设计微流体装置用于研究细胞反应在单人或共存化工/电气/剪切应力刺激
2016年8月13日
与流体组件集成的微制造设备为模拟体内微环境的细胞研究提供了一个体外平台。我们开发了基于聚甲基丙烯酸甲酯的微流体芯片，用于研究单个或共存化学/电/剪切应力刺激下的细胞反应。
该程序的总体目标是设计和制造微流体装置，用于观察细胞如何对环境刺激做出反应。这种方法可以帮助回答细胞生物学和生物医学工程中的关键问题。例如细胞在不同环境刺激（包括化学物质、剪切应力和电场）下的反应。这种技术的主要优点是管材易于制造和组装。而这些微流体装置可以使体内条件具有极强的能力。演示该程序的是我实验室的本科生 Tzu-Yuan Chou 和 Julian Chung 博士实验室的后期工作人员 Hsien-San Hou。首先，打开二氧化碳激光划线器并检查它与计算机的连接。然后将预先绘制的圣诞树图案（如此处所示的图案之一）加载到划线器中。接下来，将 PMMA 片材或双面胶带放在划线机的舞台顶部。使用校准杆和可见氦氖激光器调整二氧化碳激光器的焦距。然后将图案划到基材上。完成后，拿起图案基材，去除不需要的碎片，并使用氮气流清洁表面的任何剩余碎屑。对芯片的每个组件重复此过程。组装化学剪切应力微流控芯片。为此，使用两张划线双面胶带贴上三张化学剪切应力图案 PMMA 片材。通过对齐螺纹和孔，使用强力胶将中间带有螺纹的亚克力适配器连接到芯片最顶部的 PMMA 层。这些适配器将用作介质入口出口和盐桥连接器。然后在芯片底部再添加一块 07 毫米厚的双面胶带，并将芯片贴在直径为 10 厘米的培养皿上。组装完成后，将芯片放入真空室中过夜。现在组装化学电场芯片。用双面胶带将用于化学电场芯片的划线 PMMA 片材贴在直径为 10 厘米的培养皿上。使用芯片前，请将它们暴露在紫外线下 30 分钟以对其进行消毒。接下来，通过塑料管和手紧螺母将芯片的入口连接到 3 ml PBS 填充的注射器上。以同样的方式，将出口连接到废液管。慢慢按下注射器上的柱塞，用 PBS 灌注流体通道。来回推动注射器以去除气泡，然后将芯片放入培养箱中过夜。要设置化学电场芯片，请在管状螺母中填充 3% 的 agarous 以设置盐桥。然后将氯化银电极放入管状螺母中。在补充有 10% FBS 的 D-MEM 中培养肺癌 CL1-5 细胞，直到它们达到 90% 汇合。然后吸出培养基。用预热的 PBS 洗涤细胞一次。并将 2 ml 05% tripsin 缓冲溶液加入直径为 10 cm 的板中。将单元格放回 37
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概述
本研究介绍了集成了流体组件的微制造设备，它们创建了一个体外细胞研究平台，紧密模拟体内微环境。该研究专注于基于聚甲基丙烯酸甲酯的微流控芯片，旨在研究细胞对各种刺激的反应。
关键研究组成部分
科学领域
- 微流控
- 细胞生物学
- 体外研究
背景
- 微制造设备增强了对细胞环境的研究。
- 流体组件允许精确控制实验条件。
- 理解细胞反应对各种生物学应用至关重要。
- 聚甲基丙烯酸甲酯是微流控应用的多功能材料。
研究目的
- 开发一个平台用于在受控环境中研究细胞反应。
- 使用微流控技术模拟体内条件。
- 研究化学、电和剪切应力刺激对细胞的影响。
使用的方法
- 使用聚甲基丙烯酸甲酯设计和制造微流控芯片。
- 集成流体组件以增强实验控制。
- 应用各种刺激以评估细胞反应。
- 体外实验以模拟体内条件。
主要结果
- 成功开发了用于细胞研究的微流控芯片。
- 证明了有效控制环境条件的能力。
- 观察到对不同刺激的不同细胞反应。
- 提供了细胞与其微环境之间相互作用的见解。
结论
- 微流控平台是研究细胞行为的有价值工具。
- 研究结果有助于在受控环境中理解细胞反应。
- 未来的应用可能包括药物测试和疾病建模。
常见问题
Executive Industry Relevance
Microfluidic platforms that enable precise control over chemical, electrical, and shear stress stimuli provide biopharma R&D with a scalable approach to de-risk target validation by mimicking complex in vivo microenvironments. These PMMA-based devices increase experimental throughput for screening cellular responses under coexisting stimuli, supporting mechanistic insight in early discovery. The technology aids in prioritizing targets with higher predictive confidence before committing resources to lead identification.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of cellular responses to chemical gradients, electric fields, and shear stress to clarify pathway involvement in disease mechanisms.
