Lange Janina R, Metzner Claus, Richter Sebastian, Schneider Werner, Spermann Monika, Kolb Thorsten, Whyte Graeme, Fabry Ben
Biophysics Group, Department of Physics, Friedrich-Alexander University of Erlangen-Nuremberg, Erlangen, Germany.
Division of Molecular Genetics, German Cancer Research Center (DKFZ), Heidelberg, Germany.
Biophys J. 2017 Apr 11;112(7):1472-1480. doi: 10.1016/j.bpj.2017.02.018.
We describe a quantitative, high-precision, high-throughput method for measuring the mechanical properties of cells in suspension with a microfluidic device, and for relating cell mechanical responses to protein expression levels. Using a high-speed (750 fps) charge-coupled device camera, we measure the driving pressure Δp, maximum cell deformation ε, and entry time t of cells in an array of microconstrictions. From these measurements, we estimate population averages of elastic modulus E and fluidity β (the power-law exponent of the cell deformation in response to a step change in pressure). We find that cell elasticity increases with increasing strain ε according to E ∼ ε, and with increasing pressure according to E ∼ Δp. Variable cell stress due to driving pressure fluctuations and variable cell strain due to cell size fluctuations therefore cause significant variability between measurements. To reduce measurement variability, we use a histogram matching method that selects and analyzes only those cells from different measurements that have experienced the same pressure and strain. With this method, we investigate the influence of measurement parameters on the resulting cell elastic modulus and fluidity. We find a small but significant softening of cells with increasing time after cell harvesting. Cells harvested from confluent cultures are softer compared to cells harvested from subconfluent cultures. Moreover, cell elastic modulus increases with decreasing concentration of the adhesion-reducing surfactant pluronic. Lastly, we simultaneously measure cell mechanics and fluorescence signals of cells that overexpress the GFP-tagged nuclear envelope protein lamin A. We find a dose-dependent increase in cell elastic modulus and decrease in cell fluidity with increasing lamin A levels. Together, our findings demonstrate that histogram matching of pressure, strain, and protein expression levels greatly reduces the variability between measurements and enables us to reproducibly detect small differences in cell mechanics.
我们描述了一种定量、高精度、高通量的方法，用于使用微流控装置测量悬浮细胞的力学性能，并将细胞力学响应与蛋白质表达水平相关联。使用高速（750帧/秒）电荷耦合器件相机，我们测量微缩通道阵列中细胞的驱动压力Δp、最大细胞变形ε和进入时间t。通过这些测量，我们估计了弹性模量E和流动性β（细胞变形响应压力阶跃变化的幂律指数）的群体平均值。我们发现细胞弹性随着应变ε的增加而增加，符合E ∼ ε，并且随着压力的增加而增加，符合E ∼ Δp。因此，由于驱动压力波动导致的可变细胞应力和由于细胞大小波动导致的可变细胞应变会导致测量之间存在显著差异。为了减少测量差异，我们使用直方图匹配方法，该方法仅选择和分析来自不同测量且经历相同压力和应变的细胞。通过这种方法，我们研究了测量参数对所得细胞弹性模量和流动性的影响。我们发现细胞收获后随着时间的增加会有轻微但显著的软化。与从亚汇合培养物中收获的细胞相比，从汇合培养物中收获的细胞更软。此外，细胞弹性模量随着降低粘附性的表面活性剂普朗尼克浓度的降低而增加。最后，我们同时测量过表达绿色荧光蛋白标记的核膜蛋白lamin A的细胞的细胞力学和荧光信号。我们发现随着lamin A水平的增加，细胞弹性模量呈剂量依赖性增加，细胞流动性降低。总之，我们的研究结果表明，压力、应变和蛋白质表达水平的直方图匹配大大降低了测量之间的差异，并使我们能够可重复地检测细胞力学中的微小差异。
