1。主要关节软骨细胞的分离收获10-15全层动物关节的关节面的软骨片，从（ 如掌趾关节，骨骼发育成熟的奶牛从本地屠宰场获得）。 <ol><li>将软骨片在100毫米的陪替氏培养皿和孵育在20毫升0.5％的蛋白酶的Ham F-12（w / v的）为2小时，在37℃下冲洗三次火腿的F-12培养基中，并用20毫升0.15％胶原酶孵育A的Ham氏F-12培养基中过夜，在37℃下</li><li>过滤细胞悬浮液通过200目筛网过滤器到一个干净的菜。清洗过滤器，用5毫升联队的F-12和添加的菜。细胞悬浮液转移到50ml的锥形管中。洗净用10ml的Ham F-12的陪替氏培养皿中，并加至锥形管。 </li><li>离心管中于600-800相对离心力在室温下6-8分钟。 </li><li>吸取上清液，确保不打扰次E细胞沉淀和重新悬浮在40毫升的Ham氏F-12培养基。 </li><li>离心管在600-800区域合作框架6-8分钟。 </li><li>重复步骤1.4和1.5的两次以上的总的3倍。在此之前的第三次离心分离，通过搅拌悬浮颗粒，并获得了500微升等分进行细胞计数。 </li><li>计数活细胞，血球计数器和一个倒置相差显微镜使用台盼蓝排除方法9。 </li><li>吸取上清，并重新悬浮沉淀增加一倍所需的接种密度在一定体积的Ham氏F-12介质（ 例如，20×10 6细胞/ ml的最终浓度为10×10 6细胞/ ml后，封装）。 </li></ol> 2。软骨细胞琼脂糖凝胶封装<ol><li>烧热0.8克蒸压VII型琼脂糖在20毫升无菌，1×PBS（pH 7.4）中，设定为120℃，直至溶解在热板上。一旦溶解，降低到60℃，因为较高的温度加热西港岛线升影响细胞活力。 </li><li>在一个50毫升锥形管中，充分混合等体积的细胞悬浮液，用两倍的所需浓度的琼脂糖（ 如 4％的琼脂糖悬浮液，最后的2％琼脂糖水凝胶）。避免产生大的泡沫，因为他们可以影响细胞的活力和构建疲劳寿命。 </li><li>小心地等分试样10毫升软骨细胞琼脂糖混合物到一个直径60毫米的培养皿中，同时避免大气泡的创建。 </li><li>让在室温下静置30分钟，直至胶凝。 </li><li>获取尽可能多软骨细胞琼脂糖根据需要的水凝胶样品（通常为20-30），使用一个4毫米活检打孔。 </li><li>测量和记录的物理尺寸（直径和高度）的有代表性的样品，使用测微计。构建体应为约5毫米的高度，丢弃从样品高度方差是大于2％的区域获得的任何样品。 </li><li>将样品转移到新鲜的60毫米陪替氏培养皿和补充用10ml的完整的媒体（ 如 20％胎牛血清的Ham氏F-12培养基中的20mM HEPES，100μg/ ml的抗坏血酸，和2x抗生素/抗 ​​真菌剂）。 </li></ol> 3。机械性刺激和放射性标记的软骨细胞琼脂糖凝胶<ol><li>高压灭菌的金属组分的压缩钻机。用70％乙醇（过夜）和UV光（至少15分钟）灭菌的塑料部件。 </li><li>为了保持结构，从翻倒，使用镊子，地点12的无菌塑料护环（具有内径大于该构建直径）组（n = 6，样品和对照组）内的24孔培养板的孔中。 </li><li>使用镊子，小心转移软骨细胞 - 琼脂糖凝胶从陪替氏培养皿中的24孔板上，每一个固定在一个保持环。 </li><li>转移400μl的完整的媒体（ 例如，20％FBS的Ham氏F-12培养基中，用20mM HEPES，100μg/ ml的抗坏血酸，和2倍抗生素/抗真菌剂），每个样品。 </li><li>组装消毒压缩钻机（ 图1），并用紧定螺钉固定的压板。将压缩钻机的24孔培养板中。 </li><li>释放和重新设置的固定螺钉，以建立压板水凝胶接触（零应变状态）。 </li><li>将组装的压缩钻机到了Mach-1微机械测试系统。安全钻机垂直阶段通过定位销和锁固定螺丝的地方。删除间距夹具。 </li><li>应用动态压缩载荷在所希望的振幅（ 例如，10％的原始高度的样品的基础上）的应变振幅，频率（ 例如，1赫兹）和持续时间或数量的周期（ 例如到第60分钟的时间间隔）。 </li><li>完成后的机械性刺激，更换间隔夹具，定位销固定螺丝松动和删除压缩钻机和培养板的Mach-1微机械测试系统。 </lI&gt; <li>放射性标记的细胞的水凝胶结构用5微居里所需的同位素（ 例如 ，[3 H] -脯氨酸的蛋白标签为软骨细胞的特定的胶原蛋白的合成和[35 S] -硫蛋白聚糖的合成）通过补充媒体。 </li><li>放射性标记的构造孵育24小时，在37℃，5％CO 2 10。 </li><li>收获的放射性标记的水凝胶的结构，并在1x PBS（pH值7.4）冲洗3次，以除去任何非法人的同位素。月刊样品用木瓜蛋白酶（40微克/毫升在20mM乙酸铵中，1 mM的ethyldiaminetetraacetic的酸和2mM的二硫苏糖醇在65℃下72小时）。 </li><li>测定DNA含量Picogreen荧光染色实验检测11注册成立的同位素活性，规范化DNA含量，β-液体闪烁计数12,13紧随其后的木瓜蛋白酶消化消化。 </li></ol>注：购买和出售的放射性物质（垃圾同位素和项目接触放射性物质可能已经进入）必须遵循相关的制度（和/或政府）的政策和程序的安全处理和处置放射性物质。 4。代表性的成果牛关节软骨-琼脂糖凝胶的构造（与10×10 6细胞/ ml包裹细胞2％琼脂糖），机械刺激的振幅的10％的压缩应变，在20至60分钟（或1,200至3,600个周期的频率为1赫），并测定DNA含量和细胞外基质合成的放射性同位素成立。 DNA和细胞外基质的合成，在以剂量依赖性的方式的影响。 DNA含量表现出显着减少35％，为20或30分钟的刺激（P &lt;0.01）的结果，而没有显示出效果（ 图2）的60分钟的刺激。软骨特异性胶原蛋白和蛋白聚糖的合成（[3 H] -脯氨酸和确定[35 S] -硫掺入，分别）表现出显着增加了约60％，在响应于20分钟或30分钟的刺激（P &lt;0.01），用60分钟的刺激没有表现出可观察的效果（ 图3）。 <img src="/files/ftp_upload/4229/4229fig1.jpg" alt="图1" /> 刺激软骨细胞琼脂糖结构示意图如图1所示。定制的动态压缩钻机。：组装钻机。 右 ：爆炸视图的各个组成部分。 <img src="/files/ftp_upload/4229/4229fig2.jpg" alt="图2" /> 水凝胶机械下10％的压缩应变振幅为20分钟到第60分钟以1 Hz（均值±扫描电镜（SEM）中，n = 6/group）刺激软骨细胞-琼脂糖的DNA含量的图2的变化。 水凝胶机械刺激软骨细胞-琼脂糖的合成细胞外基质（胶原和蛋白聚糖）的图3的变化下10％压缩应变振幅为20分钟到第60分钟（均值±扫描电镜（SEM）中，n = 6/group）在1 Hz。

