title: Nano-on-micro fibrous extracellular matrices for scalable expansion of human ES/iPS cells.
abstract: Human pluripotent stem cells (hPSCs) hold great potential for industrial and clinical applications. Clinical-grade scaffolds and high-quality hPSCs are required for cell expansion as well as easy handling and manipulation of the products. Current hPSC culture methods do not fulfill these requirements because of a lack of proper extracellular matrices (ECMs) and cell culture wares. We developed a layered nano-on-micro fibrous cellular matrix mimicking ECM, named "fiber-on-fiber (FF)" matrix, which enables easy handling and manipulation of cultured cells. While non-woven sheets of cellulose and polyglycolic acid were used as a microfiber layer facilitating mechanical stability, electrospun gelatin nanofibers were crosslinked on the microfiber layer, generating a mesh structure with connected nanofibers facilitating cell adhesion and growth. Our results showed that the FF matrix supports effective hPSC culture with maintenance of their pluripotency and normal chromosomes over two months, as well as effective scaled-up expansion, with fold increases of 54.1 ± 15.6 and 40.4 ± 8.4 in cell number per week for H1 human embryonic stem cells and 253G1 human induced pluripotent stem cells, respectively. This simple approach to mimick the ECM may have important implications after further optimization to generate lineage-specific products.
authors.affiliation: Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto, 606-8501, Japan.
authors.forename: Li
authors.initials: L
authors.lastname: Liu
authors.affiliation: Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto, 606-8501, Japan. Electronic address: kkamei@icems.kyoto-u.ac.jp.
authors.forename: Ken-Ichiro
authors.initials: KI
authors.lastname: Kamei
authors.affiliation: Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto, 606-8501, Japan.
authors.forename: Momoko
authors.initials: M
authors.lastname: Yoshioka
authors.affiliation: Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto, 606-8501, Japan.
authors.forename: Minako
authors.initials: M
authors.lastname: Nakajima
authors.affiliation: Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto, 606-8501, Japan.
authors.forename: Junjun
authors.initials: J
authors.lastname: Li
authors.affiliation: Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto, 606-8501, Japan; Department of Micro Engineering, Graduate School of Engineering, Kyoto University, Kyotodaigaku-katsura, Nishikyo-ku, Kyoto, 615-8540, Japan.
authors.forename: Nanae
authors.initials: N
authors.lastname: Fujimoto
authors.affiliation: Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto, 606-8501, Japan.
authors.forename: Shiho
authors.initials: S
authors.lastname: Terada
authors.affiliation: Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto, 606-8501, Japan.
authors.forename: Yumie
authors.initials: Y
authors.lastname: Tokunaga
authors.affiliation: Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto, 606-8501, Japan.
authors.forename: Yoshie
authors.initials: Y
authors.lastname: Koyama
authors.affiliation: QOL Research Center, Gunze Limited, Kyoto, 623-8512 Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Sato
authors.affiliation: Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto, 606-8501, Japan; Institute for Stem Cell Biology and Regenerative Medicine (inStem), National Centre for Biological Sciences (NCBS), Bangalore, 560065, India.
authors.forename: Kouichi
authors.initials: K
authors.lastname: Hasegawa
authors.affiliation: Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto, 606-8501, Japan; Institute for Frontier Medical Sciences, Kyoto University, Kyoto, 606-8507, Japan.
authors.forename: Norio
authors.initials: N
authors.lastname: Nakatsuji
authors.affiliation: Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto, 606-8501, Japan; Ecole Normale Supérieure, CNRS-ENS-UPMC UMR 8640, 24 Rue Lhomond, Paris, 75005, France. Electronic address: yong.chen@ens.fr.
authors.forename: Yong
authors.initials: Y
authors.lastname: Chen
mesh_terms: D061249:Batch Cell Culture Techniques / Q000379:methods*; D040761:Biomimetic Materials / Q000737:chemistry; D002454:Cell Differentiation / Q000502:physiology; D049109:Cell Proliferation / Q000502:physiology; D002478:Cells, Cultured; D005109:Extracellular Matrix / Q000737:chemistry*; D000066449:Human Embryonic Stem Cells / Q000166:cytology / Q000502:physiology*; D006801:Humans; D057139:Nanofibers / Q000737:chemistry* / Q000648:ultrastructure; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000502:physiology*; D023822:Tissue Engineering / Q000295:instrumentation / Q000379:methods*; D054457:Tissue Scaffolds
keywords: Extracellular matrix; Human pluripotent stem cells; Nanofiber; Scaled-up culture; Self-renewal