Este trabajo fue apoyado por el Fondo de Investigación del Cáncer de ovario [conceder LT/UIC/01.2011], el Centro para la Ciencia UIC clínica y traslacional, el Centro de Cáncer de la UIC, y los Institutos Nacionales de Salud de subvención C06RR15482. Los experimentos con animales se realizaron de acuerdo con las directrices y normas establecidas por AAALAC en los protocolos aprobados por el cuidado de animales por el Cuidado de Animales y el empleo en la UIC. Los animales de este proyecto se encuentran en las salas de barrera en el Laboratorio de Biología de Recursos (BRL) de la Universidad de Illinois en Chicago. El BRL cuenta con un personal totalmente veterinario que supervisa los animales por lo menos dos veces al día y ofrece asesoramiento sobre cuidado de los animales.

1. La disección de ovario y el aislamiento de los tejidos <ol><li> Prepare una solución al 0,5% (w / v) de alginato de sodio, de preparar una versión modificada del protocolo descrito previamente 5, con PBS estéril y calentar a 37 º C. Mezclar por inversión o de rock, pero no del vórtice. Dibujar el alginato en una jeringuilla de 1 ml con una aguja de 25 G y mantener a 37 ° C. </li><li> Prepare 10 mM estéril solución de CaCl2 para la reticulación del alginato en la encapsulación de tejido y calentar a 37 º C. </li><li> Prepare 1 ml de medio de cultivo (αMEM + 5 unidades de penicilina y estreptomicina 5 mg / ml) por pocillo en una placa de 24 pocillos para cada experimento. Incluyen diversos fármacos, péptidos, o virus en el medio de cultivo. Para la transfección antes de la encapsulación de órganos, los tejidos se incuba con Lipofectamine 2000 y el ADN del plásmido durante cuatro horas a 37 º C. </li><li> Diseccionar los órganos del animal, eliminar o quitar cualquier grasa o tejido residual, tire suavemente de la membrana que rodea la bursa del ovario, y cortar los ovarios o el oviducto en cuatro pedazos por que atraviesa el órgano. Los animales fueron sacrificados por asfixia de CO 2 seguido por dislocación cervical. Para el oviducto, desenrollar suavemente el tejido tirando de las membranas de conexión y ampliación de los extremos del tubo en sentido contrario el uso de fórceps antes de cortar. </li><li> Coloque una gota de alginato solo (l ~ 1-3) en una malla de fibra de esterilización. </li><li> Con unas pinzas estériles, mover una pieza de órgano en la gota de alginato y el centro de la gota uso de fórceps o una aguja estéril. </li><li> Invertir la gota que contiene la pieza de órgano (organoide), tome el malla de fibra con pinzas estériles y se vuelve boca abajo para que la gravedad de la fuerza del alginato en una gota. </li><li> Toque la pinza en el borde de un plato de Petri que contiene 10 mM de 10 cm de CaCl2 solución para que la gota cae en la solución. </li><li> Incubar durante 2 minutos, retire con una cuchara estéril que contiene poros para el lanzamiento adicional de CaCl 2 10 mM, y la transferencia al medio de cultivo. Hasta cuatro geles de alginato se pueden transferir a un solo pozo de una placa de 24 pocillos que contienen el medio de cultivo. Cultura por un periodo de tiempo definido. </li></ol> 2. La degradación de alginato y la recolección de las células <ol><li> Para la solubilización del gel de alginato, se incuban los cultivos en alginato liasa (3,4 mg / ml) disuelto en L-15 de Leibovitz medios de comunicación a 37 ° C durante 30 minutos o hasta que el gel esté completamente disuelta. </li><li> Eliminar el tejido de la liasa de alginato. </li><li> Para la recogida de la OSE, se incuban los organoides con 500 unidades de la colagenasa en el L-15 de Leibovitz medios de comunicación durante 1 hora 8. Vortex en dos pulsos segundo (2 segundos y 2 segundos vórtice resto) a una potencia media durante 1 minuto, seguido por un segundo pulso (1 segundo vórtice y un segundos de descanso) durante 1 minuto. Deje que las piezas caen al fondo del órgano de tubo de microcentrífuga y el sobrenadante de la pipeta en un tubo nuevo de microcentrífuga estéril. Centrifugar 3 minutos a 3000 rpm en el epitelio superficial del ovario de pellets. </li><li> Para la recogida del epitelio de las trompas, cortar el tubo a lo largo y lugar en el plástico de cultivo de tejidos en DMEM con 5 unidades de penicilina y 5 mg / ml de estreptomicina y 4% (v / v) de SFB. Durante 3-5 días, las células se &quot;arrastre&quot; de la estroma de trompas y crecer en cultivo de tejidos plastic9. </li></ol> 3. La fijación de las culturas de alginato de órganos encapsulados para cortes de parafina <ol><li> Recoger el alginato encapsulado de cuentas del medio de cultivo en el punto de tiempo seleccionado. </li><li> Recrosslink el alginato para endurecer el gel por la caída de la cultura en el estéril de CaCl2 10 mM solución de dos minutos. </li><li> Fijar el órgano encapsulado en alginato paraformaldehído al 2% que contiene 50 mM de sacarosa, CaCl2 10 mM y 50 mM cacodilato de sodio para asegurar que el gel se mantiene sólido y totalmente encapsula los tejidos durante la fijación. </li><li> Después de procesamiento de tejidos, integrar todo el alginato encapsulado pieza cultivo de órganos en parafina y sección con un microtomo para generar secciones de 5 micras. </li><li> El alginato se disolverá y se retira de la diapositiva si se realiza inmunohistoquímica debido al calor de recuperación de antígeno base. El alginato se mantendrá y manchas de eosina en pie hematoxilina y eosina. </li></ol> 4. Los resultados representativos: Un ejemplo de una cultura del órgano en tres dimensiones del ovario y el oviducto se muestra en la