1. Isolamento de condrócitos articulares primárias Colheita 10-15 fatias de cartilagem de espessura completa das superfícies articulares das articulações de animais (por exemplo, a articulação metacarpo-falângica de vacas com esqueleto maduro obtido a partir de um matadouro local). <ol><li> Coloque fatias de cartilagem em uma placa de Petri de 100 mm e Incubar em 20 ml de 0,5% de protease em Ham F-12 (w / v) durante 2 horas a 37 ° C. Enxaguar três vezes em meio de Ham F-12 de cultura, e incuba-se com 20 ml de colagenase a 0,15% em meio A de Ham F-12 de cultura durante a noite a 37 ° C. </li><li> Filtra-se a suspensão de células através de um filtro de tela de 200 mesh para uma placa limpa. Lavar o filtro com 5 ml de Ham F-12 e adicionar ao prato. Transferir a suspensão de células para um tubo cónico de 50 ml. Lava-se a placa de Petri com 10 mL de Ham F-12 e adicionar ao tubo cónico. </li><li> Centrifugar o tubo de 600-800 rcf durante 6-8 minutos à temperatura ambiente. </li><li> Aspirar o sobrenadante, garantindo a não perturbar the sedimento de células e re-suspensão em 40 ml de meio de Ham F-12 de cultura. </li><li> Centrifugar o tubo de 600-800 rcf durante 6-8 min. </li><li> Repita os passos 1.4 e 1.5 vezes mais para um total de 3 vezes. Antes da centrifugação, pela terceira vez, ressuspender o sedimento através da agitação e da obtenção de uma alíquota de 500 ul para a contagem de células. </li><li> Contar as células viáveis ​​utilizando o método de exclusão de azul de Tripan 9 com um hemacitómetro e um microscópio óptico invertido. </li><li> Aspirar o sobrenadante e ressuspender o sedimento em um volume de F-12 de Ham meios para dobrar a densidade de sementeira desejada (por exemplo, 20 x 10 6 células / ml, para uma concentração final de 10 x 10 6 células / ml, após o encapsulamento). </li></ol> 2. Condrócitos-agarose Encapsulation Hydrogel <ol><li> Aquecer 0,8 g Tipo VII autoclavado agarose em 20 ml de solução estéril 1x PBS (pH 7,4) numa placa quente a 120 ° C até à dissolução. Uma vez dissolvido, reduzir o calor a 60 ° C como temperaturas mais elevadas will afetar a viabilidade celular. </li><li> Num tubo cónico de 50 ml, misturar cuidadosamente volumes iguais da suspensão de células com o dobro da concentração desejada de agarose (por exemplo, 4% de suspensão de agarose para um final de hidrogel de agarose 2%). Evitar a criação de bolhas grandes, pois podem afectar a viabilidade celular e construir a resistência à fadiga. </li><li> Cuidadosamente uma alíquota de 10 ml da mistura de condrócitos-agarose em um prato de 60 milímetros de diâmetro Petri, evitando a criação de bolhas grandes. </li><li> Deixar repousar durante 30 min à temperatura ambiente até que gelificou. </li><li> Obter o maior número de amostras de hidrogel de agarose-condrócito conforme necessário (tipicamente 20-30) utilizando um perfurador de biópsia 4 milímetros. </li><li> Medir e registrar as dimensões físicas (diâmetro e altura) de amostras representativas, utilizando um micrômetro. Construtos deve ser aproximadamente de 5 mm de altura, descartar quaisquer amostras obtidas a partir de uma região na qual a variação de altura da amostra foi superior a 2%. </li><li> Transferir as amostras para um prato 60 milímetros fresco Petri e suplementocom 10 ml de meio completo (por exemplo, 20% de soro fetal bovino em meio de Ham F-12 de cultura com 20 mM de HEPES, 100 mg de ácido ascórbico / ml, e antibióticos / antimicóticos 2x). </li></ol> 3. A estimulação mecânica e marcação radioactiva de condrócitos-agarose Hidrogéis <ol><li> Autoclavar os componentes metálicos do equipamento de compressão. Esterilizar os componentes de plástico com 70% de etanol (durante a noite) e luz ultravioleta (por, pelo menos, 15 min). </li><li> Para manter as construções de cair, utilizando fórceps, o local 12 anéis de plástico estéreis de retenção (com um diâmetro interior maior do que o diâmetro do construto) (n = 6, as amostras e controlos), dentro dos poços de uma placa de cultura de 24 cavidades. </li><li> Usando fórceps, transferir cuidadosamente os hidrogéis de condrócitos-agarose a partir de uma placa de Petri para a placa de 24 poços, assegurando cada uma em um anel de retenção. </li><li> Transferir 400 uL de meio completo (por exemplo, 