High-throughput microfluidic micropipette aspiration device to probe time-scale dependent nuclear mechanics in intact cells
High-throughput microfluidic micropipette aspiration device to probe time-scale dependent nuclear mechanics in intact cells
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DOI：
10.1039/c9lc00444k
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发表时间：
2019-11-07
期刊：
影响因子：
6.1
通讯作者：
Lammerding, Jan
中科院分区：
文献类型：
--
作者：
Davidson, Patricia M.;Fedorchak, Gregory R.;Lammerding, Jan
细胞核的机械性质在许多生物过程中越来越被认为是关键的。细胞核的可变形性决定了免疫细胞和癌细胞迁移通过组织和穿过内皮细胞层的能力，并且细胞核的机械性质的变化可以在诸如癌症进展和干细胞分化的过程中充当新的生物标志物。然而，目前的技术来测量粘弹性核机械性能往往是耗时的，仅限于探测一个细胞的时间，或需要昂贵的，高度专业化的设备。此外，许多目前的测定法不测量时间依赖性，这是粘弹性材料的特征。在这里，我们提出了一种易于使用的微流体装置，该装置应用了微管抽吸的成熟方法，适于并行测量许多细胞。该装置的设计允许快速加载和清洗细胞进行测量，并最大限度地减少大颗粒或细胞簇的堵塞。结合半自动图像分析管道，微流体装置方法能够显著提高实验吞吐量。我们验证了实验平台，通过比较计算模型的流体力学的设备与实验测量的流体流动。此外，我们对缺乏核膜蛋白核纤层蛋白A/C和野生型对照的细胞进行了实验，这些细胞具有良好的核机械特性。拟合随时间变化的核变形数据的幂律和不同的粘弹性模型显示，层蛋白A/C的损失显着改变了核的弹性和粘性的性质，从而大大增加核的变形能力。最后，为了证明设备的多功能性，我们表征了各种细胞系和实验模型系统中的粘弹性核机械性能，包括来自具有与扩张型心肌病相关的核纤层蛋白基因突变的个体的人皮肤成纤维细胞、健康对照成纤维细胞、诱导多能干细胞（iPSC）和人肿瘤细胞。总之，这些实验证明了微流体装置和自动化图像分析平台在广泛的应用中提供核机械性质（包括时间依赖性弹性和粘性行为）的稳健的高通量测量的能力。
The mechanical properties of the cell nucleus are increasingly recognized as critical in many biological processes. The deformability of the nucleus determines the ability of immune and cancer cells to migrate through tissues and across endothelial cell layers, and changes to the mechanical properties of the nucleus can serve as novel biomarkers in processes such as cancer progression and stem cell differentiation. However, current techniques to measure the viscoelastic nuclear mechanical properties are often time consuming, limited to probing one cell at a time, or require expensive, highly specialized equipment. Furthermore, many current assays do not measure time-dependent properties, which are characteristic of viscoelastic materials. Here, we present an easy-to-use microfluidic device that applies the well-established approach of micropipette aspiration, adapted to measure many cells in parallel. The device design allows rapid loading and purging of cells for measurements, and minimizes clogging by large particles or clusters of cells. Combined with a semi-automated image analysis pipeline, the microfluidic device approach enables significantly increased experimental throughput. We validated the experimental platform by comparing computational models of the fluid mechanics in the device with experimental measurements of fluid flow. In addition, we conducted experiments on cells lacking the nuclear envelope protein lamin A/C and wild-type controls, which have well-characterized nuclear mechanical properties. Fitting time-dependent nuclear deformation data to power law and different viscoelastic models revealed that loss of lamin A/C significantly altered the elastic and viscous properties of the nucleus, resulting in substantially increased nuclear deformability. Lastly, to demonstrate the versatility of the devices, we characterized the viscoelastic nuclear mechanical properties in a variety of cell lines and experimental model systems, including human skin fibroblasts from an individual with a mutation in the lamin gene associated with dilated cardiomyopathy, healthy control fibroblasts, induced pluripotent stem cells (iPSCs), and human tumor cells. Taken together, these experiments demonstrate the ability of the microfluidic device and automated image analysis platform to provide robust, high throughput measurements of nuclear mechanical properties, including time-dependent elastic and viscous behavior, in a broad range of applications.