Figura 1. Los ovarios se pueden cortar en trozos de diferente tamaño, y OSE proliferar y migrar a la reparación de la superficie que resultó herido de la incisión con el bisturí. Durante este proceso, la orientación correcta de la OSE en relación con el estroma subyacente se mantiene, con la OSE encapsular el ovario. Como se muestra en la Figura 2, la OSE de los ovarios reduce a la mitad encapsular el ovario de forma más completa, en comparación con los ovarios cortadas en cuartos u octavos. La reparación de las heridas de los ovariosla superficie puede ser estudiado en el contexto de todo el ovario y puede arrojar luz sobre cómo reparar la ovulación y la superficie del ovario induce la transformación de la superficie. El objetivo del experimento depende de la información que se requiera. La arquitectura del tejido, la morfología celular, y las tasas de crecimiento de las células son fácilmente controlables con los cortes de parafina. Por ejemplo, una sola capa de epitelio ovárico superficie se observa después de un cultivo en el suero libre αMEM durante 7 días (Figura 3), mientras que la adición de 5 mg / ml de insulina por 7 días induce hiperproliferación del epitelio superficial del ovario y la formación de múltiples capas de las células (Figura 3B). Mediante la adición de bromodesoxiuridina a los cultivos a una concentración final de 10 mM de 24 horas antes de la fijación, la proliferación de las células se puede medir (3A y 3B recuadro). De cultivos mantenidos en el suero libre αMEM, aproximadamente el 3-5% de OSE someterse a la proliferación durante el período de etiquetado de 24 horas (3A recuadro), mientras que un porcentaje mayor de la proliferación de células de la granulosa. Además de la insulina a la cultura de los medios de comunicación aumenta el porcentaje de la proliferación de OSE a aproximadamente el 30% del total de OSE (3B recuadro). Inmunofluorescencia de la proteína verde fluorescente (GFP) se pueden visualizar a través del gel de alginato con el microscopio estándar después de la transfección de cDNA (Figura 3C). Si bien la cultura proporciona alginato de órganos para el crecimiento y el análisis de la superficie de epitelio ovárico y los folículos primarios, cabe señalar que los folículos maduros, secundarios no llegan a sobrevivir en este sistema de cultivo (Figura 3D). Cultura de los folículos individuales a la madurez dentro de una matriz de hidrogel de alginato se ha descrito en otra parte 10. Alteraciones en la proteína o ARN puede ser analizada desde el órgano completo o de tipos específicos de células con la degradación enzimática (Figura 4). El tratamiento de organoides de ovario cultivadas con colagenasa deja el estroma subyacente intacto (Figura 4 C) al tiempo que permite el enriquecimiento de OSE en el sedimento celular obtenido tras el tratamiento, aunque algunas células del estroma son aislados (Figura 4). <img alt="Figura 1" src="/files/ftp_upload/2804/2804fig1.jpg" /> Figura 1. Ovario ratón tridimensional sistema de cultivo utilizado para propagar OSE después de la herida. A) Bajo el microscopio, la bolsa y la almohadilla de grasa alrededor se han eliminado. B) ovarios intactos (O) con el tubo del oviducto (T) y la almohadilla de grasa (F). C) Ovario con bolsa y la almohadilla de grasa extraída (izquierda) y desenrolló el oviducto (derecha). D) organoides ovario o del oviducto se colocaron en una gota de alginato. E) organoides alginato encerrado se deja caer en un entrecruzamiento solución de CaCl2 el alginato para formar un gel. F) El alginato encapsulado organoide de ovario. G) alginato encapsulado organoide oviducto. H) organoides alginato encapsulado incubadas en medio de cultivo. <img alt="Figura 2" src="/files/ftp_upload/2804/2804fig2.jpg" /> Figura 2. Superficie regenerada por la superficie del ovario mide por citoqueratina 8. La encapsulación de los organoides de ovario por la OSE tras ser cortadas en cuartos octavos, y mitades, cultivadas en un medio de BSA, y se mide por el análisis de inmunohistoquímica con citoqueratina 8 (CK8) de anticuerpos. <img alt="Figura 3" src="/files/ftp_upload/2804/2804fig3.jpg" /> Figura 3. Sistema de cultivo de alginato permite el paso de los factores de crecimiento y de la transfección de cDNA en el entorno de sistema operativo, pero no es compatible con un rápido crecimiento y maduración de los folículos. A) organoide ovario cultivadas durante 7 días en medio de base para analizar CK8 expresión, que marca el OSE. Sección de inserción, serial manchados de incorporación de BrdU. B) la adición de insulina al medio de cultivo induce hiperplasia de la OSE. Sección de inserción, serial manchados de incorporación de BrdU. C) organoide ovario transfectadas con cDNA de expresar las buenas prácticas agrarias. D) Sistema de alginato de la cultura apoya el crecimiento de folículos primordiales (rojo punta de flecha), pero no folículos secundarios maduros (flecha negro). <img alt="Figura 4" src="/files/ftp_upload/2804/2804fig4.jpg" /> Figura 4. OSE puede ser aislado de la cultura siguiente análisis. A) organoide cultivaron en un medio para un máximo de 2 semanas. B) organoide encapsulado se trata con alginato liasa dejar organoide intacto. Ovario se tiñe con CK8 para marcar OSE. C) organoide es tratada con colagenasa a OSE separado del estroma subyacente. Pieza restante tejido teñido con CK8 para destacar la eliminación de la superficie del ovario. D) las células obtenidas después del tratamiento se enriquecen de OSE como manchas de CK8 (rojo). Las células son contrastados con DAPI para marcar los núcleos.

