Center for Biosystems Dynamics Research (BDR), RIKEN, 1-3 Yamadaoka, Suita, Osaka565-0871, Japan.
Graduate School of Science and Technology, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara630-0192, Japan.
Anal Chem. 2022 Nov 29;94(47):16299-16307. doi: 10.1021/acs.analchem.2c02815. Epub 2022 Nov 16.
Sophisticated functions of biological tissues are supported by small biological units of cells that are localized within a region of 100 μm scale. The cells in these units secrete molecules to form their microenvironment to play a vital role in biological functions. Various microfluidic devices have been developed to analyze the microenvironment but were not designed for cells in a culture dish in a confluent condition, a typical setup for cell and tissue cultivation. This study presents a novel glass capillary-based microfluidic device for studying confluent cells in a culture dish. The multiple capillaries allow the device to confine the local flow in 100 μm or smaller scale to form two adjacent regions with different chemical properties; it can simultaneously perform local cell stimulation and collect secreted molecules from the stimulated cells. Cell removal was achieved upon trypsin stimulation from a limited area (3.8 × 10 ± 1.0 × 10 mm), which corresponded to 7.6 ± 2.0 cells, using the mouse skeletal myoblast cell line (C2C12 cells) in a confluent condition. Microenvironmental analysis was demonstrated by measuring the secreted tumor necrosis factor alpha (TNF-α) collected from the microenvironment of the stimulated and unstimulated mouse leukemic monocyte cell line (RAW264 cells) to track temporal changes in the TNF-α production. The TNF-α secreted from stimulated cells was approximately four-fold higher than that from unstimulated cells in 90 min. This device enables local cell stimulation and the collection of secreted molecules for cells under confluent conditions, which contributes to the analysis of the cellular microenvironment.
生物组织的复杂功能是由位于 100μm 尺度范围内的细胞这一小生物单元支持的。这些单元中的细胞分泌分子以形成其微环境，从而在生物功能中发挥重要作用。已经开发出各种微流控设备来分析微环境，但这些设备不是为培养皿中处于汇合状态的细胞设计的，而汇合状态是细胞和组织培养的典型设置。本研究提出了一种用于研究培养皿中汇合细胞的新型基于玻璃毛细管的微流控装置。多个毛细管允许设备将局部流动限制在 100μm 或更小的尺度内，以形成具有不同化学性质的两个相邻区域；它可以同时对局部细胞进行刺激，并从受刺激的细胞中收集分泌的分子。使用处于汇合状态的小鼠骨骼肌成肌细胞系（C2C12 细胞），通过从有限区域（3.8×10±1.0×10mm）进行胰蛋白酶刺激，可以实现细胞去除，这对应于 7.6±2.0 个细胞。通过测量从受刺激和未受刺激的小鼠白血病单核细胞系（RAW264 细胞）的微环境中收集的分泌肿瘤坏死因子α（TNF-α）来进行微环境分析，以跟踪 TNF-α产生的时间变化。刺激细胞分泌的 TNF-α在 90 分钟内大约是未刺激细胞的四倍。该装置能够在汇合条件下对局部细胞进行刺激和收集分泌的分子，有助于分析细胞的微环境。
