微型硅悬臂梁的利用率，评估细胞收缩功能<em>在体外</em
Summary
这个协议描述了用于测量肌肉细胞的收缩力在体外使用的微型硅悬臂梁的柔韧培养表面。细胞收缩引起悬臂弯曲，可以测量，记录和转换成力读数，用于测量收缩功能的体外提供一种非侵入性和可扩展的系统。
Abstract
的多个预测和生物相关的体外测定法的发展的前提是多功能的细胞培养系统，促进种子细胞的功能评价的进步。为此，微型悬臂技术提供了一个平台，用以测量各种细胞类型，包括骨骼，心脏和平滑肌细胞的收缩的功能，通过收缩引起的基板弯曲的评估。复用的悬臂阵列的应用提供了一种方式来开发中等至高通量的协议，以评估药物疗效和毒性，疾病的表型和进展，以及神经肌肉和其他细胞 – 细胞相互作用。该原稿提供的信息用于制造可靠的悬臂阵列用于此目的，并且需要在这些表面上成功地培养细胞的方法。设置在需要进行官能肛门的步骤进一步描述收缩的细胞类型ysis保持上使用一种新的激光和光检测系统的这种阵列。到这样的技术可以应用于提供亮点的精度和收缩功能可以利用本系统的分析的可再现性的有代表性的数据，以及在广泛的研究。成功的广泛采用该系统的能提供研究者的手段来在体外进行快速，低成本的功能性研究，导致了组织的性能，疾病发展和响应于新的治疗治疗更准确的预测。
Introduction
The in vitro culture of muscle cells from both human and rodent sources has been possible for decades1,2. However, while standard coverslip preparations are useful for biochemical assessment, they do not facilitate analysis of the cell’s primary functional output (contractility), and therefore are of somewhat limited value as a means to assess cellular maturation and performance. In order to maximize the amount of data obtainable from such in vitro cultures, it is necessary to advance the development of systems capable of housing such cells in configurations that permit the real-time assessment of their functional performance. The establishment of a multitude of three dimensional muscle models has made some progress toward fulfilling this need, and such systems have been used in a number of publications as a means to assess the contractile capacity of cultured muscle cells in vitro3-5. While such systems are invaluable for tissue modeling and reconstruction studies, they are limited in their applicability for studies of single cell responses. In such cases where single fiber studies are necessary, complex and labor intensive ex vivo methodologies remain the only option6-10. Furthermore, current movement toward the development of complex, multi-organ platforms for drug development and screening protocols requires the establishment of systems which are non-invasive, easily scalable and which integrate readily with supporting cells and tissue models11.
Microscale cantilevers offer a simple method for assessing the functional contractile capacity of single cells/small populations of cells12,13. The technique is based on modified Atomic Force Microscopy (AFM) technology14, and uses a laser and photo-detector system to measure microscale cantilever deflection in response to cultured myotube contractile activity. Modified Stoney’s equations are then used to calculate stress in the myotube, and the force exerted by the myotube in order to generate the observed substrate deflection15. A scanning program has been written which enables simultaneous assessment of multiplexed cantilever arrays, offering potential moderate to high through-put applications for drug toxicity/efficacy studies15,16. Such technology may prove invaluable in the development of functional, pre-clinical assays for predicting drug efficacy in vivo. Furthermore, fabrication of cantilever chips in silicon does not impede post analysis processing of cells for standard biomolecular assays such as immunostaining, western blotting and PCR.
This manuscript provides detailed instructions on the fabrication and preparation of microscale silicon cantilevers, the hardware and software set-up, and the operating guidelines for assessing the functionality of contractile cells cultured on these chips. Standard cell culture techniques can be implemented for plating and maintenance of cells on these surfaces, hence any contractile cell type for which reliable culture parameters exist should be able to integrate with this device with ease. The relatively simple 2D culture parameters utilized in this system makes integration of other cell models or addition of cell types that can interact with muscle (such as innervating neurons) straight-forward, greatly increasing the applicability of this model in the development of more complex functional in vitro assays and multi-organ models of mammalian systems.
Protocol
Representative Results
Discussion
在分析微尺度悬臂蜂窝收缩证据的关键步骤是在放置于显微镜载物台的悬臂芯片，并且激光和光检测器的阵列中的角悬臂的末端的随后对准。如果不正确地进行，则该软件将无法推断剩余的悬臂的位置的阵列中，从而可能导致假阴性积累数据收集期间。运营商应注意，以确保悬臂芯片校准激光的位置之前，躺在平齐培养皿的底部。如果芯片坐在在盘的角度，它会改变反射激光束的路径和混淆的数?…
Declarações
The authors have nothing to disclose.
Acknowledgements
该研究是由卫生部资助号R01NS050452和R01EB009429研究所。在外部进行悬臂芯片制造由合作者在位于康奈尔大学的纳米加工设施。在悬臂的制造过程中使用的所有设备被设在该设施。特别感谢小敏的Esch和Jean-马修普罗特其悬臂微加工的援助。由查尔斯·休斯，阿莱克斯Zelenin和埃里克·御从合成现实实验室在UCF的生成影视动画的悬臂功能。
Materials
| Name of material/ equipment | Company | Catalog number | Comments/ Description |
| Primary rat muscle growth medium | |||
| Neurobasal medium | Life Technologies | 21103-049 | N/A |
| B27 (50x) | Life Technologies | 17504044 | 1x |
| Glutamax (100x) | Life Technologies | 35050061 | 1x |
| G5 supplement | Life Technologies | 17503-012 | 1x |
| Glial-Derived Neurotrophic Factor | Cell sciences | CRG400B | 20 ng/ ml |
| Brain-Derived Neurotrophic Factor | Cell sciences | CRB600B | 20 ng/ ml |
| Ciliary Neurotrophic Factor | Cell sciences | CRC400A | 40 ng/ ml |
| Neurotrophin-3 | Cell sciences | CRN500B | 20 ng/ ml |
| Neurotrophin-4 | Cell sciences | CRN501B | 20 ng/ ml |
| Acidic Fibroblast Growth Factor | Life Technologies | 13241-013 | 25 ng/ ml |
| Vascular Endothelial Growth Factor | Life Technologies | P2654 | 20 ng/ ml |
| Cardiotrophin-1 | Cell sciences | CRC700B | 20 ng/ ml |
| Heparin Sulphate | Sigma | D9809 | 100 ng/ ml |
| Leukemia Inhibitory Factor | Sigma | L5158 | 20 ng/ ml |
| Vitronectin | Sigma | V0132 | 100 ng/ ml |
| Primary rat muscle differentiation medium | |||
| NB Activ 4 | Brain Bits LLC | NB4-500 | N/A |
| Equipment | |||
| Class 2 red diode laser | Newport | N/A | |
| Photo-detector | Noah Industries | N/A | |
| Model 2100 Pulse stimulator | A-M systems | N/A | |
| Multiclamp 700B Digitizer | Axon Instruments | N/A | |
| Patch clamp microscope and stage | Olympus | N/A | |
| Delta T4 culture dish controller | Bioptechs | N/A | |
| Axoscope software | Molecular Devices | N/A | |
| LabVIEW software | National Instruments | N/A | |
| 37oC, 5% CO2 incubator | NAPCO | N/A | |
| Class 2 microbiological flow hood | Labconco | N/A | |
| Pipettes and tips | Eppendorf | N/A |
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