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No hay nada que revelar.

El desarrollo de un sistema de órganos en tres dimensiones utilizando los hidrogeles de alginato de la cultura representa un método versátil para el análisis de muchos tipos diferentes de tejidos a partir de una amplia variedad de organismos. El uso de cultivos en 3D se puede extender a los campos de la ingeniería de tejidos y medicina regenerativa, ya que proporciona un andamio en el que las células pueden crecer 11. En la actualidad, nuestro laboratorio está pasando por los estudios iniciales con los...

<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> Leibovitz medio L-15 </td><td> Gibco </td><td> 11415 </td><td></td></tr><tr><td> αMEM medio </td><td> Gibco </td><td> 12000-022 </td><td></td></tr><tr><td> Alginato de sodio </td><td> NovaMatrix </td><td></td><td></td></tr><tr><td> Cacodilato de sodio </td><td> Sigma Aldrich </td><td> C0250 </td><td></td></tr><tr><td> Malla de polipropileno de fibra </td><td> McMaster Carr </td><td></td><td> 45 &quot;rollo de ancho; 0,0041&quot; apertura </td></tr><tr><td> Lipofectamine 2000 </td><td> Invitrogen </td><td> 11668 </td><td></td></tr><tr><td> Penicilina / estreptomicina </td><td> Gibco </td><td> 15070-063 </td><td></td></tr><tr><td> Alginato liasa </td><td> Sigma Aldrich </td><td> A1603 </td><td></td></tr><tr><td> A1603 </td><td> Sigma Aldrich </td><td> C9697 </td><td> De Clostridium histolyticum </td></tr><tr><td> Albúmina de suero bovino </td><td> MP Biomedicals </td><td> 103700 </td><td></td></tr><tr><td> De suero fetal bovino </td><td> Gibco </td><td> Gibco </td><td></td></tr><tr><td> SU solución </td><td> Roche </td><td> 11074547001 </td><td> Insulina / transferrina / selenito de sodio </td></tr><tr><td> Citoqueratina 8 de anticuerpos </td><td> Estudios del Desarrollo del Banco hibridoma </td><td> TROMA-1 </td><td> El uso de 1:200 </td></tr><tr><td> DAPI </td><td> Vector de Laboratorios </td><td> H1200 </td><td></td></tr><tr><td> Bromodeoxyuridine </td><td> Sigma </td><td> B50002-1G </td><td> Concentración final de 10 M </td></tr></tbody></table>

