Gracias al laboratorio de Kristi Anseth de la Universidad de Colorado, Boulder generosamente proporcionaron MIN6 células. La financiación de este proyecto ha sido proporcionado por la NSF.

1. PEGDM síntesis y PEGDM fotoactivos preparación de la solución de macrómero <ol><li> Pesar 2 g de PEG (lineal, 10.000 Da) en un vial de vidrio de 40 ml con una tapa de plástico duro. </li><li> Añadir aproximadamente 308μL de anhídrido metacrílico y libremente tapa del frasco. </li><li> Microondas en el vial de alto por 2 minutos en un horno de microondas doméstico estándar. El uso de guantes resistentes al calor, a fondo vórtice del vial, a continuación, microondas a máxima potencia durante 5 minutos. </li><li> En pocas palabras permiten al vial que se enfríe lo suficiente como para ser manejados con guantes. Destape y agregue lentamente que el cloruro de metileno, mientras que hace girar el vial. Agregar suficiente para que la solución parece clara y homogénea, agitación de la muestra, según sea necesario. El volumen total de cloruro de metileno utilizado debe ser de 1,5 - 2 ml. </li><li> PEGDM precipitado en 200 ml de frío, se agita éter dietílico mediante la adición de la solución gota a gota. </li><li> Recoge los PEGDM por filtración al vacío y deje que se seque. </li><li> Disolver el PEGDM en una cantidad adecuadade agua desionizada (típicamente 100 a 150 ml). </li><li> La solución de diálisis contra agua desionizada durante 5 días en tubos de diálisis con un peso molecular de corte de 1000 Da. Reemplazar el agua una vez al día. </li><li> Alícuota de la solución en recipientes adecuados y se congelan a -80 ° C durante la noche. Liofilizar la solución durante 4 días para producir un polvo blanco PEGDM purificada. Guarde el polvo a 4 ° C cuando no se utiliza. </li><li> Para preparar una solución PEGDM fotoactivos, en primer lugar elaborar un 0,5 por ciento en peso fotoiniciador solución madre disolviendo Irgacure 2959 en agua desionizada. Vortex la solución y guardarla en la oscuridad. </li><li> Prepare una PEGDM 10% en peso y 0,025% en peso de 2959 Irgacure solución en solución salina equilibrada de Hank (HBSS). Vortex a fondo para asegurar la disolución de la PEGDM. Nos suelen preparar 1 ml de esta solución a la vez. </li><li> Esterilizar la solución por filtrado a través de un filtro de 0,2 micras de la jeringa en un tubo de microcentrífuga de 1,8 ml. Envuelva el tubo con papel de aluminioy se almacena a 4 ° C </li></ol> 2. La cultura, la infección, y la agregación de células MIN6 <ol><li> MIN6 células se cultivan en medio RPMI 1640 suplementado con FBS al 10%, la glucosa de 7 mm, 100 unidades / ml de penicilina / estreptomicina y 0,5 mg / mL anfotericina B en un ambiente humidificado a 37 º C y 5% CO 2. </li><li> Para liberar las células de la superficie de cultivo tratada frasco, aspire el medio, lavar las células con 37 ° C de calcio / magnesio libre de HBSS, aspirar el HBSS, a continuación, agregar 2 ml de 37 ° C con tripsina-EDTA y permitir que las células incubar durante 3 minutos. </li><li> Jar firmemente del lado de la botella para golpear a las células libres, y se añaden 8 ml de 37 º C y la media pipeta vigorosamente la suspensión celular arriba y hacia abajo con cuidado de no introducir burbujas. </li><li> Pipeta las células en un tubo de centrífuga estéril 15 ml y llevar a cabo un recuento de células utilizando un hemocitómetro o celulares contar procedimiento. </li><li> En la preparación para infectar las células con el fabricantevirus, primero se determina el número total de células que están infectadas y calcular el volumen de suspensión de virus necesaria para lograr la multiplicidad deseado de infección (MOI) (50 a 100 partículas infecciosas por célula). </li><li> Diluir el virus en HBSS cuidadosamente pipeta la cantidad apropiada de suspensión de virus en un volumen pre-alícuotas de HBSS en un tubo de microcentrífuga de 1,8 ml. 50 l de HBSS