1. Establecimiento de cultivos de fibroblastos de explantes de piel <ol><li> 4 mm biopsias adquiridas de antebrazo humano se colocan en MEM suplementado 100 unidades / ml de penicilina, estreptomicina 100 mg / ml y 0,25 mg / ml Fungizone. </li><li> Recortar la grasa subcutánea y picar finamente la biopsia en trozos pequeños, colocándola un número de 24 hoja de bisturí en contra de la parte inferior de la placa de Petri. </li><li> Mezcla de tejidos en lugar de 25 cm 2 frasco de cultivo de tejidos con los medios de comunicación primario de crecimiento de fibroblastos (MEM suplementado con 10% FCS, 100 unidades / ml de penicilina, estreptomicina 100 mg / ml y 25 mg / Fungizone ml) hasta que la superficie del recipiente es cubierta, pero la profundidad del líquido es insuficiente para el tejido a flotar. </li><li> Después de tres días de incubación a 37 ° C en una atmósfera húmeda de CO2 al 5%, añadir 3 ml de medio de crecimiento de fibroblastos primarios. </li><li> Después de otros 3 días los medios de comunicación el cambio de la incubación con los medios de comunicación primaria fresca de crecimiento de fibroblastos, o si las células son casi confluentes que se puede dividir en 1:04 medio fresco. (Fungizone se puede omitir a partir de este punto). </li></ol> 2. Etapa I - Preparación de colágeno I de la cola de rata Nota: El Protocolo de aproximadamente 12 a 14 colas de rata adolescente (frescas o congeladas) <ol><li> Preparar la cola de rata por el lavado en etanol al 70% y eliminar los tendones de la siguiente manera: </li><li> Quite la piel de la cola de rata por corte en el centro de la cola de arriba a abajo con una. Bisturí y repique a lo largo de la longitud de la cola </li><li> Separar el tendón del núcleo de la región proximal de la cola. </li><li> Quitar el tendón hacia la región distal de la cola para evitar la vaina con pinzas dentadas. </li><li> Extracto de 1 g de tendón / 250 ml de 0,5 M de ácido acético por agitación a 4 º C durante 48 h. </li><li> Centrifugar (7.500 xg) durante 30 minutos y desechar el sedimento. </li><li> Añadir un volumen igual de 10% (w / v) NaCl al sobrenadante, y se agita durante 30-60 min. </li><li> Centrífuga (10.000 xg) durante 30 minutos, descartar el sobrenadante y volver a disolver el precipitado en 0,25 M de ácido acético en una proporción de 1:1 ~ por agitación durante 24 horas a 4 ° C. </li><li> Dializar la solución de colágeno en contra de 6-8 cambios de 6L ~ 17,5 mM de ácido acético (1 ml de ácido acético glacial por litro de agua fría, el cambio de 2 veces al día). </li><li> Centrífuga dializados colágeno a 30.000 xg durante 1,5 horas. </li><li> Aspirar el sobrenadante y el lugar en el frasco estéril. </li><li> Ajustar el colágeno I concentración de ~ 2 mg / ml con 0,5 mM de ácido acético a 4 ° C. </li></ol> 3. Etapa II - Creación de la matriz 3D con fibroblastos embebidos (permite contratar durante 8 días). <ol><li> Montar la mezcla con los reactivos en frío a 4 ° C en una botella de pre-enfriado. Mantenga a que el colágeno en el hielo. </li><li> Para un frasco de T75 confluente fibroblastos primarios (~ el número de células 1 x 10 6) se utiliza: <ul><li> 25 ml de cola de rata colágeno (aprox. conc 2mg/ml) </li><li> 3 ml 10xMEM </li><li> 0,22 M NaOH: en primer lugar, añadir 2 ml, y luego, gota a gota mientras se agita, hasta que el colágeno se torna naranja, </li><li> pero no de color rosa (generalmente hasta 3 ml). P aproximado H = 7,2. </li><li> Nota: Es importante asegurarse de que el medio / gel permanece neutro o ligeramente ácido, como los fibroblastos no se contrae el gel si se expone a ligeramente alcalino. </li></ul></li><li> Trypsinise los fibroblastos, giran a 400 xg durante 5 minutos y retirar el sobrenadante. </li><li> Durante 5 minutos por encima de giro: Prepare 12 x 35 mm platos de plástico - normalmente alcanzar 12 platos de un frasco de fibroblastos / mezcla de colágeno. </li><li> Vuelva a suspender los fibroblastos en 3 ml de FCS, e inmediatamente después a la mezcla de colágeno y revuelva. </li><li> Placa de aproximadamente 2,5 ml de colágeno / fibroblasto por plato tan pronto como sea posible. Trate de evitar las burbujas y la creación de colágeno. </li><li> Deje que el colágeno a 37 ° C en una atmósfera humidificada de 5% de CO 2 en el aire durante 10 minutos. </li><li> Añadir 1 ml de medio de crecimiento de fibroblastos (DMEM + 10% FCS). </li><li> Separe la matriz de colágeno / fibroblasto de los lados de la cápsula, utilizando una pipeta. </li><li> Al día siguiente: Añadir 1 ml de medio de crecimiento de fibroblastos (DMEM + 10% FCS). </li><li> Cambiar los medios de comunicación todos los días. Permitir colágeno / fibroblasto de la matriz se contraiga durante aproximadamente 8 días, hasta que encajen en una placa de 24 pocillos (contracción de ~ 3,5 cm a 1,5 cm de diámetro, ver Figura 1). </li></ol> Nota: La concentración de colágeno se debe ajustar para adaptarse a la aplicación. Cuanto más se diluye la solución de colágeno, más rápido será contratado por los fibroblastos. Pequeñas diferencias en el ritmo de contracción se experimentará con diferentes lotes de colágeno en la misma concentración. Del mismo modo, las diferentes culturas de fibroblastos se contraerá los geles de colágeno a un ritmo diferente, y presentar los fibroblastos, más rápido el ritmo de contracción. Concentración de colágeno y la densidad de los fibroblastos, por consiguiente, ajustado a modificar el ritmo de contracción,y la densidad final del gel de colágeno. 