title: Automatic fabrication of 3-dimensional tissues using cell sheet manipulator technique.
abstract: 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.
authors.affiliation: 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.
authors.forename: Tetsutaro
authors.initials: T
authors.lastname: Kikuchi
authors.affiliation: Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University (TWIns), 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Shimizu
authors.affiliation: ABLE Corporation, 7-9, Nishigoken-cho, Shinjyuku-ku, Tokyo 162-0812, Japan.
authors.forename: Masanori
authors.initials: M
authors.lastname: Wada
authors.affiliation: Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University (TWIns), 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Yamato
authors.affiliation: Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University (TWIns), 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. Electronic address: tokano@abmes.twmu.ac.jp.
authors.forename: Teruo
authors.initials: T
authors.lastname: Okano
mesh_terms: D057205:Automation, Laboratory; D002478:Cells, Cultured; D006801:Humans; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism; D032448:Myoblasts, Skeletal / Q000378:metabolism*; D013696:Temperature; D023822:Tissue Engineering / Q000379:methods*; D054457:Tissue Scaffolds / Q000737:chemistry*
keywords: 3-D fabrication; Gelatin; Myoblast; Sheet; Thermally responsive material