por pocillo de células de agregación es la adecuada. </li><li> Dispersar las células por la pipeta hacia arriba y hacia abajo en su tubo, pipeta el volumen necesario para alcanzar 400.000 células por pocillo en los pocillos de una placa de suspensión de 6 pocillos. </li><li> Añadir la cantidad apropiada de virus diluido a cada pocillo para lograr el deseado momento de inercia. </li><li> Añadir a cada pocillo medio para llevar el volumen total de hasta 1,7 ml. </li><li> Colocar la placa en un agitador orbital dentro de la incubadora. Establecer la coctelera en un nivel bajo para que el medio lava suavemente alrededor de los pozos (~ 100 rpm). Permitir que las células de la agregaciónte de 24 a 36 horas. Agregados con un diámetro aproximado de 75 a 175 micras deben ser formados. </li></ol> 3. La encapsulación de células en PEGDM <ol><li> Las células pueden ser encapsulados en forma de dispersión (paso 2.4) o la agregación siguiente (paso 2,10). Desde el precursor de hidrogel PEGDM solución es un líquido, las células tienden a establecerse antes de la reticulación y la formación del hidrogel. Si la liquidación se espera que ocurra rápidamente, por ejemplo, con agregados de mayor tamaño, puede ser beneficioso para producir el hidrogel en dos pasos para que las células se depositan en un plano central del gel. Un procedimiento de hidrogel de una etapa de síntesis adecuada de las células dispersas se muestra (Figura 2) y el procedimiento de dos pasos se muestra así (Figura 3) y se detallan a continuación (3.2-3.9). </li><li> Crear un recipiente en el que será el hidrogel formado por el corte de la punta cónica de una jeringa de plástico de 1 ml y colocarlo abertura hacia arriba en un estante. </li><li> Pipetear 20 L de PEGDM fotoactivos paralución en el extremo abierto de la jeringa sobre el émbolo de asegurarse de que el volumen cubre toda la superficie del émbolo. Ajustar el émbolo en pequeños incrementos hasta conseguir una superficie plana de líquido. </li><li> Coloque la pipeta en el soporte y la posición de la parrilla por debajo de una lámpara UV (365nm, ~ 7 mW / cm 2) durante aproximadamente 8 minutos para permitir que para el entrecruzamiento del hidrogel. Durante este tiempo, la preparación de las células puede ser iniciado </li><li> Pipeta un volumen que contiene el número deseado de células / agregados en un tubo de microcentrífuga de 1,8 mL, la tapa y centrifugar el tubo a 130 g durante 5 minutos. </li><li> Utilizando una micropipeta, retirar con cuidado los medios de comunicación sin perturbar el sedimento celular en la punta del tubo. A medida que la cabeza se aproxima a la media pastilla, puede ser útil usar una más fina, punta de la pipeta 10 mL. </li><li> Suavemente resuspender el botón celular en 20 l de solución de fotoactivos PEGDM (use una punta de pipeta de boca ancha, si se trabaja con agregados) y añadir la suspensión celular a la jeringa en la parte superior de the previamente reticulado parte de hidrogel. </li><li> Exponer la jeringa a un adicional de 8 minutos de luz UV para completar la construcción del hidrogel. Cuando haya terminado, las células deben estar completamente encapsulado dentro del hidrogel cilíndrico (Figura 3). </li><li> Expulsar el hidrogel de la jeringa y en 1 ml de HBSS en el pozo de una placa de 24 pozos para lavar brevemente el gel. Transferir el gel a 1 ml de los medios de comunicación en el pozo de una placa de 24 pozos y la cultura en las condiciones deseadas. </li></ol> 4. El seguimiento de la hipoxia <ol><li> En cualquier momento de la cultura, la imagen de las células mediante microscopía de fluorescencia para rastrear el inicio de la señalización de la hipoxia. Dependiendo de lo que se va a examinar se puede la imagen de la placa multi-bien o transferir el gel a un portaobjetos de microscopio antes de colocarlo en la platina del microscopio. Pico de excitación Ds Red se logra a 556nm y emisión máxima