4. Etapa II - Revestimiento de las células de interés en la parte superior de la matriz Nota: Esterilice todas las pinzas y el equipo con etanol antes de su uso. <ol><li> El uso de pinzas romas, mueva suavemente la matriz contratado para 24 y plato. Asegúrese de que no se pliega. </li><li> Prepare una suspensión de células de interés de aproximadamente 4 x 10 1 ml 4 / ml y una placa (después de trypsinising, girar las células para deshacerse de la tripsina) en la parte superior de la matriz. El número real de células necesarias pueden variar dependiendo del tipo de célula utilizada. Medio celular debe ser un medio de crecimiento normal de las células de interés. </li><li> Permiten a las células a crecer hasta la confluencia en la parte superior de la matriz, de aproximadamente 3-5 días. </li></ol> 5. Etapa III - La transferencia de la matriz a una red para la invasión (aproximadamente 0-21 días) <ol><li> Cortar las rejillas de acero inoxidable para crear un trípode y un autoclave antes de su uso (ver Figura 2). </li><li> Colocar malla estéril en una fuente de 6 cm, añadir medio de crecimiento a un nivel por encima de la rejilla (aprox. 10.5ml). Lugar de la matriz en la parrilla y con cuidado aspirar los medios de comunicación por lo que la parte inferior de la matriz está en contacto con los medios de comunicación, pero no sumergida. Esto se conoce como la interfase aire / líquido, lo que crea un gradiente que promueve la invasión. Vuelva a colocar medio cada dos días (ver Figura 2). </li><li> De células vivas, imágenes contextuales usando células fluorescentes se pueden visualizar en esta etapa o antes. El colágeno fibrilar contratados o que se pueden visualizar utilizando la generación de segundo armónico (SHG, ver figura 3). El uso de múltiples fotones de excitación combinada con un amplio campo de detección, encontramos que los grupos de autoayuda pueden ser reflejados por lo menos 100 m en la matriz, mientras que las células que expresan GFP citoplasma se pueden visualizar por lo menos 200 m de profundidad. </li></ol> Nota: Con respecto a la cuantificación de la invasión, el día en que matricies se colocan en las redes define el día 0. Colocación en redes genera un gradiente de medios de cultivo celular que promueve la invasión de la matriz. Las muestras se pueden visualizar en los próximos 1 a 21 días (o más) para evaluar los procesos biológicos, como la invasión, la proliferación, la supervivencia o diferenciación (Ver la lista de referencia). 6. Etapa IV - Fijación <ol><li> Añadir 5 ml de PFA al 4% en un tubo Falcon. </li><li> Transferencia de la matriz sobre una superficie plana. Corte la matriz por la mitad con un nuevo bisturí limpio (como se le mancha la sección transversal), levante la matriz con el bisturí y la transferencia de PFA al 4%, solución durante la noche. </li><li> Matrices organotípicos Ahora está listo para teñir con anticuerpos / mancha de la elección (ver Figura 3). </li></ol> 7. Los resultados representativos: <img alt="Figura 1" src="/files/ftp_upload/3089/3089fig1.jpg" /> Figura 1 Ejemplo de fibroblastos-colágeno contraído I. Mezcla de fibroblastos y el colágeno cola de rata separado de la placa de Petri y se deja más de contrato 6-8 días. <img alt="Figura 2" src="/files/ftp_upload/3089/3089fig2.jpg" /> Figura 2 progresión ensayo organotípicos. Un esquema de conjunto organotípicos y progresión. B, Ejemplo de matriz de colágeno / fibroblasto en la parte superior de acero inoxidable, malla estéril para crear interfase aire / líquido. <img alt="Figura 3" src="/files/ftp_upload/3089/3089fig3.jpg" /> Figura 3 Las solicitudes de ensayo organotípicos. A, B adenocarcinoma de páncreas no es invasivo y ductal invasivo (PDAC), las células invasoras a través del tiempo con la matriz organotípicos. C, equivalente a piel viva que muestra una epidermis estratificada en un fibroblasto contratados componente dérmico de colágeno. D, C8161 células del melanoma invasor en un fibroblasto contratada equivalente de colágeno dérmico. E, que invada las células PDAC (verde), que interactúan con los fibroblastos y la invasión (en rojo) dentro de la matriz organotípicos. F, que invada las células PDAC (verde), que interactúan con una degradación de la matriz extracelular circundante (púrpura) visualizan mediante multifotónica basado en la generación del segundo armónico.

