We used inclined lithography to fabricate a pneumatic microvalve for tall microchannels such as those used to convey large cells. The pneumatic microvalve consists of three layers. The upper layer is the actual liquid microchannel, which has a parallelogram-shaped cross section of width 500 μm, height 100 μm, and an acute angle of 53.6°. The lower layer is a pneumatic microchannel that functions as an actuator, and the middle layer is a thin polydimethylsiloxane membrane between the upper and lower layers. The operation of the pneumatic microchannel actuator causes the thin membrane to bend, resulting in the bending of the liquid microchannel and its closure. It was confirmed that the closure of the liquid microchannel completely stopped the flow of the HeLa cell suspension that was used to demonstrate the operation of the microvalve. The HeLa cells that passed through the microchannel were also observed to retain their proliferation and morphological properties.
Abstract:The uniform dispersion of cells in a microchamber is important to reproduce results in cellular research. However, achieving this is difficult owing to the laminar flow caused by the small dimensions of such a chamber. In this study, we propose a technique to achieve a uniform distribution of cells using a micropillar array inside a microchamber. The cells deform when they pass through a gap between the micropillars. The deformation causes a repetitive clog-and-release process of cells at the gaps between the micropillars. The micropillar array generates random flow inside the microchamber, resulting in the uniform distribution of the cells via cell accumulation. In the experiment, the distribution of cells in the microchamber with the micropillar array is uniform from end to end, whereas that in the microchamber without the micropillar array is centered. The deviation of the cell distribution from the ideally uniform distribution in the microchamber with the micropillar array is suppressed by 63% compared with that in the microchamber without the micropillar array. The doubling time of the cells passed through the micropillar array did not change relative to that of normal N87 cells. This technique will be helpful for reproducing results in cellular research at the micro scale or for those using microfluidic devices. OPEN ACCESSMicromachines 2015, 6 410
This paper proposes a microfluidic device for screening molecules such as aptamers, antibodies, proteins, etc. for target cell-specific binding molecules. The discovery of cancer cell-specific binding molecules was the goal of this study. Its functions include filtering non-target cell-binding molecules, trapping molecules on the surface of target cells, washing away unbound molecules, and collecting target cell-specific binding molecules from target cells. These functions were effectively implemented by using our previously developed micro pillar arrays for cell homogeneous dispersion and pneumatic microvalves for tall microchannels. The device was also equipped with serially connected filter chambers in which non-target cells were cultured to reduce the molecules binding to non-target cells as much as possible. We evaluated the performance of the device using cancer cell lines (N87 cells as target cells and HeLa cells as non-target cells) and two fluorescent dye-labeled antibodies: Anti-human epidermal growth factor receptor 2 (anti-HER2) antibody that binds to target cells and anti-integrin antibody that binds to non-target cells. The results showed that the device could reduce anti-integrin antibodies to the detection limit of fluorescent measurement and collect anti-HER2 antibodies from the target cells.
The small number of high-migratory cancer cells in a cell population make studies on high-migratory cancer cells difficult. For the development of migration assays for such cancer cells, several microfluidic devices have been developed. However, they measure migration that is influenced by microstructures and they collect not only high-migratory cells, but also surrounding cells. In order to find high-migratory cells in cell populations while suppressing artifacts and to collect these cells while minimizing damages, we developed a microfluidic high-migratory cell collector with the ability to sort cancer cells according to cellular migration and mechanical detachment. High-migratory cancer cells travel further from the starting line when all of the cells are seeded on the same starting line. The high-migratory cells are detached using a stretch of cell adhesive surface using a water-driven balloon actuator. Using this cell collector, we selected high-migratory HeLa cells that migrated about 100m in 12 h and collected the cells.
Simple microfluidic systems for handling large particles such as three-dimensional (3D) cultured cells, microcapsules, and animalcules have contributed to the advancement of biology. However, obtaining a highly integrated microfluidic device for handling large particles is difficult because there are no suitable microvalves for deep microchannels. Therefore, this study proposes a microvalve with a trapezoid-shaped cross-section to close a deep microchannel. The proposed microvalve can close a 350 μm deep microchannel, which is suitable for handling hundreds of micrometer-scale particles. A double-inclined lithography process was used to fabricate the trapezoid-shaped cross-section. The microvalve was fabricated by bonding three polydimethylsiloxane layers: a trapezoid-shaped liquid channel layer, a membrane, and a pneumatic channel layer. The pneumatic balloon, consisting of the membrane and the pneumatic channel, was located beneath a trapezoid-shaped cross-section microchannel. The valve was operated by the application of pneumatic pressure to the pneumatic channel. We experimentally confirmed that the expansion of the pneumatic balloon could close the 350 μm deep microchannel.
Peptides that can bind to normal cells should be removed in the screening of the tumor specific binding peptide. We developed a microfluidic device for screening of tumor specific binding peptide that has serially-connected chambers for normal cells and for cancer cells. The normal cell is to remove the non-specific binding peptides, and the cancer cell is to capture tumor specific binding peptides. Cell introduction into the chambers without cross-contamination and screening simulation using a fluorescence-labeled antibody are examined. In cell introduction experiment, contaminations of different types of cells were prevented. In the screening simulation, the trapped amount of the non-specific antibodies can be reduced to 46 % in the downstream chamber.
Cancer is the top leading cause of death in Japan. Anti-cancer drugs are one of the most popular treatments whereas they often have heavy side effects. Anti-cancer drugs with light or no side effects are under development using tumor specific binding peptides as guides to cancer cells. However, the number of candidate peptides for the tumor specific binding peptides are extremely large. An efficient peptide screening method is necessary to find them. For this purpose, we are developing a microfluidic device to uniformly disperse cancer cells into its micro chamber and screen the peptide form the large number of peptides. In this paper, we could uniformly disperse N87 cells into the micro chamber using a micro pillar array, and selectively trap yeasts with tumor specific binding peptides by N87 cells in the micro chamber.
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