0 día - La esterilización antes del día de experimento <ol><li> Ponga los materiales en la tina de plástico y esterilizar con alcohol al 70% durante 2 horas: <ul><li> Cámaras </li><li> Tapas </li><li> Marcos (las 3 piezas) </li><li> Tornillos </li><li> Las juntas de goma </li><li> Fórceps </li><li> Tijeras </li><li> Llaves hexagonales </li></ul></li><li> Membranas limpia con agua y jabón Aquet destilada. </li><li> Sonicar membranas en alcohol al 70% durante 10 minutos. </li><li> Coloque las membranas de plástico limpia en platos cuadrados. Si se utiliza membranas de dibujos, asegúrese de que el lado estampado quede hacia arriba (alcohol spray en un lado de la membrana y velar por el líquido a correr por los surcos). </li><li> Deja membranas cubiertos en el 70% de alcohol durante 2 horas. </li><li> Guantes de paquete en papel de aluminio para autoclave mañana por la mañana. </li><li> Haga 2% (peso / volumen) solución de gelatina (2g/100mL) en agua destilada para el autoclave mañana. </li><li> Después de la esterilización de dos horas en el 70% de alcohol, coloque todos los materiales polímeros y las membranas (no autoclavable materiales) en la capilla de UV durante la noche: <ul><li> Cámaras </li><li> Tapas </li><li> Marcos (las 3 piezas) </li><li> Membranas </li></ul></li><li> Paquete de materiales sobrantes en una bolsa de autoclave: <ul><li> Tornillos </li><li> Las juntas de goma </li><li> Fórceps </li><li> Tijeras </li><li> Llaves hexagonales </li></ul></li></ol> Día 1 - La Asamblea de las cámaras se extienden <ol><li> Autoclave pinzas, tijeras, llaves hexagonales, tornillos, juntas, guantes, y la gelatina con autoclave pequeño 240 ° F durante 20 minutos en total </li><li> Quitar las membranas de los rayos UV y tratamiento con plasma de O 2 (lado del patrón hacia arriba) de aproximadamente 1 minuto. </li><li> En la campana TC, cubrir el área de modelado de las membranas de plasma tratado con gelatina. Escudo de 30 minutos bajo los rayos UV. </li><li> Lavar cada membrana con PBS 2X. Después del lavado segunda, dejar un poco de PBS en las membranas de mantener más deslizantes, para facilitar el montaje en las cámaras. (También debe utilizar este PBS para lubricar las juntas para facilitar el montaje). </li><li> Montar las membranas en las cámaras (use guantes de autoclave durante el montaje) <ol><li> Conecte las dos piezas principales de la estructura con un solo tornillo. </li><li> Da la vuelta al cuadro una y colocar una membrana en la parte superior para que las caras laterales recubiertos de gelatina hacia el bastidor (el área de modelado debe estar en el centro). </li><li> Asegurar la membrana al marco mediante una junta en cada lado. </li><li> Presione las juntas de, con suave, aunque la presión, a fin de no rasgar la membrana. </li></ol></li><li> Adjuntar marco se reunieron para T-bar. </li><li> Voltear el marco y el lugar dentro de la cámara al revés. Parte posterior del bastidor UV y de la membrana durante 30 minutos </li><li> Voltear el marco de nuevo, y el tornillo en las cámaras (sin cambio de cámara de control, pero para la cámara de estiramiento, la necesidad de utilizar dos tornillos para sujetar la pieza final de la trama en la parte inferior de la cámara, y la necesidad de quitar el tornillo que se la celebración de las dos piezas de la estructura en conjunto). Parte frontal UV durante 30 minutos. Asegúrese de que las membranas estén completamente secas antes de proceder al siguiente paso, o de lo contrario la solución de células pueden deslizarse durante la siembra. </li><li> Las células de las semillas. Área de 1 = Área placa de 3 membranas, por lo que el uso 1 / 3 de una placa confluente por membrana. Use 1 ml de solución de células por la membrana. No más que 1,5 ml, será difícil mantener la solución en. Poner la solución única celda en el área de modelado, y el uso de la pipeta para extender la solución de todo. Cubrir las cámaras y dejar que las células conectar durante 30 minutos RT en el capó. </li><li> Mover las cámaras a la incubadora y dejar que las células de conectar por 1 hora más. Tener mucho cuidado en movimiento de las cámaras para evitar que la solución de células resbale! Las células en la cámara se arruinará si la solución se cae en este momento. </li><li> Volver a la campana de la cámara y agregar 20 ml de medio. </li><li> Cámaras en lugar de alcohol limpia tina que se cubre con papel de aluminio (también rociados con alcohol). Mover las cámaras para estirar la incubadora (10% CO 2). </li><li> Cámaras de seguro en la máquina de estiramiento. Deje que las células más adjuntar toda la noche, y empezar a estirar al día siguiente. (Nota: la hora de poner cámaras en la máquina de estirar, recuerde que debe cerrar la máquina en la posición &quot;cero&quot; antes de colocar las cámaras, y luego ajuste la banda de goma alrededor de los engranajes No te olvides de desbloquear los engranajes antes de iniciar la máquina.). </li></ol>