se produce a 586nm. Emisión es bastante intenso y por lo tanto no ha sido photobleaching observadoresed a ser problemático. Tiempo de retraso entre el inicio de la producción de proteínas y la detección de la primera señal es de aproximadamente 12 horas. La señal es lo suficientemente específico para identificar las células individuales de la señalización, pero la resolución puede ser insuficiente para determinar la localización intracelular de la señal. En las células de señalización, señales que normalmente se observa de manera uniforme por todo el citoplasma. Intensidad de la señal también puede aumentar con la exposición prolongada a la hipoxia, a pesar de que aún no han determinado si esto se debe al aumento de expresión de la proteína, la acumulación de proteínas en general, o proteínas de maduración retardada. </li></ol> 5. Los resultados representativos Un ejemplo representativo de MIN6 agregados encapsulados en un hidrogel de PEG se muestra en la Figura 4. El gel reticulado será sólida en todo, tomando la forma del recipiente en el que se llevó a cabo la reacción. Un gel con suaves superficies exteriores es preferible para la implantación de la ayuda en la prevención de un nuevo cuerpo extrañorespuesta. En el gel, los agregados de células deberían estar totalmente cerradas en la matriz y homogéneamente distribuidos para permitir un mejor transporte de nutrientes. Imágenes representativas de la señalización de hipoxia en MIN6 agregados se muestran en la Figura 5. Idéntica MIN6 agregados infectados con el virus marcador fueron cultivadas en un 20% de O 2 (5 bis) o el 1% de O 2 (5b) durante 44 horas antes de la captura de imágenes. <img alt="Figura 1" src="/files/ftp_upload/3521/3521fig1.jpg" /> Figura 1. Esquema de la actividad de nuestro sistema de marcador de hipoxia. Inserción adenoviral de la Ds Red DR gen promotor aguas arriba y educación en derechos humanos permite la hipoxia inducida por la producción de la proteína fluorescente bajo el control de HIF-1. <img alt="Figura 2" src="/files/ftp_upload/3521/3521fig2.jpg" /> Figura 2. Diagrama de flujo del procedimiento para la encapsulación de un solo paso de la dispersión de las células en un MIN6 crosslinked hidrogel PEGDM. Para cada gel, las células dispersas se suspenden en 40μL de la solución de macrómero fotoactivos PEGDM. Este se coloca en una jeringa de 1 ml y decapitado el hidrogel se forma bajo la luz UV 365 nm después de 10-12 minutos. Una vez terminado, el disco de hidrogel se retira la jeringa, se lavan y se colocan en un plato y medio llena de incubación. <img alt="Figura 3" src="/files/ftp_upload/3521/3521fig3.jpg" /> Figura 3. Diagrama de flujo del procedimiento para la encapsulación de doble paso de agregados de células MIN6 en un hidrogel reticulado PEGDM. Para cada gel, medio-gel se formó por primera vez por reticulación UV de 20μL de la solución de macrómero fotoactivos PEGDM durante 8 minutos. MIN6 agregados son cuidadosamente suspendidos en una solución adicional de 20μL fotoactivos macrómero que se añade en la parte superior de la pre-formados semi-gel. El gel completo está formado por un adicional de 8 minutos de exposición UV con agregados encapsulados en el medioplano de la gel. <img alt="Figura 4" src="/files/ftp_upload/3521/3521fig4.jpg" /> Figura 4. Imagen de un hidrogel de 40μL en 20X. MIN6 agregados (~ 400.000 células totales) se ven claramente en el gel. Diámetro es de aproximadamente 6 mm de hidrogel (barra = 1 mm). <img alt="Figura 5" src="/files/ftp_upload/3521/3521fig5.jpg" /> Hipoxia Figura 5. Fluorescentes de señalización en las células MIN6 agregados en un hidrogel PEGDM. Las células que se concentraron y se coloca en la incubación a 20% de O 2 durante 44 horas no muestran señales de hipoxia (a) mientras que las células que se concentraron luego incubadas en el 2% de O 2 por 44 horas de visualización de la señal clara, en todas partes. (Barra = 100μm) (b).

