Microfluidic perfusion system for culturing and imaging yeast cell microarrays and rapidly exchanging media
Microfluidic perfusion system for culturing and imaging yeast cell microarrays and rapidly exchanging media
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DOI：
10.1039/c004857g
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发表时间：
2010-01-01
期刊：
影响因子：
6.1
通讯作者：
Juncker, David
中科院分区：
文献类型：
--
作者：
Mirzaei, Maryam;Pla-Roca, Mateu;Juncker, David
高分辨率活细胞显微镜越来越多地用于检测对药物和化学品反应的细胞动力学，但它依赖于复杂且昂贵的液体处理设备，这限制了其更广泛的采用。在这里，我们提出了一种微流体灌注系统，该系统在不使用专门的微加工基础设施的情况下构建，使用简单，因为仅需要移液器来处理液体，并且允许快速介质交换和同步荧光显微镜成像。酵母细胞可以从培养物中引入，或者以阵列形式点在盖玻片上，并用20μm厚的径迹蚀刻膜夹在中间。第二个盖玻片和孔隙率为 120 μm 的网放置在顶部，形成微流体导管，通过毛细管效应实现溶液的横向流动。通过入口引入的溶液流过网，化学物质垂直穿过膜扩散到下面捕获的细胞。通过向进样口添加新样品来交换溶液。使用该系统，我们研究了表达 Sac6-EGFP 的活酵母中的 F-肌动蛋白（一种与称为“斑块”的离散 F-肌动蛋白结构相关的蛋白质）对药物 latrunculin A（一种众所周知的肌动蛋白聚合抑制剂）的动态响应。我们观察到斑块在 5 分钟内在 85% 的细胞中消失，并在与介质交换药物后 45 分钟内重新组装。这里介绍的灌注系统是一种简单、廉价的装置，适用于药物分析单细胞和细胞阵列的剂量反应和再生。
High resolution live cell microscopy is increasingly used to detect cellular dynamics in response to drugs and chemicals, but it depends on complex and expensive liquid handling devices that have limited its wider adoption. Here, we present a microfluidic perfusion system that is built without using specialized microfabrication infrastructure, simple to use because only a pipette is needed for liquid handling, and yet allows for rapid media exchange and simultaneous fluorescence microscopy imaging. Yeast cells may be introduced from a culture, or spotted as arrays on a coverslip, and are sandwiched with a 20 mu m thick track-etched membrane. A second coverslip and a mesh with 120 mu m porosity are placed on top, forming a microfluidic conduit for lateral flow of solutions by capillary effects. Solutions introduced through the inlet flow through the mesh and chemicals diffuse vertically across the membrane to the cells trapped below. Solutions are exchanged by adding a new sample to the inlet. Using this system, we studied the dynamic response of F-actin in living yeast expressing Sac6-EGFP-a protein associated with discrete F-actin structures called "patches''-to the drug latrunculin A, a well known inhibitor of actin polymerization. We observed that the patches disappeared in 85% of the cells within 5 min, and re-assembled in 45 min following exchange of the drug with media. The perfusion system presented here is a simple, inexpensive device suited for analysis of drug dose-response and regeneration of single cells and arrays of cells.