alginate hydrogels for three-dimensional organ culture of ovaries and oviducts

El cáncer de ovario es la quinta causa principal de muerte por cáncer en las mujeres y tiene una tasa de mortalidad del 63% en los Estados Unidos 1. El tipo de célula de origen de los cánceres de ovario sigue siendo en cuestión y podría ser el epitelio superficial del ovario (OSE) y del epitelio distal de la trompa de Falopio fimbrias 2,3. El cultivo de las células normales como cultivo primario in vitro permitirá a los científicos para modelar los cambios específicos que podrían conducir al cáncer de ovario en el epitelio distintas, lo que definitivamente la determinación del tipo de célula de origen. Esto permitirá el desarrollo de marcadores biológicos más precisos, los modelos animales con cambios en los tejidos específicos de genes, y mejores estrategias de prevención dirigidas a esta enfermedad. El mantenimiento de las células normales en los hidrogeles de alginato promueve a corto plazo en el cultivo in vitro de las células en sus tres dimensiones contexto y permite la introducción de ADN plásmido, siRNA, y moléculas pequeñas. Por los órganos de cultivo en las piezas que se derivan de los cortes estratégicos utilizando un bisturí culturas, varios de un solo órgano se pueden generar, aumentar el número de experimentos de un solo animal. Estos recortes aspectos el modelo de la ovulación que conduce a la proliferación de la OSE, que se asocia con la formación de cáncer de ovario. Tipos de células, como el entorno de sistema operativo que no crecen bien en superficies de plástico pueden ser cultivadas con este método y facilitar la investigación sobre los procesos celulares normales o los primeros eventos en la formación del cáncer 4. Hidrogeles de alginato se pueden utilizar para apoyar el crecimiento de muchos tipos de tejidos 5. El alginato es un polisacárido lineal compuesto por unidades repetidas de β-D-manurónico ácido y α-L-gulurónico ácido que puede ser reticulado con iones de calcio, lo que resulta en una acción gelificante suave que no dañan los tejidos 6,7. Al igual que otras matrices tridimensionales de cultivo celular como Matrigel, alginato proporciona soporte mecánico para los tejidos, sin embargo, las proteínas no son reactivos con la matriz de alginato, y por eso funciona como una matriz de alginato de síntesis extracelular que no inicia la señalización celular 5. El hidrogel de alginato flota en el medio de cultivo celular estándar y compatible con la arquitectura del crecimiento de los tejidos in vitro. Se presenta un método para la preparación, la separación, y la inclusión de ovario y el oviducto pedazos de órganos en los hidrogeles de alginato, que pueden mantenerse en cultivo durante un máximo de dos semanas. La liberación enzimática de las células para el análisis de proteínas y RNA de muestras de la cultura de órganos también se describe. Finalmente, el crecimiento de tipos de células primarias es posible sin la inmortalización genética de ratones y permite a los investigadores a utilizar por nocaut y en ratones transgénicos.

Watch this Scientific Journal Video about Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts at JoVE.com

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Cultivo de células normales en sus tres dimensiones contexto representa un método alternativo para estudiar los primeros eventos necesarios para la transformación celular y tumorigénesis. Este método se utiliza para producir los ovarios normales y las células del oviducto para estudiar los eventos tempranos en la formación de cáncer de ovario.

Los hidrogeles de alginato para la cultura del órgano en tres dimensiones de los ovarios y oviductos

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

alginate-hydrogels-for-three-dimensional-organ-culture-ovaries

Research

JoVE Journal

Bioengineering

21.8K vistas.  University of Illinois at Chicago. Cultivo de células normales en sus tres dimensiones contexto representa un método alternativo para estudiar los primeros eventos necesarios para la transformación celular y tumorigénesis. Este método se utiliza para producir los ovarios normales y las células del oviducto para estudiar los eventos tempranos en la formación de cáncer de ovario.