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No hay conflictos de interés declarado.

El método que aquí se presenta ofrece una técnica rápida y sencilla para la encapsulación de células en un hidrogel de PEG con un uso mínimo de condiciones no fisiológicas. PEG representa un material de encapsulación de gran utilidad para su biocompatibilidad y facilidad de modificación. Simple variación del porcentaje de PEG en la solución de fotoactivos, por ejemplo, se puede utilizar para ajustar las propiedades mecánicas, como el módulo de compresión, y las propiedades de transporte a través del tamaño de los poros. Además, el PEG es fácil de modificar por la adición de cadenas laterales. Hidrogeles de PEG, por lo tanto, representan tanto un dispositivo prometedor clínica y una plataforma flexible para la investigación in vitro Un método para el seguimiento de la hipoxia en las células encapsuladas PEG-También se ha presentado. Este método es útil para la simplicidad de la detección de la hipoxia y para evitar la necesidad de sacrificar las células de interés. La técnica se puede aplicar a una variedad de tipos de células en una variedad de condiciones lo que nos de suefulness amplio. Por ejemplo, la hipoxia como señal para la diferenciación de células madre puede ser rastreado en las culturas madre micromasa celular. Sin embargo, este método sólo puede aplicarse a sistemas dispersos de células o sistema en el que dispersa las células son luego agregados. Además, la detección de la señal fluorescente puede ser difícil en los tejidos más grandes o más densas.

<table border="1"><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional </td></tr><tr><td> PEG </td><td> Sigma-Aldrich </td><td> 309028-500G </td><td></td></tr><tr><td> El anhídrido metacrílico </td><td> Sigma-Aldrich </td><td> 276685-100ML </td><td></td></tr><tr><td> Microonda </td><td> Emerson </td><td> MW8784SB </td><td></td></tr><tr><td> Vórtice </td><td> Scientific Industries </td><td> SI-A236 </td><td></td></tr><tr><td> Cloruro de metileno </td><td> Sigma-Aldrich </td><td> D65100-1L </td><td></td></tr><tr><td> Éter dietílico </td><td> Sigma-Aldrich </td><td> 346136-1L </td><td></td></tr><tr><td> Tubo de diálisis </td><td> Espectro </td><td> 132640 </td><td></td></tr><tr><td></td><td> Laboratorios </td><td></td><td></td></tr><tr><td> Congelador </td><td></td><td></td><td></td></tr><tr><td&gt; Liofilizador </td><td> Labconco </td><td> 7670521 </td><td></td></tr><tr><td> Bomba de vacío </td><td> Welch </td><td> 8917Z-01 </td><td></td></tr><tr><td> Irgacure 2959 </td><td> Ciba-Geigy </td><td> 029891301PS04 </td><td></td></tr><tr><td> HBSS </td><td> Mediatech </td><td> 21 a 022 CV </td><td></td></tr><tr><td> Filtro de jeringa </td><td> VWR </td><td> 28145-477 </td><td></td></tr><tr><td> RPMI 1640 </td><td> Mediatech </td><td> * </td><td> * Formulación personalizada </td></tr><tr><td> FBS </td><td> PAA Laboratories </td><td> A15-351 </td><td></td></tr><tr><td> Penicilina-estreptomicina </td><td> Mediatech </td><td> 30 a 002-CI </td><td></td></tr><tr><td> Anfotericina B </td><td> Mediatech </td><td> 30 a 003-CF </td><td></td></tr><tr><td> Incubadora </td><td> Thermo Scientific </td><td> 3597 </td><td> Napco Series 8000 WJ w / O2 supresión </td></tr><tr><td> Tripsina EDTA </td><td> Mediatech </td><td> 25 a 052-CI </td><td></td></tr><tr><td> Agitador orbital</td><td> VWR </td><td> 12620-926 </td><td></td></tr><tr><td> Lámpara UV </td><td> Sanyo Denri </td><td> FLR40SBLB / M </td><td> Tiene dos 40W, 365nm bombillas de luz negra azul UV </td></tr><tr><td> Centrífugo </td><td> Eppendorf </td><td> 5811 000.010 * </td><td> * El número de orden. Modelo 5810 R </td></tr><tr><td> Microscopio </td><td> Nikon </td><td> TI-ND6-PFS </td><td> Con filterset para la excitación de 556nm / 586nm de emisión </td></tr></table>