20% de FBS em meio de Ham F-12 de cultura com 20 mM de HEPES, 100 ug / ml de ácido ascórbico, e2x antibiótico / antimicótico) a cada poço de amostra. </li><li> Monte o equipamento de compressão esterilizada (Figura 1) e fixe as placas com parafusos. Anexar o equipamento de compressão na placa de cultura de 24 cavidades. </li><li> Solte e re-assegurar os parafusos de ajuste para estabelecer cilindro hidrogel contato (zero estado de tensão). </li><li> Carregar o equipamento de compressão montados em um sistema de testes de Mach-1 micromecânico. Plataforma segura para a fase vertical via pino de localização e bloqueio no local com parafuso de fixação. Remova o gabarito de espaçamento. </li><li> Aplicar carga de compressão dinâmica da amplitude desejada (por exemplo, amplitude de deformação de 10% com base na altura original das amostras), a frequência (por exemplo, 1 Hz) e duração ou quantidade de ciclos (por exemplo, intervalos de tempo de até 60 min.) </li><li> Após a conclusão da estimulação mecânica, substituir o dispositivo de montagem de espaçamento, soltar o parafuso de fixação do pino de localização e remover o equipamento de compressão e a partir da placa de cultura de Teste do Sistema de Mach-1 micromecânico. </li&gt; <li> Radiomarcador célula-hidrogel construções, completando os meios de comunicação com 5 uCi de isótopo desejado (por exemplo, [3 H]-prolina etiqueta proteína por condrócitos específicos de síntese de colagénio e [35S]-enxofre para a síntese de proteoglicanos). </li><li> Incubar as construções marcadas radioactivamente, durante 24 horas a 37 ° C, 5% CO 2, 10. </li><li> Colheita das construções de hidrogel radiomarcados e lavar 3 vezes em 1x PBS (pH 7,4) para remover qualquer isótopo não incorporado. Digest amostras utilizando papaína (40 ug / ml em acetato de amónio 20 mM, 1 mM de ácido ethyldiaminetetraacetic e 2 mM de ditiotreitol a 65 ° C durante 72 horas). </li><li> Ensaio de digest para o conteúdo de ADN, utilizando o ensaio de PicoGreen corante 11 e incorporou actividade isótopo, normalizados para o conteúdo de ADN, por β-líquido de cintilação de contagem 12,13 digestão com papaína imediatamente a seguir. </li></ol> Nota: A compra e descarte de materiais radioativos (isótopos de resíduos e itensque possam ter estado em contacto materiais radioativos) devem seguir o institucional relevante (e / ou governamentais) políticas e procedimentos para o manuseio e eliminação de substâncias radioactivas. 4. Resultados representativos Bovina articulares construções condrócitos-agarose hidrogéis (2% de agarose com 10 x 10 6 células / ml, as células encapsuladas) foram estimulados mecanicamente a uma amplitude de 10% de deformação à compressão a uma frequência de 1 Hz por 20 a 60 minutos (ou 1200 a 3600 ciclos ) e ensaiadas para teor de ADN e síntese de matriz extracelular, por incorporação de radioisótopos. DNA e síntese de matriz extracelular foi afetado de maneira dose-dependente. O conteúdo de DNA apresentaram uma redução significativa de 35% como resultado de 20 ou 30 minutos de estimulação (p &lt;0,01), enquanto 60 minutos de estimulação não exibiu qualquer efeito (Figura 2). Cartilagem específica a síntese do colagénio e proteoglicanos (determinado por [3H]-prolina e[35S]-incorporação de enxofre, respectivamente) exibiram aumentos significativos de cerca de 60% ​​em resposta a 20 ou 30 minutos de estimulação (p &lt;0,01), com 60 minutos de estimulação que não apresente efeito observável (Figura 3). <img src="/files/ftp_upload/4229/4229fig1.jpg" alt="Figura 1" /> . Figura 1 Esquema do equipamento de compactação de custom-built dinâmica para estimular condrócitos-agarose construções esquerda:. Plataforma Montada direita:. Vista explodida mostrando componentes individuais. <img src="/files/ftp_upload/4229/4229fig2.jpg" alt="Figura 2" /> Figura 2. Alterações no conteúdo de ADN de condrócitos-agarose hidrogéis estimulada mecanicamente sob uma amplitude de deformação de compressão a 10% a 1 Hz durante 20 a 60 min (média ± SEM, n = 6/grupo). Figura 3. Alterações na síntese da matriz extracelular (colagénio e proteoglicanos) de condrócito-agarose hidrogéis mecanicamente estimulados sob amplitude de deformação à compressão a 10% a 1 Hz durante 20 a 60 min (média ± SEM, n = 6/grupo).