<ol>
	<li>Banes, A. J., Gilbert, J., Taylor, D., Monbureau, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+vacuum-operated+stress-providing+instrument+that+applies+static+or+variable+duration+cyclic+tension+or+compression+to+cells+in+vitro.">A new vacuum-operated stress-providing instrument that applies static or variable duration cyclic tension or compression to cells in vitro.</a> J Cell Sci. 75, 35-42 (1985).</li><li>Park, J. S., Chu, J. S., Cheng, C., Chen, F., Chen, D., Li, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+equiaxial+and+uniaxial+strain+on+mesenchymal+stem+cells.">Differential effects of equiaxial and uniaxial strain on mesenchymal stem cells.</a> Biotechnol. Bioeng. 88, 359-368 (2004).</li><li>Kurpinski, K., Chu, J., Hashi, C., Li, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anisotropic+mechanosensing+by+mesenchymal+stem+cells.">Anisotropic mechanosensing by mesenchymal stem cells.</a> Proc. Natl. Acad. Sci. U S A. 103, 16095-16100 (2006).</li></ol>

Banes et al. informó por primera vez el uso de un sistema de estimulación mecánica de las células in vitro mediante el uso de un sustrato de elastómero flexible para proporcionar la fuerza mecánica a las células 1. Desde entonces, muchas variaciones en este diseño han sido concebidos y utilizados. Varios sistemas de estiramiento mecánico están disponibles comercialmente bajo el nombre de &quot;Flexercell&quot; (FlexCell International Corp.), mientras que algunos laboratorios utilizan costumbre-construido dispositivos. E...

Notas sobre los medios de comunicación: Estos experimentos se extienden uniaxial son propensos a la contaminación debido a la complejidad de la configuración del biorreactor. Para combatir esto, hemos tratado de diferentes combinaciones de antibióticos y antifúngicos. El fungizone 1% suele ser necesario, junto con una combinación de antibióticos. O bien utilizar una combinación de penicilina / estreptomicina, o utilizar una combinación de kanamicina y gentamicina (pero no tratar de usar una combinación / cóctel de tres o más antibióticos juntos, ya que puede dar lugar a bacterias resistentes a los mutantes, y también puede ser perjudicial para las células) .

