Dia 0 - Esterilização antes do dia de experimento <ol><li> Colocar materiais em tubo de plástico e esterilizar com álcool 70% durante 2 horas: <ul><li> Chambers </li><li> Tampas </li><li> Quadros (todos os 3 peças) </li><li> Parafusos </li><li> Juntas de borracha </li><li> Fórceps </li><li> Tesoura </li><li> Chaves Hex </li></ul></li><li> Membranas limpo com Aquet sabão e água destilada. </li><li> Sonicate membranas em álcool 70% por 10 minutos. </li><li> Coloque as membranas limpa em plástico pratos quadrados. Se utilizando membranas padronizada, garantir que o lado padronizada é a face para cima (álcool de spray em um lado da membrana e prestar atenção para o líquido para executar as ranhuras). </li><li> Deixe membranas cobertas em álcool 70% por 2 horas. </li><li> Luvas de pacote em folha de alumínio para autoclave amanhã de manhã. </li><li> Faça 2% (peso / volume) de solução de gelatina (2g/100mL) em água destilada para autoclave amanhã. </li><li> Após a esterilização duas horas em álcool 70%, coloque todos os materiais de polímeros e membranas (não autoclavável materiais) na capa de UV durante a noite: <ul><li> Chambers </li><li> Tampas </li><li> Quadros (todos os 3 peças) </li><li> Membranas </li></ul></li><li> Pacote de materiais remanescentes na bolsa autoclavável: <ul><li> Parafusos </li><li> Juntas de borracha </li><li> Fórceps </li><li> Tesoura </li><li> Chaves Hex </li></ul></li></ol> Dia 1 - Montagem das câmaras de estiramento <ol><li> Forceps autoclave, tesouras, chaves hex, parafusos, juntas, luvas, e gelatina utilizando autoclave pequena a 240 ° F durante 20 minutos no total </li><li> Remova as membranas de UV e tratar com O 2 plasma (lado modelado para cima) para ~ 1 minuto. </li><li> TC na capa, capa área padronizada de membranas de plasma tratado com gelatina. Casaco de 30 para minutos sob UV. </li><li> Lavar cada membrana com PBS 2X. Após a lavagem 2, deixar alguns PBS nas membranas para mantê-los escorregadios, para facilitar a montagem para as câmaras. (Deve também usar este PBS para lubrificar as juntas para facilitar a montagem). </li><li> Montar membranas em câmaras (usar luvas autoclavado durante a montagem) <ol><li> Ligar as duas principais peças do quadro usando um único parafuso. </li><li> Virar o quadro e coloque uma membrana na parte superior de modo que as faces de gelatina revestido lateral em direção à estrutura (área padrão deve estar no centro). </li><li> Assegurar a membrana para o quadro usando uma junta em cada lado. </li><li> Pressione a juntas de, usando gentil, mesmo pressão, para não rasgar a membrana. </li></ol></li><li> Anexar quadro montado para T-bar. </li><li> Virar o quadro e colocar em câmara de cabeça para baixo. Verso da moldura UV e membrana por 30 minutos </li><li> Vire a moldura novamente, e parafuso em câmaras (sem mudança para câmara de controle, mas para câmara de estiramento, precisa usar dois parafusos para fixar a peça final do quadro para o fundo da câmara, e precisa remover o único parafuso que é segurando as duas peças do quadro juntos). Frente UV por 30 min. Certifique-se que as membranas estejam completamente secas antes de prosseguir para a próxima etapa, ou então a solução da célula pode escapar durante a semeadura. </li><li> Células da semente. Área de 1 = Área da placa de 3 membranas, portanto, use 1 / 3 de uma placa confluentes por membrana. Use 1 mL de solução por célula de membrana. Mais do que 1,5 ml será difícil manter em solução. Coloque solução única célula na área de estampados, e usar a ponta da pipeta para espalhar a solução ao redor. Cubra e deixe câmaras anexar células por 30 minutos RT no capô. </li><li> Mover câmaras para incubadora e vamos anexar células por mais 1 hora. Ter muito cuidado movendo as câmaras de evitar que solução da célula escorregar! As células em que câmara será arruinado se a solução cai neste momento. </li><li> Retorno câmara para capô e adicionar 20 mL de mídia. </li><li> Câmaras lugar em álcool limpo banheira que é coberto com folha de alumínio (também pulverizadas com álcool). Mover câmaras para esticar incubadora (10% CO 2). </li><li> Câmaras de seguro para uma máquina de estiramento. Vamos anexar células mais durante a noite, e começar a esticar no dia seguinte. (Nota: quando colocar câmaras em máquina de esticar, lembre-se de bloquear a máquina na posição &quot;zero&quot; antes de colocar câmaras, e então aperte o elástico em volta das engrenagens Não se esqueça de desbloquear as engrenagens antes de iniciar a 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. relatada pela primeira vez o uso de um sistema de estimulação mecânica das células in vitro, utilizando um substrato flexível elastomérica para entregar a força mecânica para as células 1. Desde então, muitas variações sobre este projeto ter sido concebido e utilizado. Vários sistemas de estiramento mecânico estão disponíveis comercialmente sob o nome &quot;Flexercell&quot; (FlexCell International Corp), enquanto que alguns laboratórios usam custom-built dispositivos. Neste protocolo de vídeo que de...