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<html> O método descrito para a aplicação de estímulos mecânicos controlados de hidrogéis de agarose de células-seeded permite a investigação directa sobre os efeitos de forças de compressão dinâmica sobre o metabolismo dos condrócitos. A utilização do equipamento de teste de costume em conjunto com os anéis de retenção fornecido restrição lateral para as construções, para evitar potenciais problemas de amostra de inflexão. O uso de placas mortos ponderados carregamento garantidos pelas tripulaçõ...

<table border="1"><tbody><tr><td> Nome do reagente ou do equipamento </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários (opcional) </td></tr><tr><td> Ham F-12 </td><td> Thermo Fisher Scientific </td><td> SH3001002 </td><td></td></tr><tr><td> A colagenase </td><td> Sigma Aldrich Ltd. </td><td> C0130 </td><td></td></tr><tr><td> Protease </td><td> Sigma Aldrich Ltd. </td><td> P5147 </td><td></td></tr><tr><td> Soro Fetal Bovino </td><td> Sigma Aldrich Ltd. </td><td> F1051 </td><td></td></tr><tr><td> Ascorbato </td><td> Sigma Aldrich Ltd. </td><td> A4034 </td><td></td></tr><tr><td> Antibióticos / antimicóticos </td><td> Sigma Aldrich Ltd. </td><td> A5955 </td><td></td></tr><tr><td> HEPES </td><td> Bioshop Canada Ltd. </td><td> HEP001 </td><td></td></tr><tr><td> Trypan azul </td><td> Sigma Aldrich Ltd. </td><td> 93595 </td><td></td></tr><tr><td> Reichert Brilhante-Line Hemocitômetro </td><td> Hausser Scientific </td><td> 1490 </td><td></td></tr><tr><td> Quant-iT PicoGreen </td><td> Invitrogen </td><td> P7589 </td><td></td></tr><tr><td> Papaína a partir do látex </td><td> Sigma Aldrich Ltd. </td><td> P3125 </td><td></td></tr><tr><td> Acetato de amónio </td><td> Sigma Aldrich Ltd. </td><td> A1542 </td><td></td></tr><tr><td> Ácido Ethyldiaminetetraacetic </td><td> Sigma Aldrich Ltd. </td><td> E9884 </td><td></td></tr><tr><td> DL-Ditiotreitol </td><td> Sigma Aldrich Ltd. </td><td> 43819 </td><td></td></tr><tr><td> Baixo ponto de fusão de agarose, Tipo VII </td><td> Sigma Aldrich Ltd. </td><td> A9045 </td><td></td></tr> &lt;tr&gt; <td> Tela de malha (200) Filtrar </td><td> Sigma Aldrich Ltd. </td><td> S4145 </td><td></td></tr><tr><td> Mach-1 Tester micromecânico </td><td> Biomomentum Inc. </td><td> V500cs </td><td></td></tr><tr><td> Carregando compressão Jig </td><td> Custom-built </td><td></td><td> Produto similar pode ser fornecida por Biomomentum Inc. </td></tr><tr><td> Falcon 24 Placa Cultura Bem </td><td> Thermo Fisher Scientific </td><td> B353047 </td><td></td></tr><tr><td> β-Liquid Scintillation Counter </td><td> Beckman Coulter </td><td> LS6500 </td><td></td></tr><tr><td> [3H] prolina </td><td> Perkin-Elmer </td><td> NET323005MC </td><td></td></tr><tr><td> [35S] Enxofre </td><td> Perkin-Elmer </td><td> NEX041005MC </td><td></td></tr></tbody></table>

