新型微流控芯片中单细胞趋化运动及其影响机制研究

Concentration gradient generation in microfluidic system is of great importance in the biological and chemical areas, such as chemotaxis, nerve growth cone guidance, and nucleation and growth of crystals. Chemotaxis is the directed, active movement of living cells according to the concentration gradient of chemicals in their environment. Therefore, a well-defined concentration gradient with temporal and spatial stability is very important for the in vitro analysis. Current investigations conclude that the microfluidic system is a promising platform for chemotaxis analysis for its stability. However, quantification of the cell chemotaxis is still unreliable because of the tremendous errors in determine the actual concentration gradient around the cell and the actual chemotactic movement component. These tremendous errors are majorly originated from the cell's chemokinetic movement, the fluid viscous force, and the cell's active disturbance on the attractant's concentration field. These factors are not considered in any of the current researches. As a consequence, current chemotactic assays exhibit widely scattered results which are barely redeemed as quantified chemotactic assays. Therefore, we proposed a new microfluidic quantification system. Firstly, a chemotactic chamber is designed to be separated from the fluids mixing chamber, so that the fluid viscous force can be controlled independent to the concentration field. Optimization can be performed by numerical simulations. Hence, we can largely reduce the influence of the fluid viscous force on the chemotactic movement. Then, a chemokinetic assay for the same cell is proposed as a control assay, so that the chemokinetic movement under a uniform concentration of attractant can be obtained. However, the true concentration gradient around the cell is disturbed by the actual cell movement, which can hardly be traced simultaneously with the cell's movement. Therefore, numerical simulation is proposed in computing the true concentration gradient evolving with the cell's active movement. To accomplish the numerical simulation, a 3D theoretical model will be developed to numerically simulate solid-liquid interacted mass transfer processes in microfluidic chips based on actual cell's activities. This model consists of a set of governing equations, which includes (i) Navier-Stokes equation describing the flow field in microchannels, and (ii) a mass transfer equation describing the sample concentration field. Solid-liquid interactions can be solved by the ALE method. In this way, a quantified relation with known errors can be established between cell chemotactic movement and its true surrounding environment. This quantification system would be a bridge to reveal the cell responses on the molecular level, and would be an essential platform to researches such as the healing process and tumor migration.

单细胞的趋化运动往往受实验中的多种因素影响而无法得到定量的结果，因此研究单细胞的趋化机制首要解决的是建立一套重复性高、精确度高的定量实验系统和方法，来确定细胞周边的实际浓度梯度和实际趋化运动分量。本项研究的主要目标即是建立和完善一种利用微流控系统定量研究单细胞趋化运动及其影响因素的方法。首先，设计一新型系统使得浓度梯度相对独立于Peclet数，从而控制流动对趋化运动的影响。其次，利用实验和数值模拟有机结合的方法分别研究化学增活和流动剪切与该细胞运动及对引物传质的相互影响，探索准确、科学的测量体系和评价方法来揭示单细胞的趋化运动机制及其与细胞化学微环境之间的复杂关系。这对进一步阐明细胞感知能力和行为方式及其分子动力学机制有重要的意义，为体外研究组织发炎、伤口愈合、癌细胞转移提供先进的模拟平台和理论及实验基础。

许多活细胞在引物浓度梯度的影响下能发生自主的定向运动，即趋化运动。例如，自由生活微生物、白细胞、精子细胞、内皮细胞、成纤维细胞、癌细胞等，这些细胞在炎症反应、受精、器官生长、伤口愈合、癌症迁移等过程中普遍存在趋化运动。揭示细胞在低雷诺数剪切流中的趋化运动影响因素及机制，可以细胞趋化运动的定量化测量提供新理论概念和新的测试平台。目前，关于细胞趋化效应的研究主要着眼于利用微流体技术建立线性或非线性的浓度梯度，观测细胞对不同浓度梯度的反应。这些研究大都忽略细胞运动对浓度场的扰动及剪切流对细胞趋化的影响，这些都将导致对实际细胞趋化运动的失常测算。本项目着眼于数值模拟与实验观测相结合，研究在微通道内细胞的直线运动及细胞的翻滚对周围的剪切流场、引物浓度场的影响，探讨了在微通道中非线性引物梯度场中细胞运动对引物浓度场的耦合影响机制。研究结果表明，在细胞平动运动中，细胞的运动对周围浓度梯度场有明显影响，忽略细胞的运动是不合适的。平动的细胞相比静止的细胞可以感受到更大的浓度差，因而实际的趋化运动动力也更大。而趋化方向则相差不大。在细胞转动过程中，其周围引物浓度场在一定时间内发生变化，但很快达到稳态；顺时针与逆时针转动的细胞对微环境的搅动效果相反；增加转动线速度可提高搅动效果，导致趋化偏转角增大，而趋化动力减小。综上所述，细胞的自主趋化运动与其周围的引物剪切流之间存在耦合效应，细胞的两种运动（平动与转动）对引物的剪切流影响机制不同。细胞的平动将增强趋化运动，而对趋化方向影响较小；细胞的转动将促进细胞的趋化方向的调整，而使得趋化动力急剧减小。这与前人及本项目实验中观察到的现象一致。