使用高通量微流体设备生成动态环境条件
Summary
我们介绍了一个用于复杂生命机械高吞吐量研究的微流体系统，该系统由 1500 个培养单元、一系列增强的永久泵和现场混合模组组成。微流体芯片可分析体内高度复杂和动态的微环境条件。
Abstract
模仿体内环境条件对于复杂生命机械的体外研究至关重要。然而，目前针对活细胞和器官的技术要么非常昂贵，比如机器人技术，要么在液体操作中缺乏纳米线体积和毫秒时间精度。我们介绍一个微型流体系统的设计和制造，该系统由 1，500 个培养单元、一系列增强的永久泵和现场混合模组组成。为了证明微流体装置的能力，神经干细胞（NSC）球体在拟议的系统中保持。我们观察到，当 NSC 球体在第 1 天接触 CXCL，在第 2 天接触 EGF 时，圆形构象维护良好。6 种药物输入顺序的变化导致 NSC 球体的形态变化和 NSC 茎的表达水平代表标记（即 Hes5 和 Dcx）。结果表明，动态复杂的环境条件对NSC分化和自我更新有重大影响，建议的微流体装置是复杂生命机械高吞吐量研究的适宜平台。
Introduction
高吞吐量技术对生物医学和临床研究至关重要。通过同时进行数百万次化学、遗传或活细胞和器官测试，研究人员可以快速识别调节生物分子通路的基因，并根据自己的特定需求定制顺序药物输入。机器人1和微流体芯片与设备控制程序相结合，允许复杂的实验程序自动化，包括细胞/组织操作，液体处理，成像和数据处理/控制2，3。因此，根据所需的吞吐量4，5，可以在一个芯片上维持成百上千的实验条件。
在此协议中，我们描述了微流体装置的设计和制造过程，该装置由 1500 个培养单元、一系列增强的围流泵和现场混合模组组成。二级细胞培养室可防止在中等交换过程中不必要的剪切，从而确保长期活细胞成像不受干扰的文化环境。研究表明，拟议中的微流体装置是研究复杂生命机械的高吞吐量的合适平台。此外，微流体芯片的先进功能允许在体内自动重组高度复杂和动态的微环境条件，如不断变化的细胞因子和配体组成6，7，其完成需要几个月的传统平台，如96井板。
Protocol
1. 微流体芯片设计 设计由 18 个入口组成的微流体多路复用器，每个入口都由单个阀门和永久泵控制。为了增加每个泵周期驱动的液体体积，由 3 个控制通道（特意加宽至 200 μm）和 10 条连接的流线组成。 设计无剪切文化室。二级培养单元的复制由较低细胞培养室（400μm x 400 μm x 150 μm）和较高的缓冲层（400μm x 400 μm x 75 μm）组成，可防止在中等交换期间对细胞产生不必要的剪切?…
Representative Results
传统的芯片上渗透泵最初由斯蒂芬·奎克在2000年描述，使用这种渗透是由模式101，100，110，010，011，0018，10激活的。 数字 0 和 1 表示 3 个水平控制线的”打开”和”关闭”。使用超过3个阀门（例如，5个）的研究也报告了11个。尽管由 3 条控制线和 3 条流线组成的永久泵提供了纳米线精度，但传输速度太慢，无法养活 1，500 个培养室。为了?…
Discussion
各种微流体装置已开发出来，以执行多路复用和复杂的实验17，18，19，20。例如，由一系列拓扑凹槽制成的微井可以捕获单个细胞，而无需使用外部力，表现出有利的特性，包括样本量小、平行、材料成本更低、响应更快、灵敏度高 21、22、23、24。<…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢陈氏仪器（中国）有限公司程志峰的技术支持。这项工作得到了资助（中国国家自然科学基金委员会，51927804）。
Materials
| 2713 Loker Avenue West | Torrey pines scientific | ||
| AZ-50X | AZ Electronic Materials, Luxembourg | ||
| Chlorotrimethylsilane(TMCS) 92360-25mL | Sigma | ||
| CO2 Incubator HP151 | Heal Force | ||
| Desktop Hole Puncher for PDMS chips WH-CF-14 | Suzhou Wenhao Microfluidic Technology Co., Ltd. | ||
| DMEM(L-glutamine, High Glucose, henol Red) | Invitrogen | ||
| Electronic Balance UTP-313 Max:600g, e:0.1g, d:0.01g | Shanghai Hochoice Apparatus Manufacturer Co.,LTD. | ||
| FBS | Sigma | ||
| Fibronection 0.25 mg/mL | Millipore, Austria | ||
| Glutamax 100x | Gibco | ||
| Heating Incubator BGG-9240A | Shanghai bluepard instruments Co.,Ltd. | ||
| Nikon Model Eclipse Ti2-E | Nikon | ||
| Pen/Strep 10 Units/mL Penicillin 10 ug/mL Streptomycin | Invitrogen | ||
| Plasma cleaner PDC-002 | Harrick Plasma | ||
| polydimethylsiloxane(PDMS) | Momentive | ||
| polylysine 0.01% | Sigma | ||
| Spin coater ARE-310 | Awatori Rentaro | ||
| Spin coater TDZ5-WS | Cence | ||
| Spin coater WH-SC-01 | Suzhou Wenhao Microfluidic Technology Co., Ltd. | ||
| SU-8 3025 | MicroChem, Westborough, MA, USA | ||
| SU-8 3075 | MicroChem, Westborough, MA, USA |
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Cite This Article
Che, B., Zhu, J., Sun, D., Feng, X., Zhang, C. Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device. J. Vis. Exp. (170), e61735, doi:10.3791/61735 (2021).