional.

1. Unidades de Preparación organoide <ol><li> Instrumentos apropiados para la disección del ratón (tijeras y pinzas) debe ser esterilizado por autoclave. </li><li> Humanamente sacrificar al ratón donante de acuerdo con los protocolos locales IACUC. Asegúrese de que el animal está muerto antes de continuar. </li><li> Hacer una incisión de línea media para tener acceso a la cavidad peritoneal. Los colgajos de piel se puede reflejar como sea necesario para mejorar la exposición. </li><li> Eviscerar el intestino delgado y se divide justo distal al ligamento de Treitz. Separe el intestino delgado de su mesenterio utilizando disección roma fuerte y suave. Identificar la unión ileocecal y dividir el intestino delgado 5 mm proximal a este. </li><li> Usando tijeras, abrir el intestino longitudinalmente a lo largo del borde antimesentérico en una placa de Petri con 10 ml 4 ° C, la solución salina tamponada estéril de Hanks (HBSS, Invitrogen, Carlsbad CA) / 1X antibiótico-antimicótico (anti-anti, Invitrogen) solución. Borrar la materia fecal desde el intestino se abrió conagitación suave a continuación, transferir intestino se abrió a un tubo de centrífuga de 15 ml con 10 ml de 4 ° C, HBSS estéril / 1X anti-anti. </li><li> Se lava el intestino se abrió tres veces en 10 ml de 4 ° C, 1X HBSS estéril / anti anti-en un tubo de ensayo. Cada lavado se puede realizar con agitación suave del tubo de 15 ml por 30 seg. Después de agitar, el tejido intestinal se hunde hasta el fondo del tubo. Deseche el material flotante, que es basura mesenquimal. Quitar la solución de lavado cuidadosamente con una pipeta. </li><li> Picar el intestino lavado en una placa de Petri con 10 ml de 4 ° C, 1X HBSS estéril / anti anti-a menos de piezas de 1 mm cuadrados usando unas tijeras. Reúna el material triturado con una pipeta automática y colocarlo en un tubo de ensayo. </li><li> Centrifugar el tubo a 500 rpm durante 8 min. Eliminar el sobrenadante, que contiene grasa y el mesénquima. </li><li> Se digiere el material triturado, se lavó con 10 ml de HBSS estéril / 1X anti-anti más 0,125 mg / ml de dispasa (Invitrogen) y 800 unidades / ml colagenasa tipo 1 (Worthington, Lakewood NJ). Para preparar 40 ml de la solución de digestión, pesar 5 mg de dispasa, 142 mg de colagenasa, y añadir HBSS estéril hasta un volumen de 40 ml. Preparar esta solución recién cada unidad de tiempo organoides se preparan, y mantener a 4 º C hasta su utilización. Añadir la solución de digestión directamente a la pastilla del paso 1,8. </li><li> Incubar el tubo de ensayo que contiene el material triturado, se lavó con la solución de digestión a 37 ° C durante 20 min. </li><li> Recuperar el tubo de ensayo y además perturbar el tejido digerido por trituración con una pipeta de 10 ml. Repita entre 20 a 50 veces hasta una apariencia uniforme. </li><li> Centrifugar el tubo de ensayo durante 5 min a 800 rpm. Eliminar el sobrenadante, que contiene las células individuales. </li><li> Detener la reacción de digestión con 10 ml de 4 ° C, Modificado de Dulbecco estéril medio Eagle (DMEM, Invitrogen) más 10% v / v inactivado por calor suero fetal bovino (HI-FBS, Invitrogen). Resuspender el pellet y agitar el tubo. </li><li> Centrifugar el tubo de ensayo durante 5 min a 800 rpm. Eliminar el sobrenadante cuidadosamente con una pipeta automática hasta las últimas gotas. Usar una pipeta de plástico desechable para las últimas gotas para evitar resuspendiendo el sedimento. </li></ol> 2. La carga de ácido poliglicólico Andamio <ol><li> Formar 2-mm de largo, 5-mm andamios diámetro exterior cilíndrico de ácido poliglicólico no tejido (2 mm de espesor de chapa, 60 mg cm-3 densidad, porosidad&gt; 95%, Fibras Concordia, Coventry RI) como se describe en la ref. 4. </li><li> Recorte el distal 2 mm de una pipeta de microlitro desechable 1.000 inclinar con tijeras preparadas con 70% de etanol en agua destilada. </li><li> Coloque el andamio en una placa de cultivo de 4 pocillos. Cargar las unidades de organoides al andamio con la pipeta de microlitro 1.000, primero en el lumen y, a continuación sobre la superficie exterior. Utilice una pinza para asegurar el revestimiento del lumen. No interrumpir o romper la forma cilíndrica del polímero. </li&gt; </ol> 3. Implantación en ratones Host <ol><li> Utilice un ratón singénico anfitrión en el mismo fondo que el donante si están disponibles. De lo contrario, emplean un diabéticos no obesos inmunocomprometidos / grave inmunodeficiencia combinada o NOD / SCID animal (Jackson Laboratories, Sacramento CA). </li><li> Inducir la anestesia general con isoflurano. Afeitado, preparación y cubrir el abdomen del ratón. </li><li> Haga una incisión de 5 mm de la línea media para ganar la entrada a la cavidad peritoneal. Identificar y destripar cuidadosamente el epiplón mayor. Coloque el polímero cargado en el epiplón y se envuelve con el tejido. No rompa el epiplón. </li><li> Asegure que el polímero el epiplón con una sutura 5-0 monocryl. Suavemente reemplazar el epiplón con el polímero envuelto en su posición anatómica. </li><li> Cierre la incisión abdominal en capas con suturas de vicryl 4-0. Ejecute el cierre del músculo y tener cuidado de no lesionar las vísceras abdominales por debajo de la incisión. Use suturas interrumpidas para la piel. </li><li> Administrar analgesia postoperatoria con 2 mg / kg de ketoprofeno (Ketofen, Fort Dodge Animal Health) en agua estéril como una pápula subcutánea adyacente a la incisión. Los animales deben ser evaluados diariamente y si el animal está demostrando signos de dolor o angustia, una dosis adicional de ketoprofeno puede ser administrado el día después de la operación 2. En el tercer día postoperatorio, el animal debe estar completamente recuperado sin evidencia de dolor o angustia. Si el dolor o molestia persiste en el día 3 después de la operación, se considera anormal y debería ser tratado de acuerdo con los protocolos del IACUC instalaciones y cuidado de los animales. </li><li> Permitir el ratón para recuperar y el intestino de tejido de ingeniería crecer durante cuatro semanas. Dar acceso a la ad libitum a los animales roedores chow (Lab Diet 5001, PMI Nutrition, St. Louis MO) y el agua con Septra 200 mg / 40 mg por 5 ml (Hi-Tech Pharmacal, Amityville NY) a una dilución 1:100. </li></ol> 4. Cosecha <ol><li> Humanely sacrificar al animal huésped cuatro semanas después de la implantación. </li><li> Vuelva a abrir la incisión original y reflejan la piel cefálica a facilitar el acceso a la cavidad peritoneal. </li><li> Abra la capa muscular e identificar el constructo de tejido modificado como un globo de tejido. </li><li> Tome las adherencias a la construcción de las vísceras intraabdominales mediante disección cortante. </li><li> Fijar el constructo en formalina para posterior parafina montaje o el uso del tejido fresco para ensayos bioquímicos tales como la PCR en tiempo real o aislamiento de las proteínas. </li></ol>