<ol>
	<li>Yamada, K. M., Cukierman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+tissue+morphogenesis+and+cancer+in+3D.">Modeling tissue morphogenesis and cancer in 3D.</a> Cell. 130, 601-610 (2007).</li><li>Edward, M., Gillan, C., Micha, D., Tammi, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour+regulation+of+fibroblast+hyaluronan+expression:+a+mechanism+to+facilitate+tumour+growth+and+invasion.">Tumour regulation of fibroblast hyaluronan expression: a mechanism to facilitate tumour growth and invasion.</a> Carcinogenesis. 26, 1215-1223 (2005).</li><li>Patsialou, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+of+human+breast+cancer+cells+in+vivo+requires+both+paracrine+and+autocrine+loops+involving+the+colony-stimulating+factor-1+receptor.">Invasion of human breast cancer cells in vivo requires both paracrine and autocrine loops involving the colony-stimulating factor-1 receptor.</a> Cancer Res. 69, 9498-9506 (2009).</li><li>Serrels, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+study+of+E-cadherin+and+membrane+dynamics+in+living+animals:+implications+for+disease+modeling+and+drug+development.">Real-time study of E-cadherin and membrane dynamics in living animals: implications for disease modeling and drug development.</a> Cancer Res. 69, 2714-2719 (2009).</li><li>Timpson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+Regulation+of+RhoA+Activity+during+Pancreatic+Cancer+Cell+Invasion+Driven+by+Mutant+p53.">Spatial Regulation of RhoA Activity during Pancreatic Cancer Cell Invasion Driven by Mutant p53.</a> Cancer Res. 71, 747-757 (2011).</li><li>Edward, M., Quinn, J. A., Pasonen-Seppanen, S. M., McCann, B. A., Tammi, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=4-Methylumbelliferone+inhibits+tumour+cell+growth+and+the+activation+of+stromal+hyaluronan+synthesis+by+melanoma+cell-derived+factors.">4-Methylumbelliferone inhibits tumour cell growth and the activation of stromal hyaluronan synthesis by melanoma cell-derived factors.</a> Br. J. Dermatol. 162, 1224-1232 (2010).</li><li>Fusenig, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+differentiation+characteristics+of+transformed+keratinocytes+from+mouse+and+human+skin+in+vitro+and+in+vivo.">Growth and differentiation characteristics of transformed keratinocytes from mouse and human skin in vitro and in vivo.</a> J. Invest. Dermatol. 81, 168s-175s (1983).</li><li>Nystrom, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+quantitative+method+to+analyse+tumour+cell+invasion+in+organotypic+culture.">Development of a quantitative method to analyse tumour cell invasion in organotypic culture.</a> J. Pathol. 205, 468-475 (2005).</li><li>Sabeh, F., Shimizu-Hirota, R., Weiss, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protease-dependent+versus+-independent+cancer+cell+invasion+programs:+three-dimensional+amoeboid+movement+revisited.">Protease-dependent versus -independent cancer cell invasion programs: three-dimensional amoeboid movement revisited.</a> J. Cell. Biol. 185, 11-19 (2009).</li><li>Amjad, S. B., Carachi, R., Edward, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keratinocyte+regulation+of+TGF-beta+and+connective+tissue+growth+factor+expression:+a+role+in+suppression+of+scar+tissue+formation.">Keratinocyte regulation of TGF-beta and connective tissue growth factor expression: a role in suppression of scar tissue formation.</a> Wound Repair Regen. 15, 748-755 (2007).</li><li>Gaggioli, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast-led+collective+invasion+of+carcinoma+cells+with+differing+roles+for+RhoGTPases+in+leading+and+following+cells.">Fibroblast-led collective invasion of carcinoma cells with differing roles for RhoGTPases in leading and following cells.</a> Nat. Cell. Biol. 9, 1392-1400 (2007).</li></ol>

