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Production and Separation of Micrometer-size Extracellular Vesicles Using Microhole Arrays

  • Sakurada Kazuho
    Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology
  • Yasuda Takashi
    Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology

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Other Title
  • 微小孔アレイを用いたミクロンサイズ細胞外ベシクルの生成分離
  • ビショウコウ アレイ オ モチイタ ミクロンサイズ サイボウ ガイ ベシクル ノ セイセイ ブンリ

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Abstract

<p>We developed a technique for producing and separating micrometer-size vesicles from human lymphocytes using microholes. Using cell-anchoring molecules, lymphocytes were immobilized on a microhole array formed in a silicon nitride (SiN) membrane. Then, the cells were chemically stimulated with sodium butyrate, and the vesicle production was induced near the microholes with the assistance of the cell-anchoring molecules. This resulted in the vesicle production inside the microholes, and the vesicles finally passed through the microholes. We tested microholes having 3 different diameters of 2, 4, and 7.5 µm, and counted the number of fluorescently-stained vesicles and cells which passed through microholes of each diameter. The number of vesicles increased with increasing diameter of microholes, but the number of invading cells also increased. In case of microholes 4 µm in diameter, a relatively large number of vesicles were obtained, and the cell invasion rate was below 1%, which was the best result in the present experiment. Also, we succeeded in drastically increasing the number of vesicles in case of microholes 2 µm in diameter by preventing vesicles from adhering to the back side of a SiN membrane.</p>

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