tracking hypoxic signaling within encapsulated cell aggregates

In Diabetes mellitus type 1, autoimmune destruction of the pancreatic &beta;-cells results in loss of insulin production and potentially lethal hyperglycemia. As an alternative treatment option to exogenous insulin injection, transplantation of functional pancreatic tissue has been explored1,2. This approach offers the promise of a more natural, long-term restoration of normoglycemia. Protection of the donor tissue from the host's immune system is required to prevent rejection and encapsulation is a method used to help achieve this aim.
Biologically-derived materials, such as alginate3 and agarose4, have been the traditional choice for capsule construction but may induce inflammation or fibrotic overgrowth5 which can impede nutrient and oxygen transport. Alternatively, synthetic poly(ethylene glycol) (PEG)-based hydrogels are non-degrading, easily functionalized, available at high purity, have controllable pore size, and are extremely biocompatible,6,7,8. As an additional benefit, PEG hydrogels may be formed rapidly in a simple photo-crosslinking reaction that does not require application of non-physiological temperatures6,7. Such a procedure is described here. In the crosslinking reaction, UV degradation of the photoinitiator, 1-[4-(2-Hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propane-1-one (Irgacure 2959), produces free radicals which attack the vinyl carbon-carbon double bonds of dimethacrylated PEG (PEGDM) inducing crosslinking at the chain ends. Crosslinking can be achieved within 10 minutes. PEG hydrogels constructed in such a manner have been shown to favorably support cells7,9, and the low photoinitiator concentration and brief exposure to UV irradiation is not detrimental to viability and function of the encapsulated tissue10. While we methacrylate our PEG with the method described below, PEGDM can also be directly purchased from vendors such as Sigma.
An inherent consequence of encapsulation is isolation of the cells from a vascular network. Supply of nutrients, notably oxygen, is therefore reduced and limited by diffusion. This reduced oxygen availability may especially impact &beta;-cells whose insulin secretory function is highly dependent on oxygen11-13. Capsule composition and geometry will also impact diffusion rates and lengths for oxygen. Therefore, we also describe a technique for identifying hypoxic cells within our PEG capsules. Infection of the cells with a recombinant adenovirus allows for a fluorescent signal to be produced when intracellular hypoxia-inducible factor (HIF) pathways are activated14. As HIFs are the primary regulators of the transcriptional response to hypoxia, they represent an ideal target marker for detection of hypoxic signaling15. This approach allows for easy and rapid detection of hypoxic cells. Briefly, the adenovirus has the sequence for a red fluorescent protein (Ds Red DR from Clontech) under the control of a hypoxia-responsive element (HRE) trimer. Stabilization of HIF-1 by low oxygen conditions will drive transcription of the fluorescent protein (Figure 1). Additional details on the construction of this virus have been published previously15. The virus is stored in 10% glycerol at -80&deg; C as many 150 &mu;L aliquots in 1.5 mL centrifuge tubes at a concentration of 3.4 x 1010 pfu/mL. 
Previous studies in our lab have shown that MIN6 cells encapsulated as aggregates maintain their viability throughout 4 weeks of culture in 20% oxygen. MIN6 aggregates cultured at 2 or 1% oxygen showed both signs of necrotic cells (still about 85-90% viable) by staining with ethidium bromide as well as morphological changes relative to cells in 20% oxygen. The smooth spherical shape of the aggregates displayed at 20% was lost and aggregates appeared more like disorganized groups of cells. While the low oxygen stress does not cause a pronounced drop in viability, it is clearly impacting MIN6 aggregation and function as measured by glucose-stimulated insulin secretion15. Western blot analysis of encapsulated cells in 20% and 1% oxygen also showed a significant increase in HIF-1&alpha; for cells cultured in the low oxygen conditions which correlates with the expression of the DsRed DR protein.