No tenemos nada que revelar.

Aquí presentamos un método para la producción de una matriz de tres dimensiones adecuadas para el estudio de la migración de células invasivas 6-8. La matriz está formada por colágeno tipo 1 fibrillas, que son contratados por un período de varios días por fibroblastos primarios epidérmico humano. El colágeno es extraído el ácido, la digestión no enzimática, que conserva la reactividad en los extremos de poli-péptido y promueve el entrecruzamiento de las fibrillas de colágeno en grandes agregados en la neutralización de 9 de las condiciones de amortiguación. Esto se traduce en un gel reconstituido que más se asemeja mucho a las características de colágeno en vivo y tiene importantes consecuencias para la capacidad invasora de las células cultivadas en los geles. No importa si el material de partida fresca o congelada se utiliza, pero el uso de colas de ratas adolescentes es importante porque la reticulación del colágeno es más lábil en animales más jóvenes. Colágeno I de los animales más jóvenes por lo tanto, es más fácil de extraer y re-constituye con mayor fidelidad. La matriz de colágeno pueden ser fijadas y teñidas con anticuerpos dirigidos contra cualquiera de las células tumorales invasoras o los fibroblastos del estroma 2,10 y son muy útiles para probar la eficacia de los fármacos que pueden inhibir la invasión 5,6. Aunque este protocolo sugiere el uso de primaria fibroblastos de piel humana, es factible el uso de fibroblastos primarios de otros tipos de tejidos, de acuerdo con el tipo de tejido bajo investigación. También es posible establecer cultivos con fibroblastos inmortalizados, incluyendo las células que han sido transfectadas establemente con los conjugados proteína fluorescente. De esta manera, las células del estroma y los tumores pueden ser etiquetados para visualizar interacciones célula-célula durante la invasión 11.