mechanical stimulation of chondrocyte-agarose hydrogels

A cartilagem articular sofre de uma capacidade limitada de reparação quando danificadas por insulto mecânico ou degradada pela doença, tais como a osteoartrite. Para sanar esta deficiência, várias intervenções médicas têm sido desenvolvidos. Um tal método é a ressurgir a área danificada com o tecido de cartilagem artificial, no entanto, a engenharia de tecidos tipicamente não tem as propriedades bioquímicas e durabilidade da cartilagem nativa, questionar a sua sobrevivência a longo prazo. Isto limita a aplicação de engenharia de tecidos de cartilagem para a reparação de pequenos defeitos focais, baseando-se o tecido circundante para proteger o material implantado. Para melhorar as propriedades do tecido desenvolvido, estimulação mecânica é um método popular utilizado para aumentar a síntese de matriz extracelular cartilaginosa, assim como as propriedades mecânicas dos resultantes da engenharia de tecidos. Estimulação mecânica aplica forças ao tecido constrói análoga àquelas experimentadas in vivo. Estebaseia-se na premissa de que o ambiente mecânico, em parte, regula o desenvolvimento e manutenção de 1,2 tecido nativo. A forma mais vulgarmente aplicado de estimulação mecânica na engenharia de tecidos de cartilagem é de compressão dinâmica em estirpes fisiológicas de aproximadamente 5-20%, com uma frequência de 1 Hz 1,3. Vários estudos investigaram os efeitos de compressão dinâmica e têm mostrado que para ter um efeito positivo sobre o metabolismo dos condrócitos e a biossíntese, em última análise afectar as propriedades funcionais do tecido desenvolvido 4-8. Neste artigo, vamos ilustrar o método para estimular mecanicamente construções condrócitos-agarose hidrogel sob compressão dinâmica e analisar as alterações na biossíntese através de testes bioquímicos e radioisótopo. Este método também pode ser facilmente modificado para avaliar as alterações potencialmente induzidos em resposta celular, como resultado de estímulos mecânicos.

Watch this Scientific Journal Video about Mechanical Stimulation of Chondrocyte-agarose Hydrogels at JoVE.com

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

A biossíntese da matriz extracelular cartilaginosa por condrócitos podem ser afectadas pela aplicação de estímulos mecânicos. Este método descreve a técnica de aplicação de dinâmicas tensões compressivas para condrócitos encapsulados em construções em 3D e de avaliação de alterações induzidas no metabolismo dos condrócitos.

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-of-chondrocyte-agarose-hydrogels

Research

JoVE Journal

Biology

11.6K visualizações.  Queen's University. A biossíntese da matriz extracelular cartilaginosa por condrócitos podem ser afectadas pela aplicação de estímulos mecânicos. Este método descreve a técnica de aplicação de dinâmicas tensões compressivas para condrócitos encapsulados em construções em 3D e de avaliação de alterações induzidas no metabolismo dos condrócitos.

Vídeo: Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Ex vivo Mechanical Loading of Tendon

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace. Most are associated with repetitive, high force hand activities. The mechanisms of cellular and structural damage due to cyclical loading are not well known. The purpose of this video is to present a new system that can simultaneously load four tendons in tissue culture. The video describes the methods of sterile tissue harvest and how the tendons are loaded onto a clamping system that is subsequently immersed into media and maintained at 37&deg;C. One clamp is fixed while the other one is moved with a linear actuator. Tendon tensile force is monitored with a load cell in series with the mobile clamp. The actuators are controlled with a LabView program. The four tendons can be repetitively loaded with different patterns of loading, repetition rate, rate of loading, and duration. Loading can continue for a few minutes to 48 hours. At the end of loading, the tendons are removed and the mid-substance extracted for biochemical analyses. This system allows for the investigation of the effects of loading patterns on gene expression and structural changes in tendon. Ultimately, mechanisms of injury due to overuse can be studies with the findings applied to treatment and prevention.