Notas sobre a mídia: Estes experimentos estiramento uniaxial são propensos à contaminação devido à complexidade de configuração de biorreatores. Para combater isso, tentamos várias combinações de antibióticos e antifúngicos. O Fungizone 1% normalmente necessário, juntamente com uma combinação de antibióticos. Ou use uma combinação Pen / Strep, ou usar uma combinação de canamicina e gentamicina (mas não tente usar uma combinação / cocktail de três ou mais antibióticos em conjunto, como pode levar a bactérias resistentes a mutante, e também pode ser prejudicial para as células) .

mechanical stimulation of stem cells using cyclic uniaxial strain

O papel das forças mecânicas no desenvolvimento e manutenção dos tecidos biológicos é bem documentado, incluindo vários fenômenos mecanicamente regulamentadas, tais como remodelação óssea, hipertrofia muscular, e da plasticidade das células musculares lisas. No entanto, as forças envolvidas são extremamente complexas e difíceis de monitorar e controlar in vivo. Para melhor investigar os efeitos de forças mecânicas sobre as células, nós desenvolvemos um método in vitro para a aplicação de deformação de tração uniaxial cíclicos de células aderentes cultivadas em membranas elásticas. Este método utiliza um biorreator personalizados com um sistema motorizado cam-rotor para aplicar a força desejada. Aqui apresentamos um protocolo de vídeo passo-a-passo demonstrando como montar os vários componentes de cada &quot;câmara de estiramento&quot;, incluindo, neste caso, uma membrana de silicone com micropatterned topografia para orientar as células com a direção da tensão. Descrevemos também procedimentos para a esterilização das câmaras, a semeadura em células da membrana, trancando a câmara para o biorreator, e ajustando os parâmetros mecânicos (ou seja, magnitude e taxa de deformação). Os procedimentos descritos neste protocolo particular, são específicos para a semeadura de células-tronco mesenquimais em membrana de silicone com 10 mM canais amplo orientado paralelamente à direção de tensão. No entanto, os métodos e materiais apresentados neste sistema são flexíveis o suficiente para acomodar uma série de variações sobre este tema: taxa de deformação, magnitude, duração, tipo de célula, topografia de membrana, revestimento de membrana, etc podem ser adaptados para a aplicação desejada ou resultado. Este é um método robusto para investigar os efeitos da deformação de tração uniaxial aplicada às 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

É amplamente entendido que forças mecânicas no corpo pode influenciar a diferenciação e proliferação celular. Aqui apresentamos um protocolo de vídeo demonstrando o uso de um biorreator custom-built para a entrega de deformação de tração uniaxial cíclicos às células-tronco cultivadas em substratos flexíveis micropatterned.

A estimulação mecânica de Células-Tronco Usando Strain Uniaxial Cíclico

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 visualizações.  University of California, Berkeley. É amplamente entendido que forças mecânicas no corpo pode influenciar a diferenciação e proliferação celular. Aqui apresentamos um protocolo de vídeo demonstrando o uso de um biorreator custom-built para a entrega de deformação de tração uniaxial cíclicos às células-tronco cultivadas em substratos flexíveis micropatterned.

Vídeo: 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.

Derivação de Células-Tronco Hematopoéticas murino de Células-Tronco Embrionárias

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 ...

Biologia

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.

Padronização de Células-Tronco Embrionárias Usando o Chip Bio Virar

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

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.

Resposta celular ao vivo para Estimulação mecânicas estudadas por Microscopia de Força Atômica e Óptica Integrada

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.

Análise de células-tronco pluripotentes usando criosecções dos corpos embrióides

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.

Quantificar a freqüência de Tumor-propagação células usando Transplante de Células Limitando diluição em singênicos 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 ...

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-Mecânica Caracterização do tecido pulmonar Usando Microscopia de Força Atômica

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.

Ensaios biofísicos para investigar as propriedades mecânicas do núcleo da célula Interphase: Aplicação de substrato Strain e Manipulação de microagulhas

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.

Reprogramação de células somáticas humanas em células tronco pluripotentes induzidas (iPSCs) usando vetor retroviral com GFP

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.

A estimulação mecânica de condrócitos-agarose Hidrogéis

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

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

A estimulação mecânica induzida por cálcio Propagação de Ondas em monocamadas de células: O Exemplo de Bovinos endotélio corneano

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

Methods Article

A estimulação mecânica de Células-Tronco Usando Strain Uniaxial Cíclico