Watch this Scientific Journal Video about Tracking Hypoxic Signaling within Encapsulated Cell Aggregates at JoVE.com

Tracking Hypoxic Signaling within Encapsulated Cell Aggregates

Un método para la foto-encapsulación de células en un hidrogel de PEG reticulado se describe. Señalización de hipoxia en insulinoma murino encapsulado (MIN6) agregados se realiza un seguimiento mediante un sistema de marcadores fluorescentes. Este sistema permite el examen de serie de células dentro de un hidrogel de andamios y la correlación de las señales de hipoxia con los cambios en el fenotipo celular.

Seguimiento de señalización dentro de los agregados de células hipóxicas encapsulado

In Diabetes mellitus type 1, autoimmune destruction of the pancreatic &beta;-cells results in loss of insulin production and potentially lethal ...

tracking-hypoxic-signaling-within-encapsulated-cell-aggregates

Research

JoVE Journal

Bioengineering

10.5K vistas.  University of South Carolina. Un método para la foto-encapsulación de células en un hidrogel de PEG reticulado se describe. Señalización de hipoxia en insulinoma murino encapsulado (MIN6) agregados se realiza un seguimiento mediante un sistema de marcadores fluorescentes. Este sistema permite el examen de serie de células dentro de un hidrogel de andamios y la correlación de las señales de hipoxia con los cambios en el fenotipo celular.

Video: Tracking Hypoxic Signaling within Encapsulated Cell Aggregates

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

Rastreo Orbital 3D en un microscopio de dos fotones Modificado: una aplicación para el Seguimiento de vesículas intracelulares

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

Investigación

Bioingeniería

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 ...

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions are likely affected by the mechanical environment in which binding and signaling take place. A recent study demonstrated that mechanical force can regulate antigen recognition by and triggering of the T-cell receptor (TCR). This was made possible by a new technology we developed and termed fluorescence biomembrane force probe (fBFP), which combines single-molecule force spectroscopy with fluorescence microscopy. Using an ultra-soft human red blood cell as the sensitive force sensor, a high-speed camera and real-time imaging tracking techniques, the fBFP is of ~1 pN (10-12 N), ~3 nm and ~0.5 msec in force, spatial and temporal resolution. With the fBFP, one can precisely measure single receptor-ligand binding kinetics under force regulation and simultaneously image binding-triggered intracellular calcium signaling on a single live cell. This new technology can be used to study other membrane receptor-ligand interaction and signaling in other cells under mechanical regulation.

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

Fluorescencia Biomembranas Fuerza Sonda: La cuantificación simultánea de Cinética-Receptor ligando y encuadernación inducida Señalización Intracelular en una sola célula

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions ...

Transport Properties of Ibuprofen Encapsulated in Cyclodextrin Nanosponge Hydrogels: A Proton HR-MAS NMR Spectroscopy Study

The chemical cross-linking of &#946;-cyclodextrin (&#946;-CD) with ethylenediaminetetraacetic dianhydride (EDTA) led to branched polymers referred to as cyclodextrin nanosponges (CDNSEDTA). Two different preparations are described with 1:4 and 1:8 CD-EDTA molar ratios. The corresponding cross-linked polymers were contacted with 0.27 M aqueous solution of ibuprofen sodium salt (IP) leading to homogeneous, colorless, drug loaded hydrogels.
The systems were characterized by high resolution magic angle spinning (HR-MAS) NMR spectroscopy. Pulsed field gradient spin echo (PGSE) NMR spectroscopy was used to determine the mean square displacement (MSD) of IP inside the polymeric gel at different observation times td. The data were further processed in order to study the time dependence of MSD: MSD = f(td). The proposed methodology is useful to characterize the different diffusion regimes that, in principle, the solute may experience inside the hydrogel, namely normal or anomalous diffusion. The full protocols including the polymer preparation and purification, the obtainment of drug-loaded hydrogels, the NMR sample preparation, the measurement of MSD by HR-MAS NMR spectroscopy and the final data processing to achieve the time dependence of MSD are here reported and discussed. The presented experiments represent a paradigmatic case and the data are discussed in terms of innovative approach to the characterization of the transport properties of an encapsulated guest within a polymeric host of potential application for drug delivery.

