Goncharova Valentina, Khaldoyanidi Sophia K
Torrey Pines Institute for Molecular Studies.
J Vis Exp. 2013 Jul 15(77):e50959. doi: 10.3791/50959.
Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.
循环细胞从血液中渗出在许多生理和病理生理过程中起着核心作用，包括干细胞归巢和肿瘤转移。三维流动腔装置（以下简称3D装置）是一种新型的体外技术，它能重现生理剪切应力，并能对细胞渗出级联反应的每一步进行量化。3D装置由一个上部腔室和一个下部腔室组成，上部腔室中感兴趣的细胞在剪切应力下循环，下部腔室是含有感兴趣趋化因子的静态孔板。两个腔室由涂有单层内皮细胞（EC）的多孔插入物隔开。一个可选的带有感兴趣微环境细胞的第二插入物可以直接放置在EC层下方。气体交换单元可维持最佳的二氧化碳张力，并在实验过程中提供一个添加或取出细胞或化合物的接入点。测试细胞在蠕动泵控制的所需剪切应力（流速）下在上部腔室中循环。实验结束时，收集循环和迁移的细胞进行进一步分析。3D装置可用于检测在剪切应力下细胞在EC上的滚动和黏附、对趋化因子梯度的迁移、对剪切应力的抗性、簇形成和细胞存活。此外，可选的第二插入物允许检测EC与微环境细胞之间的串扰效应。3D装置的转化应用包括测试靶向细胞迁移的候选药物以及预测静脉注射后细胞在体内的行为。因此，新型3D装置是研究介导细胞渗出的分子机制的一种通用且廉价的工具。