A new in vitro system for simultaneously loading four tendons in culture is described.

Ex Carregando vivo mecânica do tendão

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace.  Most are associated with ...

Biologia

Mechanical Stimulation of Stem Cells Using Cyclic Uniaxial Strain

The role of mechanical forces in the development and maintenance of biological tissues is well documented, including several mechanically regulated phenomena such as bone remodeling, muscular hypertrophy, and smooth muscle cell plasticity. However, the forces involved are often extremely complex and difficult to monitor and control in vivo. To better investigate the effects of mechanical forces on cells, we have developed an in vitro method for applying uniaxial cyclic tensile strain to adherent cells cultured on elastic membranes. This method utilizes a custom-designed bioreactor with a motorized cam-rotor system to apply the desired force. Here we present a step-by-step video protocol demonstrating how to assemble the various components of each "stretch chamber", including, in this case, a silicone membrane with micropatterned topography to orient the cells with the direction of the strain. We also describe procedures for sterilizing the chambers, seeding cells onto the membrane, latching the chamber into the bioreactor, and adjusting the mechanical parameters (i.e. magnitude and rate of strain). The procedures outlined in this particular protocol are specific for seeding human mesenchymal stem cells onto silicone membranes with 10 &micro;m wide channels oriented parallel to the direction of strain. However, the methods and materials presented in this system are flexible enough to accommodate a number of variations on this theme: strain rate, magnitude, duration, cell type, membrane topography, membrane coating, etc. can all be tailored to the desired application or outcome. This is a robust method for investigating the effects of uniaxial tensile strain applied to cells in vitro.

It is widely understood that mechanical forces in the body can influence cell differentiation and proliferation. Here we present a video protocol demonstrating the use of a custom-built bioreactor for delivering uniaxial cyclic tensile strain to stem cells cultured on flexible micropatterned substrates.

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 ...

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

O encapsulamento celular em Hidrogéis 3D para Engenharia de Tecidos

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

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 ...

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 ...

Preparation of DNA-crosslinked Polyacrylamide Hydrogels

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, the effect of tissue elasticity on cell function is a major area of mechanobiology research because tissue stiffness modulates with disease, development, and injury. Static tissue-mimicking materials, or materials that cannot alter stiffness once cells are plated, are predominately used to investigate the effects of tissue stiffness on cell functions. While information gathered from static studies is valuable, these studies are not indicative of the dynamic nature of the cellular microenvironment in vivo. To better address the effects of dynamic stiffness on cell function, we developed a DNA-crosslinked polyacrylamide hydrogel system (DNA gels). Unlike other dynamic substrates, DNA gels have the ability to decrease or increase in stiffness after fabrication without stimuli. DNA gels consist of DNA crosslinks that are polymerized into a polyacrylamide backbone. Adding and removing crosslinks via delivery of single-stranded DNA allows temporal, spatial, and reversible control of gel elasticity. We have shown in previous reports that dynamic modulation of DNA gel elasticity influences fibroblast and neuron behavior. In this report and video, we provide a schematic that describes the DNA gel crosslinking mechanisms and step-by-step instructions on the preparation DNA gels.

Our laboratory has developed DNA-crosslinked polyacrylamide hydrogels, a dynamic hydrogel system, to better understand the effects of modulating tissue stiffness on cell function. Here, we provide schematics, descriptions, and protocols to prepare these hydrogels.

Preparação de ADN de poliacrilamida reticulada-hidrogéis

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, ...