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没有利益冲突的声明。

<html>所描述的方法应用控制的机械刺激细胞种子的琼脂糖凝胶可以对软骨细胞代谢的动态压力的影响，直接调查。护环配合使用的定制试验台结构的横向约束的样品给小费，以避免潜在的问题。尽管潜在的差异，样品高度与结构，由一组人员死加权加载压板固定使用，确保直接接触。放射性标记刺激后，测量细胞的生物合成，降低污染的可能性，允许一个单一的测试平台，以用于多个应用程序的?...

<table border="1"><tbody><tr><td> 的试剂或设备的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>联队的F-12 </td><td>赛默飞世尔科技</td><td> SH3001002 </td><td></td></tr><tr><td>胶原酶A </td><td> Sigma Aldrich公司有限公司</td><td> C0130 </td><td></td></tr><tr><td>蛋白酶</td><td> Sigma Aldrich公司有限公司</td><td> P5147 </td><td></td></tr><tr><td>胎牛血清</td><td> Sigma Aldrich公司有限公司</td><td> F1051 </td><td></td></tr><tr><td>抗坏血酸</td><td> Sigma Aldrich公司有限公司</td><td> A4034 </td><td></td></tr><tr><td>抗生素/抗真菌剂</td><td> Sigma Aldrich公司有限公司</td><td> A5955 </td><td></td></tr><tr><td> HEPES </td><td> Bioshop加拿大有限公司</td><td> HEP001 </td><td></td></tr><tr><td>台盼蓝</td><td> Sigma Aldrich公司有限公司</td><td> 93595 </td><td></td></tr><tr><td>赖克特亮线 血球</td><td>豪塞尔科学</td><td> 1490 </td><td></td></tr><tr><td>定量，它PicoGreen </td><td> Invitrogen公司</td><td> P7589 </td><td></td></tr><tr><td>木瓜木瓜乳胶</td><td> Sigma Aldrich公司有限公司</td><td> P3125 </td><td></td></tr><tr><td>醋酸铵</td><td> Sigma Aldrich公司有限公司</td><td> A1542 </td><td></td></tr><tr><td> Ethyldiaminetetraacetic酸</td><td> Sigma Aldrich公司有限公司</td><td> E9884 </td><td></td></tr><tr><td> DL-二硫苏糖醇</td><td> Sigma Aldrich公司有限公司</td><td> 43819 </td><td></td></tr><tr><td>低熔点琼脂糖，第七类</td><td> Sigma Aldrich公司有限公司</td><td> A9045 </td><td></td></tr> &lt;TR&gt; <td>筛网过滤器（200） </td><td> Sigma Aldrich公司有限公司</td><td> S4145 </td><td></td></tr><tr><td> MACH-1微机械测试仪</td><td> Biomomentum公司</td><td> V500cs </td><td></td></tr><tr><td>压缩荷载跳汰机</td><td>定制</td><td></td><td>同类产品可以提供的Biomomentum公司</td></tr><tr><td>猎鹰24孔培养板</td><td>赛默飞世尔科技</td><td> B353047 </td><td></td></tr><tr><td> β-液体闪烁计数器</td><td> Beckman Coulter公司</td><td> LS6500 </td><td></td></tr><tr><td> [3H]脯氨酸</td><td> Perkin-Elmer公司</td><td> NET323005MC </td><td></td></tr><tr><td> [35]硫</td><td> Perkin-Elmer公司</td><td> NEX041005MC </td><td></td></tr></tbody></table>

mechanical stimulation of chondrocyte-agarose hydrogels

机械侮辱或退化性疾病，如骨关节炎关节软骨受到损坏时，从有限的修复能力。为了弥补这方面的不足，一些医疗干预措施已经制定出来。一种方法是复出与组织工程化软骨的受损区域，然而，组织工程通常缺乏本地软骨的生化特性和耐用性，质疑它的长期生存能力。这限制了小灶缺陷修复软骨组织工程的应用，依靠对周围组织的保护植入材料。为了改善性能的发达的组织，机械性刺激是一种流行的方法，利用增强软骨细胞外基质的合成，以及所得的机械性能的组织工程。机械性刺激，适用于力量的组织构造类似于那些有经验的体内 。这是基于的前提是机械的环境中，在某种程度上，调节的开发和维护的固有组织1,2。在软骨组织工程的机械性刺激中最常采用的形式是在生理菌株在1,3 1赫兹的频率的约5-20％的动态压缩。一些研究调查的影响，动态压缩，并已显示出它有一个积极的影响软骨细胞的代谢和生物合成，最终影响功能特性的开发组织4-8。在本文中，我们说明了该方法机械地刺激软骨细胞琼脂糖凝胶结构下的动态压缩和生物合成的变化进行分析，通过生化和放射性同位素检测。此方法也可以很容易地修改，作为一个结果，机械刺激，以评估任何可能引起的变化在细胞反应。

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Mechanical Stimulation of Chondrocyte-agarose Hydrogels

软骨细胞的软骨细胞外基质的生物合成的，可以通过施加机械刺激的影响。该方法描述了技术应用动态压缩应变包埋软骨细胞的三维结构和诱导软骨细胞代谢的变化的评价。

机械性刺激软骨细胞琼脂糖凝胶

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

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JoVE Journal

Biology

11.6K 次观看.  Queen's University. 软骨细胞的软骨细胞外基质的生物合成的，可以通过施加机械刺激的影响。该方法描述了技术应用动态压缩应变包埋软骨细胞的三维结构和诱导软骨细胞代谢的变化的评价。

视频: 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.

体外机械装载肌腱

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

科研

生物学

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.

采用循环单轴应变的干细胞的机械性刺激

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.

组织工程细胞在三维凝胶封装

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.

机械性刺激，集成光学和原子力显微镜研究活细胞的反应

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.

机械刺激引起的钙波传播的细胞单层：牛角膜内皮细胞的例子

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.

DNA交联聚丙烯酰胺水凝胶的制备

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.

多功能，微管为基础的法团细菌机械敏感离子通道与刺激的液滴界面双层膜

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成像的三维水凝胶

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.

对急性机械刺激的网柄反应的评估

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.

蛋白酶降解藻酸盐水凝胶和疏水性微生物反应器，用于猪卵母细胞封装

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