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	<li>Grikscheit, T. C., Siddique, A., Ochoa, E. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+small+intestine+improves+recovery+after+massive+small+bowel+resection.">Tissue-engineered small intestine improves recovery after massive small bowel resection.</a> Ann. Surg. 240, 748-754 (2004).</li><li>Wales, P. W., Christison-Lagay, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+bowel+syndrome:+epidemiology+and+etiology.">Short bowel syndrome: epidemiology and etiology.</a> Sem. Ped. Surg. 19, 3-9 (2010).</li><li>Evans, G. S., Flint, N., Somers, A. 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Hepatol. 7, 183-191 (2009).</li><li>Klempnauer, J., Grothues, F., Bektas, H., Pichlmayr, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+results+after+surgery+for+acute+mesenteric+ischemia.">Long-term results after surgery for acute mesenteric ischemia.</a> Surgery. , 121-239 (1997).</li><li>Fitzgibbons, S. C., Jones, B. A., Hull, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+biopsy-proven+parenteral+nutrition-associated+liver+fibrosis+and+biochemical+cholestasis+in+children+with+short+bowel+syndrome.">Relationship between biopsy-proven parenteral nutrition-associated liver fibrosis and biochemical cholestasis in children with short bowel syndrome.</a> J. Ped. Surg. 45, 95-99 (2010).</li><li>Spencer, A. U., Kovacevich, D., McKinney-Barnett, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pediatric+short+bowel+syndrome:+the+cost+of+comprehensive+care.">Pediatric short bowel syndrome: the cost of comprehensive care.</a> Am. J. Clin. Nutr. 88, 1552-1559 (2008).</li><li>Kato, T., Tzakis, A. G., Selvaggi, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+and+multivisceral+transplantation+in+children.">Intestinal and multivisceral transplantation in children.</a> Ann. Surg. 243, 756-766 (2006).</li><li>Reyes, J., Bueno, J., Kocoshis, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+status+of+intestinal+transplantation+in+children.">Current status of intestinal transplantation in children.</a> J. Ped. Surg. 33, 243-254 (1998).</li></ol>