mechanical stimulation of stem cells using cyclic uniaxial strain

El papel de las fuerzas mecánicas en el desarrollo y mantenimiento de los tejidos biológicos está bien documentado, entre ellos varios fenómenos mecánicos regulados, como la remodelación ósea, la hipertrofia muscular, y la plasticidad de células musculares lisas. Sin embargo, las fuerzas implicadas son a menudo extremadamente complejos y difíciles de supervisar y controlar en vivo. Para investigar mejor los efectos de las fuerzas mecánicas de las células, hemos desarrollado un método in vitro para la aplicación de tensión uniaxial de tracción cíclica de las células adherentes cultivadas en membranas elásticas. Este método utiliza un biorreactor de diseño personalizado con un motor de leva-rotor del sistema para aplicar la fuerza deseada. Aquí se presenta un protocolo de vídeo paso a paso que muestra cómo ensamblar los distintos componentes de cada una &quot;cámara de estiramiento&quot;, incluyendo, en este caso, una membrana de silicona con la topografía micropatterned para orientar a las células con la dirección de la cepa. También se describen los procedimientos para la esterilización de las cámaras, la siembra de las células en la membrana, con retención de la cámara en el biorreactor, y el ajuste de los parámetros mecánicos (es decir, la magnitud y la velocidad de deformación). Los procedimientos descritos en este protocolo particular son específicos para la siembra de células madre mesenquimales humanas en las membranas de silicona con 10 canales de micras de ancho orientada paralela a la dirección de la tensión. Sin embargo, los métodos y materiales que se presentan en este sistema son lo suficientemente flexibles para dar cabida a una serie de variaciones sobre este tema: la velocidad de deformación, magnitud, duración, tipo de célula, la topografía de membrana, la membrana de recubrimiento, etc todo se puede adaptar a la aplicación deseada o resultado. Este es un método robusto para la investigación de los efectos de la deformación por tracción uniaxial aplicada a las células in vitro.

Watch this Scientific Journal Video about Mechanical Stimulation of Stem Cells Using Cyclic Uniaxial Strain at JoVE.com

Mechanical Stimulation of Stem Cells Using Cyclic Uniaxial Strain

En general, se entiende que las fuerzas mecánicas en el cuerpo puede influir en la diferenciación y proliferación celular. Aquí se presenta un protocolo de vídeo que demuestra el uso de un biorreactor a medida para la entrega de tensión uniaxial de tracción cíclica a las células madre cultivadas en substratos flexibles micropatterned.

La estimulación mecánica de células madre utilizando tensión uniaxial cíclica

The role of mechanical forces in the development and maintenance of biological tissues is well documented, including several mechanically regulated ...

mechanical-stimulation-of-stem-cells-using-cyclic-uniaxial-strain

Research

JoVE Journal

Biology

12.7K vistas.  University of California, Berkeley. En general, se entiende que las fuerzas mecánicas en el cuerpo puede influir en la diferenciación y proliferación celular. Aquí se presenta un protocolo de vídeo que demuestra el uso de un biorreactor a medida para la entrega de tensión uniaxial de tracción cíclica a las células madre cultivadas en substratos flexibles micropatterned.

Video: Mechanical Stimulation of Stem Cells Using Cyclic Uniaxial Strain

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

Derivación de células madre hematopoyéticas de células madre embrionarias murinas

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Investigación

Biología

Patterning of Embryonic Stem Cells Using the Bio Flip Chip

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such as tumor growth, stem cell differentiation, and stem cell self-renewal. A number of methods currently exist to modulate cell signaling in vitro. One method of modulating the total amount of diffusible signaling is to vary the cell seeding density during culture. Due to the random nature of cell seeding, this results in considerable variation in the actual cell-cell spacing and amount of cell-cell contact, and cannot prescribe the local environment. A more specific approach for modulating cell signaling is to use molecular inhibitors or genetic approaches to knock down specific signaling proteins, but both of these methods are best suited to manipulating small numbers of molecules. Here, we demonstrate a new approach to modulating cell-cell signaling that modulates the local environment of a cluster of cells by placing different numbers of cells at desired locations on a substrate. This method provides a complementary way to control the local diffusible and juxtacrine signaling between cells. Our method makes use of the Bio Flip Chip (BFC), a microfabricated silicone chip containing hundreds-to-thousands of microwells, each sized to hold either a single cell or small numbers of cells. We load the chip with cells simply by pipetting them onto the array of wells and washing unloaded cells off the array. The chip is then flipped onto a substrate, whereby the cells fall out of the wells and onto the substrate, maintaining their patterning. After the cells have attached, the chip can be removed (or left on). This approach to cell patterning is unique in that it: 1) doesn't alter the chemistry of the substrate, thus allowing cells to proliferate and migrate; 2) allows patterning onto any substrate, including tissue-culture polystyrene, glass, matrigel, and even feeder cell layers; and 3) is compatible with traditional microcontact printing, allowing the creation of extracellular matrix islands with cells placed inside those islands. In this video, we demonstrate the patterning of mouse embryonic stem cells onto tissue-culture polystyrene using the BFC.