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios (opcional) </th></tr><tr><td> MEM 10 veces </td><td> Gibco </td><td> 21430 </td><td> - </td></tr><tr><td> NaOH </td><td> Sigma </td><td> 367176-500G </td><td> Prepare 0,22 M de valores en el agua </td></tr><tr><td> FCS </td><td> PAA Laboratories </td><td> A15-101 </td><td> - </td></tr><tr><td> Placas de 35 mm </td><td> Halcón </td><td> 353001 </td><td> Para el paso 3.4 </td></tr><tr><td> Placas de 60 mm </td><td> Halcón </td><td> 353004 </td><td> Para el paso 5.2 </td></tr><tr><td> Primavera pinza roma </td><td> Samco </td><td> E003/02 </td><td> Dientes, no sin problemas </td></tr><tr><td> 16% de paraformaldehído </td><td> Electron Microscopy Servicios </td><td> 15710 </td><td> Diluir al 4% en PBS antes de su uso </td></tr><tr><td> Pantallas de cd-1, el tamaño de malla de 40 </td><td> Sigma </td><td> S07707-5EA </td><td> Rejillas de acero inoxidable, 5 por paquete </td></tr><tr><td> Tubos de diálisis </td><td> Medicell Internacional </td><td> 7607 2295 </td><td> 12 a 14 kD </td></tr><tr><td> PBS </td><td> Oxoid </td><td> BR0014G </td><td> - </td></tr><tr><td> Ácido Acético </td><td> Sigma </td><td> 242853 </td><td> - </td></tr><tr><td> 24 platos y </td><td> Halcón </td><td> 353047 </td><td> Para el paso 4.1 </td></tr><tr><td> Fungizone </td><td> En Vitrogen </td><td> 15290018 </td><td> - </td></tr></tbody></table>

organotypic collagen i assay: a malleable platform to assess cell behaviour in a 3-dimensional context

Migración de las células es fundamental para muchos aspectos de la biología, incluyendo el desarrollo de la cicatrización de heridas, las respuestas celulares del sistema inmunológico, y la metástasis de las células tumorales. La migración se ha estudiado en cubreobjetos de vidrio a fin de que las dinámicas celulares susceptibles de investigación por microscopía de luz. Sin embargo, ha quedado claro que muchos aspectos de la migración de las células dependen de las características del entorno local, incluyendo su elasticidad, la composición de proteínas, y el tamaño de los poros, que no están fielmente representados por dos sustratos rígidos dimensiones como el vidrio y de plástico de 1. Además, la interacción con otros tipos celulares, incluyendo fibroblastos del estroma 2 y 3 células del sistema inmune, se ha demostrado que desempeñan un papel fundamental en la promoción de la invasión de las células cancerosas. Investigación a nivel molecular ha demostrado que cada vez más dinámica molecular, incluyendo la respuesta al tratamiento farmacológico, de células idénticas son significativamente diferentes en comparación in vitro e in vivo 4. Idealmente, sería mejor para estudiar la migración de células en su contexto natural en los organismos vivos, sin embargo, esto no siempre es posible. Intermedio sistemas de cultivo de tejidos, tales como la matriz de células derivadas, matrigel, la cultura organotípicos (descrito aquí) explantes de tejidos, organoides, y xenoinjertos, son importantes intermedios experimental. Estos sistemas aproximada ciertos aspectos de un entorno en vivo, pero son más susceptibles a la manipulación experimental, como el uso de líneas celulares transfectadas de forma estable, los regímenes de tratamiento de drogas, a largo plazo y de imágenes de alta resolución. Tales sistemas intermedios son especialmente útiles como demostrando los motivos para validar las sondas y establecer los parámetros necesarios para la imagen de la respuesta dinámica de las células y los reporteros fluorescentes antes de realizar imágenes en vivo 5. Como tales, pueden desempeñar un papel importante en la reducción de la necesidad de que los experimentos en animales vivos.

