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Automatic fabrication of 3-dimensional tissues using cell sheet manipulator technique

机译：使用细胞片操纵器技术自动制造3维组织

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摘要

Automated manufacturing is a key for tissue-engineered therapeutic products to become common-place and economical. Here, we developed an automatic cell sheet stacking apparatus to fabricate 3-dimensional tissue-engineered constructs exploiting our cell sheet manipulator technique, where cell sheets harvested from temperature-responsive culture dishes are stacked into a multilayered cell sheet. By optimizing the stacking conditions and cell seeding conditions, the apparatus was eventually capable of reproducibly making five-layer human skeletal muscle myoblast (HSMM) sheets with a thickness of approximately 70-80μm within 100min. Histological sections and confocal topographies of the five-layer HSMM sheets revealed a stratified structure with no delamination. In cell counts using trypsinization, the live cell numbers in one-, three- and five-layer HSMM sheets were equivalent to the seeded cell numbers at 1h after the stacking processes; however, after subsequent 5-day static cultures, the live cell numbers of the five-layered HSMM sheets decreased slightly, while one- and three-layer HSMM sheets maintained their live cell numbers. This suggests that there are thickness limitations in maintaining tissues in a static culture. We concluded that by combining our cell sheet manipulator technique and industrial robot technology we can create a secure, cost-effective manufacturing system able to produce tissue-engineered products from cell sheets.
机译：自动化制造是组织工程治疗产品变得司空见惯和经济的关键。在这里，我们开发了一种自动细胞片堆叠设备，以利用我们的细胞片操纵器技术来制造3维组织工程化的构建体，其中将从对温度敏感的培养皿中收获的细胞片堆叠到多层细胞片中。通过优化堆叠条件和细胞接种条件，该设备最终能够在100min内可重复制作厚度约70-80μm的五层人体骨骼肌成肌细胞（HSMM）片。五层HSMM片材的组织学切片和共聚焦形貌显示出分层结构，没有分层。在使用胰蛋白酶消化的细胞计数中，一层，三层和五层HSMM薄板中的活细胞数等于堆叠过程后1h的接种细胞数。然而，在随后的5天静态培养之后，五层HSMM薄板的活细胞数略有下降，而一层和三层HSMM薄板保持了活细胞数。这表明在静态培养中维持组织存在厚度限制。我们得出的结论是，通过将细胞片操纵器技术与工业机器人技术相结合，我们可以创建一个安全，具有成本效益的制造系统，该系统能够从细胞片中生产组织工程产品。

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