No hay conflictos de interés declarado.

Se presenta un protocolo para la producción de tejido de ingeniería en el intestino del ratón utilizando un organoide unidades-on-andamio enfoque. Los pasos más importantes son los de la preparación de unidades organoide. Se debe tener cuidado de limpiar adecuadamente y procesar mecánicamente el tejido, pero el mismo cuidado debe ser tomado para no overdigest o overtriturate las unidades de organoides después de la digestión se lleva a cabo (paso 1,11). Si se hace esto, las unidades de organoides se puede reduci...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td> HBSS </td><td> Gibco </td><td> 114170-112 </td><td></td></tr><tr><td> Antibiótico-antimicótico 100X </td><td> Invitrogen </td><td> 15240-062 </td><td></td></tr><tr><td> Dispasa </td><td> Gibco </td><td> 17105-041 </td><td></td></tr><tr><td> Colagenasa de tipo 1 </td><td> Worthington </td><td> LS004194 </td><td></td></tr><tr><td> DMEM alto en glucosa 1X </td><td> Gibco </td><td> 11995-065 </td><td></td></tr><tr><td> FBS inactivado por calor </td><td> Invitrogen </td><td> 16140-071 </td><td></td></tr><tr><td> Biofelt 100% PGA </td><td> Médico Concordia </td><td> FELT01-1005 </td><td> Para la preparación del polímero como en la ref.4 </td></tr><tr><td> Poli-L-láctico </td><td> Durect </td><td> B6002-1 </td><td> Para la preparación del polímero como en la ref. 4 </td></tr><tr><td> El colágeno tipo I, cola de rata </td><td> Sigma-Aldrich </td><td> C3867-1VL </td><td> Para la preparación del polímero como en la ref. 4 </td></tr><tr><td> Ketoprofeno 100 mg / ml </td><td> Fort Dodge Animal Health </td><td> 71-KETOI-100-50 </td><td></td></tr><tr><td> LabDiet 5001 comida para roedores </td><td> LabDiet </td><td> 5001 </td><td></td></tr><tr><td> Septra 200 mg / 40 mg por 5 ml, USP </td><td> Hi-Tech Pharmacal </td><td> 50383-824-16 </td><td></td></tr><tr><td> USP isoflurano, </td><td> Phoenix Pharmaceuticals </td><td> 57319-507-06 </td><td></td></tr></tbody></table>

