基于双向浓度梯度的微流控芯片系统对肿瘤细胞侵袭能力的多重分析

doi:10.7503/cjcu20190206

摘要/Abstract

摘要：

利用微流控芯片易于模拟体内生理环境、流体控制精确及易于集成等优势, 将基于扩散原理的浓度梯度形成结构与经典的圣诞树形浓度梯度发生器相集成, 建立了在垂直和水平方向上形成连续、双向浓度梯度的微流控芯片系统, 采用该系统对不同类型细胞(HEK-293, MCF-7, SGC-7901)的侵袭力进行了定量分析; 通过在垂直方向上施加血清浓度梯度, 在水平方向上施加抗肿瘤药物十字孢碱浓度梯度, 分析了在连续药物浓度作用下的人胃癌SGC-7901细胞侵袭能力被抑制的情况, 同时观察并定量评价了伴随细胞侵袭力变化过程中细胞增殖能力受抑制的情况. 研究结果表明, 该系统可形成稳定的双向物质浓度梯度; 在血清浓度梯度存在情况下, 伴随十字孢碱浓度梯度的升高, 肿瘤细胞侵袭(P<0.0001)和增殖能力(P<0.001)均呈现浓度依赖性的连续降低. 建立的双向浓度梯度微流控芯片系统可用于评价复杂环境对细胞的多重影响, 也为研究细胞间相互作用、多种药物联用及药物筛选等提供了良好的研究平台.

关键词: 微流控芯片, 浓度梯度, 肿瘤, 侵袭, 药物筛选

Abstract:

A simple integrated microfluidic system with continuous 2D concentration gradient for multiple evaluation of tumor invasion was developed. The continuous bi-directional gradient was established vertically by solute diffusion and horizontally by Christmas tree-shaped network. The microfluidic system managed to quantitatively evaluate different invasive potentials of different cell lines(HEK-293, MCF-7, SGC-7901) and effects on tumor invasion by multiple factors. Human gastric cancer SGC-7901 cells were driven to invade into matrix under vertical FBS(Fetal bovine serum) gradient and revealed an increased invasion distance with the obvious concentration gradient. Given the continuous bi-directional gradient of FBS and Staurosporine, the invasion distance and increased number of human gastric cancer SGC-7901 cells were gradually inhibited along with the increase of drug concentration. In conclusion, the established bi-directional gradient microfluidic system provides multifunctional application in investigation of tumor invasion, cell interaction under complex environmental factors or combined chemotherapy.

Key words: Microfluidic, Concentration gradient, Tumor, Invasion, Drug screening

中图分类号:

O657

TrendMD:

王洁,张宇,于敏,方瑾. 基于双向浓度梯度的微流控芯片系统对肿瘤细胞侵袭能力的多重分析. 高等学校化学学报, 2019, 40(12): 2494.

Jie WANG,Yu ZHANG,Min YU,Jin FANG. Multiple Analyses of Tumor Invasion Based on an Integrated Microfluidic System with Bi-directional Solute Concentration Gradient ^†. Chem. J. Chinese Universities, 2019, 40(12): 2494.

图/表 8

Fig.1 Bi-directional gradient microfluidic system (A) The schematic illustration of microfluidic device; (B) generation principle of the bi-directional gradient device.

Fig.2 Formation and maintenance of concentration gradients at different flow rates Red, blue water-soluble dye and clear ultrapure water were pumped into device simultaneously. Concentration gradient images were taken under stereomicroscope at a series of time points.

Fig.3 Horizontal and vertical concentration gradient at the flow rate of 3 μL/h Gray value of horizontal red(A), horizontal blue(B) and vertical(C) concentration gradient at 24 h was converted by Image J. 1. Cell culture channel; 2. matrix channel; 3. conditional medium channel.

Fig.4 Quantitative evaluation of horizontal and vertical concentration gradient at the flow rate of 3 μL/h (A) Horizontal red; (B) horizontal blue; (C) vertical.

Fig.5 Invasiveness detection of different cell lines Three types of cell lines(HEK-293, MCF-7, SGC-7901) were cultured in the Matrigel-loaded device under a 0—10% FBS gradient.

Fig.6 Invasiveness evaluation of different cell lines Invasiveness of different cell lines(HEK-293, MCF-7, SGC-7901) was assessed quantitatively according to the invasion distance of leading cells.

Fig.7 Invasion profile of tumor cells under bi-directional gradient (A) Human gastric cancer SGC-7901 cells were cultured and driven to invade into matrix by bi-directional FBS gradient. The invasion was imaged at sequential sites. (B) Under horizontal Staurosporine gradient and vertical FBS gradient, cultured SGC-7901 cells were driven to invade into matrix. The invasion profile was sequential imaged.

Fig.8 Quantitative invasion analysis of tumor cells under bi-directional gradient (A) Quantitative analysis of invasion distances at different sites over a 7 d period of time. The corresponding drug concentrations at different sites were also demonstrated. (B) Invasion distances at different sites were quantified on the third day and the fifth day. (C) Cell numbers within channels were also quantified accordingly for proliferation evaluation. (A) Drug concentration gradient/(nmol·L-1): a. 14; b. 16; c. 25; d. 45; e. 50; f. 60; g. 80; h. 90; i. 95; j. 110; k. 125; l. 135; m. 140; n. 160; o. 170; p. 200.

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