Video: Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Impresión plana y tridimensional de tintas conductoras

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Investigación

Bioingeniería

Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture

The intervertebral disc (IVD) is the joint of the spine connecting vertebra to vertebra. It functions to transmit loading of the spine and give flexibility to the spine. It composes of three compartments: the innermost nucleus pulposus (NP) encompassing by the annulus fibrosus (AF), and two cartilaginous endplates connecting the NP and AF to the vertebral body on both sides. Discogenic pain possibly caused by degenerative intervertebral disc disease (DDD) and disc herniations has been identified as a major problem in our modern society. To study possible mechanisms of IVD degeneration, in vitro organ culture systems with live disc cells are highly appealing. The in vitro culture of intact bovine coccygeal IVDs has advanced to a relevant model system, which allows the study of mechano-biological aspects in a well-controlled physiological and mechanical environment. Bovine tail IVDs can be obtained relatively easy in higher numbers and are very similar to the human lumbar IVDs with respect to cell density, cell population and dimensions. However, previous bovine caudal IVD harvesting techniques retaining cartilaginous endplates and bony endplates failed after 1-2 days of culture since the nutrition pathways were obviously blocked by clotted blood. IVDs are the biggest avascular organs, thus, the nutrients to the cells in the NP are solely dependent on diffusion via the capillary buds from the adjacent vertebral body. Presence of bone debris and clotted blood on the endplate surfaces can hinder nutrient diffusion into the center of the disc and compromise cell viability. Our group established a relatively quick protocol to "crack"-out the IVDs from the tail with a low risk for contamination. We are able to permeabilize the freshly-cut bony endplate surfaces by using a surgical jet lavage system, which removes the blood clots and cutting debris and very efficiently reopens the nutrition diffusion pathway to the center of the IVD. The presence of growth plates on both sides of the vertebral bone has to be avoided and to be removed prior to culture. In this video, we outline the crucial steps during preparation and demonstrate the key to a successful organ culture maintaining high cell viability for 14 days under free swelling culture. The culture time could be extended when appropriate mechanical environment can be maintained by using mechanical loading bioreactor. The technique demonstrated here can be extended to other animal species such as porcine, ovine and leporine caudal and lumbar IVD isolation.

This protocol illustrates a harvesting technique for coccygeal bovine intervertebral discs for organ culture for in vitro organ culture.

Preparación de la cola intacta discos intervertebrales bovina para la Cultura de Órganos

The intervertebral disc (IVD) is the joint of the spine connecting  vertebra to vertebra. It functions to transmit loading of the spine and give  ...

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 &mu;m s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.

Tridimensional de la célula modelo de cultivo para medir los efectos del flujo del fluido intersticial en la invasión de células tumorales

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

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 ...

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 ...

Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper composition, targeted modulus, and well-defined architectural features. Biomaterials that recapitulate the intrinsic architecture of in vivo tissue are vital for studying diseases as well as to facilitate the regeneration of lost and malformed soft tissue. A novel biofabrication technique was developed which combines state of the art imaging, three-dimensional (3D) printing, and selective enzymatic activity to create a new generation of biomaterials for research and clinical application. The developed material, Bovine Serum Albumin rubber, is reaction injected into a mold that upholds specific geometrical features. This sacrificial material allows the adequate transfer of architectural features to a natural scaffold material. The prototype consists of a 3D collagen scaffold with 4 and 3 mm channels that represent a branched architecture. This paper emphasizes the use of this biofabrication technique for the generation of natural constructs. This protocol utilizes a computer-aided software (CAD) to manufacture a solid mold which will be reaction injected with BSA rubber followed by the enzymatic digestion of the rubber, leaving its architectural features within the scaffold material.

An innovative biofabrication technique was developed to engineer three-dimensional constructs that resemble the architectural features, components, and mechanical properties of in vivo tissue. This technique features a newly developed sacrificial material, BSA rubber, which transfers detailed spatial features, reproducing the in vivo architectures of a wide variety of tissues.

Tridimensional Tecnología Biomimética: Novel biorubber Crea Definido micro y macro escala Arquitecturas de colágeno hidrogeles

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

La fabricación de cristal invertida coloidal poli (etilenglicol) Andamios: Una célula Plataforma de cultivo tridimensional para ingeniería de tejidos de hígado

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

Las construcciones en capas de alginato: Una plataforma para el co-cultivo de poblaciones celulares heterogéneas

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Formación luz mediada y el patrón de hidrogeles para aplicaciones de cultivo celular

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Los hidrogeles sintonizable de la matriz extracelular pulmonar por la 3D de Cultivos Celulares

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...