Watch this Scientific Journal Video about Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context at JoVE.com

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Se describe un método para la preparación de una matriz de tres dimensiones que consiste en colágeno tipo I y fibroblastos primarios humanos. Este gel organotípicos sirve como un sustrato útil para evaluar la migración de células invasivas debido a que imita las características básicas del estroma y se presta a muchas formas de microscopía.

El colágeno organotípicos I del ensayo: Una plataforma maleable para evaluar el comportamiento celular en un contexto de 3 dimensiones

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

organotypic-collagen-i-assay-malleable-platform-to-assess-cell

Research

JoVE Journal

Bioengineering

22.3K vistas.  University of Glasgow. Se describe un método para la preparación de una matriz de tres dimensiones que consiste en colágeno tipo I y fibroblastos primarios humanos. Este gel organotípicos sirve como un sustrato útil para evaluar la migración de células invasivas debido a que imita las características básicas del estroma y se presta a muchas formas de microscopía.

Video: Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

Un método para la encapsulación de los folículos ováricos y la Cultura en un proteolíticamente degradables tres dimensiones del sistema

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

Investigación

Bioingeniería

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

La creación de dos dimensiones con dibujos sustratos para el confinamiento de proteínas y células

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

Cultivo de Células progenitoras neurales en un hidrogel de péptidos 3-dimensional de auto-montaje

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Los andamios de autoinforme para el 3-Dimensional Cultivo Celular

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

Un método mejorado para la preparación de colágeno tipo I De Piel

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

Análisis de la migración de células dentro de una matriz de colágeno tridimensional

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

Planar Sistema de Difusión de degradado para Investigar quimiotaxis en una matriz de colágeno 3D

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

Tejido picado en colágeno comprimido: Un Bio Transplant que contiene células para la reparación reconstructiva-puesta en escena individual

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.

Recombinante colágeno péptido Microcarriers para la extensión celular y su posible uso como sistema de células en un biorreactor modelo

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

Rodent Behavioral Testing to Assess Functional Deficits Caused by Microelectrode Implantation in the Rat Motor Cortex

Medical devices implanted in the brain hold tremendous potential. As part of a Brain Machine Interface (BMI) system, intracortical microelectrodes demonstrate the ability to record action potentials from individual or small groups of neurons. Such recorded signals have successfully been used to allow patients to interface with or control&#160;computers, robotic limbs, and their own limbs. However, previous animal studies have shown that a microelectrode implantation in the brain not only damages the surrounding tissue but can also result in functional deficits. Here, we discuss a series of behavioral tests to quantify potential motor impairments following the implantation of intracortical microelectrodes into the motor cortex of a rat. The methods for open field grid, ladder crossing, and grip strength testing provide valuable information regarding the potential complications resulting from a microelectrode implantation. The results of the behavioral testing are correlated with endpoint histology, providing additional information on the pathological outcomes and impacts of this procedure on the adjacent tissue.

We have shown that a microelectrode implantation in the motor cortex of rats causes immediate and lasting motor deficits. The methods proposed herein outline a microelectrode implantation surgery and three rodent behavioral tasks to elucidate potential changes in the fine or gross motor function due to implantation-caused damage to the motor cortex.