Multifunctional, Micropipette-based Method for Incorporation And Stimulation of Bacterial Mechanosensitive Ion Channels in Droplet Interface Bilayers

MscL, a large conductance mechanosensitive channel (MSC), is a ubiquitous osmolyte release valve that helps bacteria survive abrupt hypo-osmotic shocks. It has been discovered and rigorously studied using the patch-clamp technique for almost three decades. Its basic role of translating tension applied to the cell membrane into permeability response makes it a strong candidate to function as a mechanoelectrical transducer in artificial membrane-based biomolecular devices. Serving as building blocks to such devices, droplet interface bilayers (DIBs) can be used as a new platform for the incorporation and stimulation of MscL channels. Here, we describe a micropipette-based method to form DIBs and measure the activity of the incorporated MscL channels. This method consists of lipid-encased aqueous droplets anchored to the tips of two opposing (coaxially positioned) borosilicate glass micropipettes. When droplets are brought into contact, a lipid bilayer interface is formed. This technique offers control over the chemical composition and the size of each droplet, as well as the dimensions of the bilayer interface. Having one of the micropipettes attached to a harmonic piezoelectric actuator provides the ability to deliver a desired oscillatory stimulus. Through analysis of the shapes of the droplets during deformation, the tension created at the interface can be estimated. Using this technique, the first activity of MscL channels in a DIB system is reported. Besides MS channels, activities of other types of channels can be studied using this method, proving the multi-functionality of this platform. The method presented here enables the measurement of fundamental membrane properties, provides a greater control over the formation of symmetric and asymmetric membranes, and is an alternative way to stimulate and study mechanosensitive channels.

Bacterial mechanosensitive channels can be used as mechanoelectrical transducers in biomolecular devices. Droplet interface bilayers (DIBs), cell-inspired building blocks to such devices, represent new platforms to incorporate and stimulate mechanosensitive channels. Here, we demonstrate a new micropipette-based method of forming DIBs, allowing the study of mechanosensitive channels under mechanical stimulation.

Método multifuncional, baseado em Micropipeta de Incorporação e estimulação do bacteriana Mechanosensitive canais iônicos em gotículas de interface Bicamadas

MscL, a large conductance mechanosensitive channel (MSC), is a ubiquitous osmolyte release valve that helps bacteria survive abrupt hypo-osmotic shocks. ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

FRET Imaging no hidrogéis tridimensionais

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Here we describe methods for assessing cellular response to acute mechanical stimulation. In the microscopy-based assay, we examine localization of fluorescently-labeled biosensors following brief stimulation with shear flow. We also test activation of various proteins of interest in response to acute mechanical stimulation biochemically.

Avaliação de Dictyostelium discoideum resposta à estimulação mecânica aguda

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...

Proteolytically Degraded Alginate Hydrogels and Hydrophobic Microbioreactors for Porcine Oocyte Encapsulation

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of assisted reproduction techniques such as oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and cloning of domestic animals by nuclear transfer from somatic cell. IVM is the method particularly of significance. It is the platform technology for the supply of mature, good quality oocytes for applications such as reduction of the generation interval in commercially important or endangered species, research concerning in vitro human reproduction, and production of transgenic animals for cell therapies. The term oocyte quality includes its competence to complete maturation, be fertilized, thereby resulting in healthy offspring. This means that oocytes of good quality are paramount for successful fertilization including IVF procedures. This poses many difficulties to develop a reliable culture method that would support growth not only of human oocytes but also of other large mammalian species. The first step in IVM is the in vitro culture of oocytes. This work describes two protocols for the 3D culture of porcine oocytes. In the first, 3D model cumulus-oocyte complexes (COCs) are encapsulated in a fibrin-alginate bead interpenetrating network, in which a mixture of fibrin and alginate are gelled simultaneously. In the second one, COCs are suspended in a drop of medium and encapsulated with fluorinated ethylene propylene (FEP; a copolymer of hexafluoropropylene and tetrafluoroethylene) powder particles to form microbioreactors defined as Liquid Marbles (LM). Both 3D systems maintain the gaseous in vitro culture environment. They also maintain COCs 3D organization by preventing their flattening and consequent disruption of gap junctions, thereby preserving the functional relationship between the oocyte, and surrounding follicular cells.

Presented here are two protocols for the encapsulation of porcine oocytes in 3D culture conditions. In the first, cumulus-oocyte complexes (COCs) are encapsulated in fibrin-alginate beads. In the second, they are enclosed with fluorinated ethylene propylene powder particles (microbioreactors). Both systems ensure optimal conditions to maintain their 3D organization.

Hidrogéis alginatos desordeiros descabidos proteolíticos e microbiorreatores hidrofóbicos para encapsulamento de oócito suíno

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of ...