在机械拉伸活细胞成像
Summary
A novel imaging protocol was developed using a custom motor-driven mechanical actuator to allow the measurement of real time responses to mechanical strain in live cells. Relevant to mechanobiology, the system can apply strains up to 20% while allowing near real-time imaging with confocal or atomic force microscopy.
Abstract
There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in their capability for measuring responses in real time in live cells or viable tissue. A protocol was developed with the use of a cell actuator to distend live cells grown on or tissues attached to an elastic substrate while imaging with confocal and atomic force microscopy (AFM). Preliminary studies show that tonic stretching of human bronchial epithelial cells caused a significant increase in the production of mitochondrial superoxide. Moreover, using this protocol, alveolar epithelial cells were stretched and imaged, which showed direct damage to the epithelial cells by overdistention simulating one form of lung injury in vitro. A protocol to conduct AFM nano-indentation on stretched cells is also provided.
Introduction
细胞进行机械载荷在许多组织中，而这种机械刺激已经显示出促进基因表达的变化，生长因子，细胞因子或细胞外基质的重塑释放的模式和细胞骨架1-4。从这样的机械刺激转导的胞内信号，以通过机械传导5-7的过程发生。在呼吸系统，机械传导的一个结果是增加的活性氧（ROS）的肺上皮细胞中的环状拉伸应变的存在下8,9-和促炎细胞因子10。强有力的证据也表明，过量的拉伸应变导致直接伤害到肺泡上皮中，除了元件11-14的生化反应。虽然这里的重点主要是对肺细胞的机械变形的响应，诱发力传导通路起到BAS关键作用在人体内许多组织，包括血管紧张15的调节和生长板16的发展的集成电路功能。
的在机械传导日益增长的兴趣已经导致许多装置开发用于生理学相关的机械负载的应用，培养细胞和组织。特别是，施加拉伸应变，这是机械负荷经历组织的常见形式，设备是受欢迎11,17-19。然而，许多现有的装置被设计或者作为生物反应器用于组织工程应用或不利于实时成像伸展。因此，有必要开发工具和方法，可以可视化的细胞和组织中的张力，以促进机械传导的途径的调查。
此处，面内机械拉伸装置被设计和协议被开发应用米到组织，同时允许在实时（ 图1A-D）的生物化学和机械响应的成像ultiple形式株和细胞。该装置利用布置在圆周抓住一个柔性膜和施加在平面内，径向膨高达约20％（ 图1B）6均匀间隔夹具。所述致动装置可放置在细胞培养孵化器在延长的时间周期，而电机（ 图1C）位于所述培养箱外并通过由电动机供应商所提供的专用软件控制。电动机被连接到线性驱动器，其旋转一个内凸轮，驱动六担架夹子均匀地拉伸和放松。
除了机械设备，定制柔性膜由市售细胞培养准备膜产生的机械系统中使用。然后圆形壁（具有直径约28毫米）作了并附着到柔性膜使细胞只能在良好描述应变轮廓的这个区域进行培养。为了确定所述致动装置内的放置这些膜是否会提供均匀的和各向同性的应变在弹性膜的中心，有限元分析，使用市售的软件（ 图1E-F）进行的。灵活的膜仿照对称的边界条件与利用网格的所有四边形单元。见于最大主应变的图1F所示的等高线图的同心圆环指示菌株的各向同性分布。
所经历的膜的应变是由通过负载（ 图2）的记录标记的图像进行测量。 图2D示出了在径向和轴向方向测得的平均膜应力为近似线性相对于所施加的电动机向上计数到20％的最大线性应变。有腹胀期间与回缩回到静止位置时相比，测量测得的应变水平之间没有差异显著。接着，人支气管上皮细胞（16HBE），核上的定制柔性膜培养的位移进行测定。在16HBE细胞的荧光标记的吲哚（DAPI）细胞核使用共聚焦显微镜下一个20X物镜成像，而全细胞位移的测定使用记录用数字显微镜相衬图像。正如在图3中 ，由核位移测量的应变是类似于由斑纹的位移对膜测得的，高达约20％的线性应变。这证实了施加到膜的应变被传递到贴壁细胞。描述在传统显微镜的使用自定义设备的协议和原子力microscope被在下面的步骤提供。
Protocol
Representative Results
Discussion
对于在机械拉伸活细胞成像的独特装置的开发;这个装置是用在协议研究肺上皮细胞的力学生物学。在初步研究中，人们发现，单个保持伸展刺激生产在支气管上皮细胞线粒体超氧化物。此外，证实了机械应变水平的增加造成直接损伤肺泡上皮细胞的一个单层的完整性。
进行这些初步实验中，该装置被第一校准，然后将其结果表明，在膜的应变被传递到细胞中（ 图2-3）?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
作者谨感谢那个技术学院联邦快递在孟菲斯大学的支持。作者要感谢学生在机械工程系高级设计项目组在孟菲斯（大卫·巴特勒，卡特成龙，多米尼克骑士，雅各布谢弗）的大学，丹尼尔·科恩来自孟菲斯大学的工程技术部为电机控制和腾斌和Charlean Luellen女士的细胞培养博士的帮助。这项工作是由K01 HL120912（ER）和R01 HL123540（CMW）的支持。
Materials
| SmartMotor NEMA 34: 3400 Series | MOOG Animatics | SM3416D | Integrated motor, controller, amplifier, encoder and communications bus |
| Flexcell Membrane (Collagen I coated) | Flexcell International Corp | SM2-1010C | 3.5×5.25×0.020" |
| Sylgard 184 | Dow Corning Corporation | 10:1 | |
| Hoechst 33342 | Sigma-Aldrich | H1399 | DAPI stain |
| MitoSOX | Sigma-Aldrich | M36008 | |
| Tiron | Sigma-Aldrich | D7389 | mitochondrial superoxide label |
| DMEM | superoxide inhibitor | ||
| FBS | |||
| HEPES | |||
| 50 ml tubes | Fisher Scientific | 06-443-19 | Any centriguge tube can be used to create an area for imaging. |
| Hybridization oven | Bellco Glass | ||
| MLE12 Cells | ATCC | CRL-2110 | Mouse Lung Epithelial Cells |
| 16HBE cells | ATCC | CRL-2741 | Human Bronchial Epithelial Cells |
| AFM Indentation Experiments | |||
| Cantilever Beams for Nano-indentation | Budget Sensors | Si-Ni30 | |
| AFM | Asylum Research | MFP3D | |
| Olympus microscope | Olympus | IX-71 | Inverted microscope with 20X and 40X objectives. |
| AFM Leg Extenders | Asylum Research | Not available | AFM microscope |
| Finite Element Analyses | |||
| ABAQUS | Simulia | 6.12 | |
| Software | |||
| ImageJ | NIH | ||
| Microscopes | |||
| Digital microscope | Life Technologies | EVOS XL Core | Initially a self standing company, now owned by Life Technologies. |
| Confocal microscope | Zeiss | LSM 710 | 2-photon upright microscope |
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