- Operational Value: PMMA substrates allow low-cost, rapid fabrication via laser ablation, supporting iterative design in target hypothesis testing.
- Predictive Value: Quantifies reactive oxygen species production and cell migration under defined stimuli, providing functional readouts for target de-risking.
Screening & Assay Development
- Scientific Value: Generates five concentration gradients with concurrent shear stress or electric field gradients, enabling multiparametric screening in a single device.
- Operational Value: Reduces experimental repeats by integrating multiple stimuli conditions, improving assay efficiency for compound or siRNA library screening.
- Scalability: Compatible with standard microscopy and incubation workflows, facilitating adoption in screening cascades.
Translational & Preclinical Research
- Translational Continuity: Uses lung cancer CL1-5 cells as a disease-relevant model to study microenvironment-driven phenotypes.
- Mechanistic De-risking: Isolates contributions of chemical, electrical, and mechanical stimuli to cellular responses, informing combination therapy strategies.
- Preclinical Alignment: Outputs such as migration and oxidative stress correlate with hallmarks relevant to metastasis and invasiveness.
Pipeline & Workflow Integration
The microfluidic chips function as a discovery-stage assay platform that bridges target validation and early lead screening by providing quantitative, stimulus-responsive cellular phenotypes.
- Discovery Biology: Supports hypothesis testing on how microenvironmental cues modulate cellular behavior in cancer and other disease models.
- Screening: Enables parallel testing of cellular responses across chemical concentration and physical stimulus gradients, increasing data density per experiment.
- Analytics: Generates measurable outputs including fluorescence-based ROS detection and time-lapse migration metrics for objective comparison.
- Translational Research: Uses a metastatic lung cancer line to link microenvironmental responses to disease-relevant invasiveness phenotypes.
- Enterprise Reuse: PMMA-based design allows replication across labs and projects for consistent stimulus delivery in mechanobiology or pharmacology studies.
Operational & Enterprise Impact
- Scientific Value: Reduces mechanistic ambiguity by enabling controlled application of single or coexisting stimuli to isolate variable contributions.
- Operational Value: Standardizes stimulus delivery through reproducible gradient generation, minimizing variability across replicates.
- Strategic Value: Improves go/no-go decisions by providing multiparametric efficacy and toxicity readouts under physiologically relevant conditions.
- Portfolio Impact: Supports risk-adjusted target prioritization by identifying compounds that modulate stimulus-specific cellular responses.
Implementation Considerations
- Requires expertise in microfluidic design, laser ablation of PMMA, and fluidic interface assembly.
- Depends on access to CO2 laser scriber, UV sterilization, and fluidic priming equipment such as syringes and tubing.
- Necessitates standardization of cell seeding, stimulus application timing, and readout normalization across teams.
- Adaptation to other cell types may require optimization of channel dimensions and flow rates to maintain shear stress and gradient integrity.
- Practical limitations include bubble formation during priming and electrode delamination in electric field setups, as noted in the protocol.