The motion regimes of ibuprofen encapsulated in &#946;-cyclodextrin nanosponges polymer network are investigated using pulsed-field-gradient spin-echo (PGSE) NMR technique. Synthesis, purification, drug loading, implementation of the NMR pulse sequence and data analysis to work out the mean square displacement of the drug at several observation times are described in detail.

Propiedades de transporte de ibuprofeno encapsulado en ciclodextrina nanoesponja hidrogeles: un protón HR-MAS RMN Espectroscopia de Estudio

The chemical cross-linking of &#946;-cyclodextrin (&#946;-CD) with ethylenediaminetetraacetic dianhydride (EDTA) led to branched polymers referred to as ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

Ingeniería tridimensionales epiteliales tejidos embebidos dentro de la matriz extracelular

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

Targeted membrana plasmática Entrega de cargo encapsulado hidrofóbico en un cristal líquido portador de nanopartículas

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

Montaje y Seguimiento de Desarrollo Comunitario Microbiano dentro de una Plataforma de Matriz de Micropedulos

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

Caracterización de la migración de la célula en tres dimensiones In Vitro herida ambientes

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

A Protocol for Real-time 3D Single Particle Tracking

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although various RT-3D-SPT methods have been put forward in recent years, tracking high speed 3D diffusing particles at low photon count rates remains a challenge. Moreover, RT-3D-SPT setups are generally complex and difficult to implement, limiting their widespread application to biological problems. This protocol presents a RT-3D-SPT system named 3D Dynamic Photon Localization Tracking (3D-DyPLoT), which can track particles with high diffusive speed (up to 20 &#181;m2/s) at low photon count rates (down to 10 kHz). 3D-DyPLoT employs a 2D electro-optic deflector (2D-EOD) and a tunable acoustic gradient (TAG) lens to drive a single focused laser spot dynamically in 3D. Combined with an optimized position estimation algorithm, 3D-DyPLoT can lock onto single particles with high tracking speed and high localization precision. Owing to the single excitation and single detection path layout, 3D-DyPLoT is robust and easy to set up. This protocol discusses how to build 3D-DyPLoT step by step. First, the optical layout is described. Next, the system is calibrated and optimized by raster scanning a 190 nm fluorescent bead with the piezoelectric nanopositioner. Finally, to demonstrate real-time 3D tracking ability, 110 nm fluorescent beads are tracked in water.

This protocol details the construction and operation of a real-time 3D single particle tracking microscope capable of tracking nanoscale fluorescent probes at high diffusive speeds and low photon count rates.

Un protocolo para el seguimiento en tiempo real 3D sola partícula

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based method is one of the simplest techniques for the mass production of cellular aggregates, but it does not protect the cultures against over-aggregation, which occurs when they gather at the bottom center of the culture vessel. To solve this problem, we developed an O-shaped dish and an O-shaped bag, neither of which contains a central region. Aggregates grown in either O-shaped culture vessel were noticeably more uniform in size than aggregates grown in conventional vessels. Histological analyses showed that aggregates in conventional culture dishes contained necrotic cores most likely caused by a poor oxygen supply. In contrast, aggregates that were grown in the O-shaped bag, even those with similar diameters to aggregates in conventional culture dishes, did not show necrotic cores. These results suggest that the O-shaped bag provides sufficient oxygen to the aggregates due to the oxygen permeability of the bag material. We, therefore, propose that this novel gas-permeable O-shaped culture bag is suitable for the mass production of uniform aggregates that are necessary in various biotechnological fields.

Here we present a protocol for using O-shaped vessels, specialized for suspension cultures of cellular aggregates, with orbital shaking. The HEK293 cells grown in this bag form more homogeneous aggregates than those grown in conventional culture vessels.

Un Orbital agitación cultivo de células mamíferas en vasos en forma de O para producir agregados uniforme

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based ...