We demonstrate a simple method for placing cells at desired locations on a substrate. This method patterns cells by flipping a silicone chip containing microwells filled with cells onto the substrate. This method provides a new way to modulate diffusible and juxtacrine signaling between cells.

Los patrones de las células madre embrionarias usando el Flip Chip Bio

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

Aislamiento y suero animal expansión libre de cordón umbilical humano derivado de células mesenquimales del estroma (MSC) y endoteliales formadoras de colonias Células Progenitoras (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

This paper aims to instruct the reader in the operation of an integrated atomic force-optical imaging microscope for mechanical stimulation of live cells in culture. A step-by-step protocol is presented. A representative data set that shows live cell response to mechanical stimulation is presented.

Respuesta de células vivas a la estimulación mecánica estudiados por microscopía óptica integrada la Fuerza Atómica y

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

Analysis of Pluripotent Stem Cells by using Cryosections of Embryoid Bodies

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage early mammalian embryos 1. A crucial stage in the differentiation of ES cells is the formation of embryoid bodies (EBs) aggregates 2, 3. EB formation is based on spontaneous aggregation when ES cells are cultured in non adherent plates. Three-dimensional EB recapitulates many aspects of early mammalian embryogenesis and differentiate into the three germ layers: ectoderm, mesoderm and endoderm 4.
Immunofluorescence and in situ hybridization are widely used techniques for the detection of target proteins and mRNA present in cells of a tissue section 5, 6, 7. Here we present a simple technique to generate high quality cryosections of embryoid bodies. This approach relies on the spatial orientation of EB embedding in OCT followed by the cryosection technique. The resulting sections can be subjected to a wide variety of analytical procedures in order to characterize populations of cells containing certain proteins, RNA or DNA. In this sense, the preparation of EB cryosections (10&mu;m) are essential tools for histology staining analysis (e.g. Hematoxilin and Eosin, DAPI), immunofluorescence (e.g. Oct4, nestin) or in situ hybridization. This technique can also help to understand aspects of embryogenesis with regards to the maintenance of the tri-dimensional spherical structure of EBs.

Pluripotent stem cells growing in suspension differentiate into embryoid bodies (EBs). Here we demonstrate how to obtain high quality EB cryosections useful for studying cellular and molecular aspects of embryogenesis, while preserving their organization as aggregates.

El análisis de las células madre pluripotentes utilizando criosecciones de cuerpos embrioides

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage early mammalian embryos 1. A crucial stage in the ...

Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as tumor-propagating cells, or tumor-initiating cells. It is thought that targeting these self-renewing cells for destruction will block tumor progression and stop relapse, greatly improving patient prognosis1. The most common way to determine the frequency of self-renewing cells within a tumor is a limiting dilution cell transplantation assay, in which tumor cells are transplanted into recipient animals at increasing doses; the proportion of animals that develop tumors is used the calculate the number of self-renewing cells within the original tumor sample2, 3. Ideally, a large number of animals would be used in each limiting dilution experiment to accurately determine the frequency of tumor-propagating cells. However, large scale experiments involving mice are costly, and most limiting dilution assays use only 10-15 mice per experiment. 
Zebrafish have gained prominence as a cancer model, in large part due to their ease of genetic manipulation and the economy by which large scale experiments can be performed. Additionally, the cancer types modeled in zebrafish have been found to closely mimic their counterpart human disease4. While it is possible to transplant tumor cells from one fish to another by sub-lethal irradiation of recipient animals, the regeneration of the immune system after 21 days often causes tumor regression5. The recent creation of syngeneic zebrafish has greatly facilitated tumor transplantation studies 6-8. Because these animals are genetically identical, transplanted tumor cells engraft robustly into recipient fish, and tumor growth can be monitored over long periods of time. Syngeneic zebrafish are ideal for limiting dilution transplantation assays in that tumor cells do not have to adapt to growth in a foreign microenvironment, which may underestimate self-renewing cell frequency9, 10. Additionally, one-cell transplants have been successfully completed using syngeneic zebrafish8 and several hundred animals can be easily and economically transplanted at one time, both of which serve to provide a more accurate estimate of self-renewing cell frequency.
Here, a method is presented for creating primary, fluorescently-labeled T-cell acute lymphoblastic leukemia (T-ALL) in syngeneic zebrafish, and transplanting these tumors at limiting dilution into adult fish to determine self-renewing cell frequency. While leukemia is provided as an example, this protocol is suitable to determine the frequency of tumor-propagating cells using any cancer model in the zebrafish.

Limiting dilution cell transplantation assays are used to determine the frequency of tumor-propagating cells. This protocol describes a method for generating syngeneic zebrafish that develop fluorescently-labeled leukemia and details how to isolate and transplant these leukemia cells at limiting dilution into the peritoneal cavity of adult zebrafish.

Cuantificar la frecuencia de tumores se propagan las células usando trasplante de células limitación de dilución en el pez cebra singénico

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as ...

Micro-Mechanical Characterization of Lung Tissue Using Atomic Force Microscopy

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3. On the macro scale the stiffness of tissues and organs within the human body span several orders of magnitude4. Much less is known about how stiffness varies spatially within tissues, and what the scope and spatial scale of stiffness changes are in disease processes that result in tissue remodeling. To better understand how changes in matrix stiffness contribute to cellular physiology in health and disease, measurements of tissue stiffness obtained at a spatial scale relevant to resident cells are needed. This is particularly true for the lung, a highly compliant and elastic tissue in which matrix remodeling is a prominent feature in diseases such as asthma, emphysema, hypertension and fibrosis. To characterize the local mechanical environment of lung parenchyma at a spatial scale relevant to resident cells, we have developed methods to directly measure the local elastic properties of fresh murine lung tissue using atomic force microscopy (AFM) microindentation. With appropriate choice of AFM indentor, cantilever, and indentation depth, these methods allow measurements of local tissue shear modulus in parallel with phase contrast and fluorescence imaging of the region of interest. Systematic sampling of tissue strips provides maps of tissue mechanical properties that reveal local spatial variations in shear modulus. Correlations between mechanical properties and underlying anatomical and pathological features illustrate how stiffness varies with matrix deposition in fibrosis. These methods can be extended to other soft tissues and disease processes to reveal how local tissue mechanical properties vary across space and disease progression.

The stiffness of the extracellular matrix strongly influences multiple behaviors of adherent cells. Matrix stiffness varies spatially throughout a tissue, and undergoes modification in various disease conditions. Here we develop methods to characterize spatial variations in stiffness in normal and fibrotic mouse lung tissue using atomic force microscopy microindentation.

Micro-Mecánica Caracterización del tejido pulmonar con microscopía de fuerza atómica

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3.  On the macro scale the stiffness of tissues and organs ...

Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the physical properties of the nucleus contribute significantly to the overall biomechanical behavior of cells under physiological and pathological conditions. For example, in migrating neutrophils and invading cancer cells, nuclear stiffness can pose a major obstacle during extravasation or passage through narrow spaces within tissues.1 On the other hand, the nucleus of cells in mechanically active tissue such as muscle requires sufficient structural support to withstand repetitive mechanical stress. Importantly, the nucleus is tightly integrated into the cellular architecture; it is physically connected to the surrounding cytoskeleton, which is a critical requirement for the intracellular movement and positioning of the nucleus, for example, in polarized cells, synaptic nuclei at neuromuscular junctions, or in migrating cells.2 Not surprisingly, mutations in nuclear envelope proteins such as lamins and nesprins, which play a critical role in determining nuclear stiffness and nucleo-cytoskeletal coupling, have been shown recently to result in a number of human diseases, including Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, and dilated cardiomyopathy.3 To investigate the biophysical function of diverse nuclear envelope proteins and the effect of specific mutations, we have developed experimental methods to study the physical properties of the nucleus in single, living cells subjected to global or localized mechanical perturbation. Measuring induced nuclear deformations in response to precisely applied substrate strain application yields important information on the deformability of the nucleus and allows quantitative comparison between different mutations or cell lines deficient for specific nuclear envelope proteins. Localized cytoskeletal strain application with a microneedle is used to complement this assay and can yield additional information on intracellular force transmission between the nucleus and the cytoskeleton. Studying nuclear mechanics in intact living cells preserves the normal intracellular architecture and avoids potential artifacts that can arise when working with isolated nuclei. Furthermore, substrate strain application presents a good model for the physiological stress experienced by cells in muscle or other tissues (e.g., vascular smooth muscle cells exposed to vessel strain). Lastly, while these tools have been developed primarily to study nuclear mechanics, they can also be applied to investigate the function of cytoskeletal proteins and mechanotransduction signaling.

We present two independent, microscope-based tools to measure the induced nuclear and cytoskeletal deformations in single, living adherent cells in response to global or localized strain application. These techniques are used to determine nuclear stiffness (i.e., deformability) and to probe intracellular force transmission between the nucleus and the cytoskeleton.

Los ensayos biofísicos investigará las propiedades mecánicas del núcleo celular Interfase: Aplicación del sustrato deformación y manipulación de Microneedle

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

La reprogramación de células somáticas humanas en células madre pluripotentes inducidas (iPSCs) utilizando el vector retroviral con las buenas prácticas agrarias

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy this deficiency, several medical interventions have been developed. One such method is to resurface the damaged area with tissue-engineered cartilage; however, the engineered tissue typically lacks the biochemical properties and durability of native cartilage, questioning its long-term survivability. This limits the application of cartilage tissue engineering to the repair of small focal defects, relying on the surrounding tissue to protect the implanted material. To improve the properties of the developed tissue, mechanical stimulation is a popular method utilized to enhance the synthesis of cartilaginous extracellular matrix as well as the resultant mechanical properties of the engineered tissue. Mechanical stimulation applies forces to the tissue constructs analogous to those experienced in vivo. This is based on the premise that the mechanical environment, in part, regulates the development and maintenance of native tissue1,2. The most commonly applied form of mechanical stimulation in cartilage tissue engineering is dynamic compression at physiologic strains of approximately 5-20% at a frequency of 1 Hz1,3. Several studies have investigated the effects of dynamic compression and have shown it to have a positive effect on chondrocyte metabolism and biosynthesis, ultimately affecting the functional properties of the developed tissue4-8. In this paper, we illustrate the method to mechanically stimulate chondrocyte-agarose hydrogel constructs under dynamic compression and analyze changes in biosynthesis through biochemical and radioisotope assays. This method can also be readily modified to assess any potentially induced changes in cellular response as a result of mechanical stimuli.

The biosynthesis of cartilaginous extracellular matrix by chondrocytes can be affected by application of mechanical stimuli. This method describes the technique of applying dynamic compressive strains to chondrocytes encapsulated in 3D constructs and the evaluation of induced changes in chondrocyte metabolism.

La estimulación mecánica de condrocitos-agarosa hidrogeles

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy ...