Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University (TWIns), 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan; CellSeed Incorporated, Katsura-Bldg. 4F, 3-61, Haramachi, Shinjuku-ku, Tokyo 162-0053, Japan.
Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University (TWIns), 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan.
Biomaterials. 2014 Mar;35(8):2428-35. doi: 10.1016/j.biomaterials.2013.12.014. Epub 2013 Dec 23.
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 70-80 μm within 100 min. 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 1 h 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.
自动化制造是使组织工程治疗产品变得常见且经济实惠的关键。在这里，我们开发了一种自动细胞片层堆叠设备，利用我们的细胞片层操纵技术来制造三维组织工程构建体，其中从温度响应培养皿收获的细胞片层被堆叠成多层细胞片层。通过优化堆叠条件和细胞接种条件，该设备最终能够在 100 分钟内可重复地制造出具有约 70-80μm 厚度的五层人骨骼肌成肌细胞（HSMM）片层。五层 HSMM 片层的组织学切片和共聚焦形貌显示出具有分层结构而无分层的结构。通过胰蛋白酶消化进行细胞计数，在堆叠过程后 1 小时，单层、三层和五层 HSMM 片层中的活细胞数与接种细胞数相当；然而，在随后的 5 天静态培养后，五层 HSMM 片层中的活细胞数略有减少，而单层和三层 HSMM 片层保持其活细胞数。这表明在静态培养中维持组织存在厚度限制。我们得出结论，通过结合我们的细胞片层操纵技术和工业机器人技术，我们可以创建一个安全、具有成本效益的制造系统，能够从细胞片层生产组织工程产品。
