title: Development of vascularized iPSC derived 3D-cardiomyocyte tissues by filtration Layer-by-Layer technique and their application for pharmaceutical assays.
abstract: In vitro development of three-dimensional (3D) human cardiomyocyte (CM) tissues derived from human induced pluripotent stem cells (iPSCs) has long been desired in tissue regeneration and pharmaceutical assays. In particular, in vitro construction of 3D-iPSC-CM tissues with blood capillary networks have attracted much attention because blood capillaries are crucial for nutrient and oxygen supplies for CMs. Blood capillaries in 3D-iPSC-CM tissues will also be important for in vitro toxicity assay of prodrugs because of the signaling interaction between cardiomyocytes and endothelial cells. Here, we report construction of vascularized 3D-iPSC-CM tissues by a newly-discovered filtration-Layer-by-Layer (LbL) technique for cells, instead of our previous centrifugation-LbL technique. The filtration-LbL allowed us to fabricate nanometer-sized extracellular matrices (ECM), fibronectin and gelatin (FN-G), films onto iPSC-CM surfaces without any damage and with high yield, although centrifugation-LbL induced physical stress and a lower yield. The fabricated FN-G nanofilms interacted with integrin molecules on the cell membrane to construct 3D-tissues. We found that the introduction of normal human cardiac fibroblasts (NHCFs) into the iPSC-CM tissues modulated organization and synchronous beating depending on NHCF ratios. Moreover, co-culture with normal human cardiac microvascular endothelial cells (NHCMECs) successfully provided blood capillary-like networks in 3D-iPSC-CM tissues, depending on NHCF ratios. The vascularized 3D-iPSC-CM tissues indicated significantly different toxicity responses as compared to 2D-iPSC-CM cells by addition of doxorubicin as a model of a toxic drug. The constructed vascularized 3D-iPSC-CM tissues would be a promising tool for tissue regeneration and drug development.
  
  
  CONCLUSIONS
  In vitro fabrication of vascularized three-dimensional (3D) human cardiomyocyte (CM) tissues derived from human induced pluripotent stem cells (iPSCs) has attracted much attention owing to their requirement of much amount of nutrition and oxygen, but not yet published. In this manuscript, we report construction of vascularized 3D-iPSC-CM tissues by a newly-discovered filtration-Layer-by-Layer (LbL) technique. The filtration-LbL fabricates nanometer-sized fibronectin and gelatin (FN-G) films onto iPSC-CM surfaces. The FN-G nanofilms induce cell-cell interactions via integrin molecules on cell surfaces, leading to construction of 3D-tissues. The constructed vascularized 3D-iPSC-CM tissues would be a promising tool for tissue regeneration and drug development. We believe that this manuscript has a strong impact and offers important suggestions to researchers concerned with biomaterials and tissue engineering.
authors.affiliation: Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Yuto
authors.initials: Y
authors.lastname: Amano
authors.affiliation: Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Akihiro
authors.initials: A
authors.lastname: Nishiguchi
authors.affiliation: Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Michiya
authors.initials: M
authors.lastname: Matsusaki
authors.affiliation: Department of Cardiovascular Surgery, Graduate School of Medicine E1, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Hiroko
authors.initials: H
authors.lastname: Iseoka
authors.affiliation: Department of Cardiovascular Surgery, Graduate School of Medicine E1, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Shigeru
authors.initials: S
authors.lastname: Miyagawa
authors.affiliation: Department of Cardiovascular Surgery, Graduate School of Medicine E1, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Sawa
authors.affiliation: Advanced Technology Research & Development Center, Ricoh Institute of Technology, RICOH Company Ltd., 16-1 Shinei-cho, Tsuzuki-ku, Yokohama, Kanagawa 224-0035, Japan.
authors.forename: Manabu
authors.initials: M
authors.lastname: Seo
authors.affiliation: Advanced Technology Research & Development Center, Ricoh Institute of Technology, RICOH Company Ltd., 16-1 Shinei-cho, Tsuzuki-ku, Yokohama, Kanagawa 224-0035, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Yamaguchi
authors.affiliation: Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; Building Block Science Joint Research Chair, Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address: akashi@chem.eng.osaka-u.ac.jp.
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Akashi
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002452:Cell Count; D002498:Centrifugation; D004317:Doxorubicin / Q000494:pharmacology*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects; D005374:Filtration / Q000379:methods*; D005434:Flow Cytometry; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D018613:Microscopy, Confocal; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000187:drug effects; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D051381:Rats; D023822:Tissue Engineering / Q000379:methods*
chemical_list: D004317:Doxorubicin
keywords: 3D-tissues; Cardiomyocyte; Drug development; Layer-by-Layer assembly; Tissue engineering; iPS cells