tissue engineering of the intestine in a murine model

De tejido de ingeniería intestino delgado (TESI) ha sido utilizado con éxito para rescatar a ratas Lewis después de la resección masiva del intestino delgado, resultando en retorno a pesos preoperatorios en 40 días. 1 En los seres humanos, resección masiva del intestino delgado puede resultar en el síndrome del intestino corto, una malabsorción funcional Estado que confiere una significativa morbilidad, mortalidad y costes sanitarios, incluyendo la dependencia de nutrición parenteral, la insuficiencia hepática y cirrosis, y la necesidad de un trasplante de órganos multivisceral. 2 En este trabajo se describen y documentan nuestro protocolo para la creación de tejido modificado intestino en un modelo de ratón con un organoide multicelular unidades-on-andamio enfoque. Unidades de organoides son agregados multicelulares derivados del intestino que contienen elementos tanto de la mucosa y mesenquimales, 3 la relación entre el que conserva el nicho de células madre intestinal. 4 En la investigación en curso y futuros, la transición de nuestra técnica en laratón permitirá la investigación de los procesos que intervienen durante la formación de TESI mediante la utilización de las herramientas transgénicas disponibles en esta especie. 5 La disponibilidad de las cepas de ratón inmunocomprometidos también nos permitirá aplicar la técnica de tejido intestinal humano y optimizar la formación de TESI humana como un de ratón con xenoinjerto antes de su transición a los seres humanos. Nuestro método utiliza buenas prácticas de fabricación (GMP) reactivos y materiales que ya han sido aprobados para su uso en pacientes humanos, y por lo tanto ofrece una ventaja significativa sobre los enfoques que se basan en los tejidos animales descelularizados. El objetivo final de este método es su traducción a los seres humanos como una estrategia de medicina regenerativa terapéutico para el síndrome de intestino corto.

La Figura 1 muestra un esquema general para el protocolo documentado aquí. El resultado final de este protocolo es una estructura de globo esférico o de tejido de ingeniería intestino murino con un lumen, mucosa, submucosa, y rodea muscularis. Figura 2A muestra un globo típico en comparación con un polímero de partida andamio. Figura 2B muestra el mismo constructo bruscamente bivalvo para revelar su lumen. Figura 3 demuestra un hematoxilina / eosi...

Watch this Scientific Journal Video about Tissue Engineering of the Intestine in a Murine Model at JoVE.com

Tissue Engineering of the Intestine in a Murine Model

Este artículo y el vídeo de acompañamiento presentar nuestro protocolo para la generación de tejido de ingeniería intestino en el ratón, utilizando un organoide unidades-on-andamio enfoque.

Ingeniería de tejidos del intestino en un modelo murino

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

tissue-engineering-of-the-intestine-in-a-murine-model

Research

JoVE Journal

Bioengineering

14.8K vistas.  Keck School of Medicine of the University of Southern California. Este artículo y el vídeo de acompañamiento presentar nuestro protocolo para la generación de tejido de ingeniería intestino en el ratón, utilizando un organoide unidades-on-andamio enfoque.

Video: Tissue Engineering of the Intestine in a Murine Model

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

La encapsulación de los cardiomiocitos en un hidrogel de fibrina para ingeniería de tejidos cardíacos

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

Investigación

Bioingeniería

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

Postproducción de procesamiento de las Fibras electrospun de Ingeniería de Tejidos

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

Los tejidos de ingeniería músculo esquelético de células mioblastos murinos progenitoras y aplicación de estimulación eléctrica

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

Diseño de un Carga mecánica Biaxial biorreactor para Ingeniería de Tejidos

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Ingeniería de tejidos de un humano 3D<em&gt; In vitro</em&gt; Sistema de Prueba de Tumores

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

Generación de una tridimensional grosor de la piel completa y automatizada Equivalente Heridas

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Ingeniería 3D Cellularized colágeno Geles para Vascular Regeneración Tisular

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

Ingeniería de tejidos por intrínseca Vascularización en una<em&gt; En Vivo</em&gt; Cámara Ingeniería de Tejidos

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.

El asa arteriovenosa (AV) en un modelo de pequeños animales para estudiar la angiogénesis e Ingeniería tejido vascularizado

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in ...

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been &#39;conditioned&#39; by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

We present a model of ligament tissue in which three-dimensional constructs are treated with the human exercise-conditioned serum and analyzed for collagen content, function, and cellular biochemistry.

Tratamiento de las estructuras de ligamentos con suero acondicionado con ejercicio: Un modelo de ingeniería de tejidos traslacional

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, ...