title: Controlled Growth and the Maintenance of Human Pluripotent Stem Cells by Cultivation with Defined Medium on Extracellular Matrix-Coated Micropatterned Dishes.
abstract: Here, we introduce a new serum-free defined medium (SPM) that supports the cultivation of human pluripotent stem cells (hPSCs) on recombinant human vitronectin-N (rhVNT-N)-coated dishes after seeding with either cell clumps or single cells. With this system, there was no need for an intervening sequential adaptation process after moving hPSCs from feeder layer-dependent conditions. We also introduce a micropatterned dish that was coated with extracellular matrix by photolithographic technology. This procedure allowed the cultivation of hPSCs on 199 individual rhVNT-N-coated small round spots (1 mm in diameter) on each 35-mm polystyrene dish (termed "patterned culture"), permitting the simultaneous formation of 199 uniform high-density small-sized colonies. This culture system supported controlled cell growth and maintenance of undifferentiated hPSCs better than dishes in which the entire surface was coated with rhVNT-N (termed "non-patterned cultures"). Non-patterned cultures produced variable, unrestricted cell proliferation with non-uniform cell growth and uneven densities in which we observed downregulated expression of some self-renewal-related markers. Comparative flow cytometric studies of the expression of pluripotency-related molecules SSEA-3 and TRA-1-60 in hPSCs from non-patterned cultures and patterned cultures supported this concept. Patterned cultures of hPSCs allowed sequential visual inspection of every hPSC colony, giving an address and number in patterned culture dishes. Several spots could be sampled for quality control tests of production batches, thereby permitting the monitoring of hPSCs in a single culture dish. Our new patterned culture system utilizing photolithography provides a robust, reproducible and controllable cell culture system and demonstrates technological advantages for the mass production of hPSCs with process quality control.
authors.affiliation: R&D Center for Cell Therapy, Foundation for Biomedical Research and Innovation, TRI#308, 1-5-4 Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo 650-0043, Japan.
authors.forename: Chiemi
authors.initials: C
authors.lastname: Takenaka
authors.affiliation: Product Development Division, Kyokuto Pharmaceutical Industrial Co., Ltd., 3333-26, Aza-Asayama, Kamitezuna Takahagi-shi, Ibaraki, 318-0004, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Miyajima
authors.affiliation: Technology Development Center, Tokyo Electron Limited, Akasaka Biz Tower, 5-3-1 Akasaka, Minato-Ku, Tokyo, 107-6325, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Yoda
authors.affiliation: R&D Center for Cell Therapy, Foundation for Biomedical Research and Innovation, TRI#308, 1-5-4 Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo 650-0043, Japan.
authors.forename: Hideo
authors.initials: H
authors.lastname: Imazato
authors.affiliation: R&D Center for Cell Therapy, Foundation for Biomedical Research and Innovation, TRI#308, 1-5-4 Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo 650-0043, Japan.
authors.forename: Takako
authors.initials: T
authors.lastname: Yamamoto
authors.affiliation: Technology Development Center, Tokyo Electron Limited, Akasaka Biz Tower, 5-3-1 Akasaka, Minato-Ku, Tokyo, 107-6325, Japan.
authors.forename: Shinichi
authors.initials: S
authors.lastname: Gomi
authors.affiliation: Technology Development Center, Tokyo Electron Limited, Akasaka Biz Tower, 5-3-1 Akasaka, Minato-Ku, Tokyo, 107-6325, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Ohshima
authors.affiliation: Technology Development Center, Tokyo Electron Limited, Akasaka Biz Tower, 5-3-1 Akasaka, Minato-Ku, Tokyo, 107-6325, Japan.
authors.forename: Kenichi
authors.initials: K
authors.lastname: Kagawa
authors.affiliation: Product Development Division, Kyokuto Pharmaceutical Industrial Co., Ltd., 3333-26, Aza-Asayama, Kamitezuna Takahagi-shi, Ibaraki, 318-0004, Japan.
authors.forename: Tetsuji
authors.initials: T
authors.lastname: Sasaki
authors.affiliation: R&D Center for Cell Therapy, Foundation for Biomedical Research and Innovation, TRI#308, 1-5-4 Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo 650-0043, Japan.
authors.forename: Shin
authors.initials: S
authors.lastname: Kawamata
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods; D002454:Cell Differentiation; D002460:Cell Line; D049109:Cell Proliferation; D000066673:Cell Self Renewal; D015925:Cryopreservation; D003470:Culture Media; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D061252:Feeder Cells; D042822:Genomic Instability; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism; D007621:Karyotyping; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism
chemical_list: D003470:Culture Media