title: Ultrastructure of blood and lymphatic vascular networks in three-dimensional cultured tissues fabricated by extracellular matrix nanofilm-based cell accumulation technique.
abstract: Cell accumulation technique is an extracellular matrix (ECM) nanofilm-based tissue-constructing method that enables formation of multilayered hybrid culture tissues. In this method, ECM-nanofilm is constructed using layer-by-layer assembly of fibronectin and gelatin on culture cells. The ECM-nanofilm promotes cell-to-cell interaction; then the three-dimensional (3D) multilayered tissue can be constructed with morphological change of the cells mimicking living tissues. By using this method, we have successfully produced tubular networks of human umbilical venous endothelial cells (HUVECs) and human dermal lymphatic endothelial cells (HDLECs) in 3D multilayered normal human dermal fibroblasts (NHDFs). This study demonstrated morphological characteristics of the vascular networks in the engineered tissues by using light and electron microscopy. In light microscopy, HUVECs and HDLECs formed luminal structures such as native blood and lymphatic capillaries, respectively. Electron microscopy showed distinct ultrastructural aspects of the vasculature of HUVECs or HDLECs with intermediated NHDFs and abundant ECM. The vasculature constructed by HUVECs exhibited structures similar to native blood capillaries, involving overlapping endothelial connections with adherens junctions, abundant vesicles in the endothelial cells and basement membrane-like structure. The detection of laminin around HUVEC-constructed vessels supported the presence of perivascular basal lamina. The vasculature constructed by HDLECs showed some ultrastructural characteristics similar to those of native lymphatic capillaries such as irregular vascular shape, loose adhesive connection and gap formation between endothelial cells. In conclusion, our novel vascular network models fabricated by the cell accumulation technique provide highly organized blood and lymphatic capillary networks mimicking the vasculatures in vivo.
authors.affiliation: Department of Neuroanatomy, Cell Biology and Histology, Hirosaki University Graduate School of Medicine, Hirosaki 036-8562, Japan asanoy@cc.hirosaki-u.ac.jp hshimoda@cc.hirosaki-u.ac.jp.
authors.forename: Yoshiya
authors.initials: Y
authors.lastname: Asano
authors.affiliation: Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamada-oka, 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 Yamada-oka, Suita, Osaka 565-0871, Japan.
authors.forename: Michiya
authors.initials: M
authors.lastname: Matsusaki
authors.affiliation: Department of Neuroanatomy, Cell Biology and Histology, Hirosaki University Graduate School of Medicine, Hirosaki 036-8562, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Okano
authors.affiliation: Department of Neuroanatomy, Cell Biology and Histology, Hirosaki University Graduate School of Medicine, Hirosaki 036-8562, Japan.
authors.forename: Erina
authors.initials: E
authors.lastname: Saito
authors.affiliation: Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamada-oka, Suita, Osaka 565-0871, Japan.
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Akashi
authors.affiliation: Department of Neuroanatomy, Cell Biology and Histology, Hirosaki University Graduate School of Medicine, Hirosaki 036-8562, Japan asanoy@cc.hirosaki-u.ac.jp hshimoda@cc.hirosaki-u.ac.jp.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Shimoda
mesh_terms: D001808:Blood Vessels / Q000166:cytology / Q000648:ultrastructure*; D002196:Capillaries / Q000166:cytology / Q000648:ultrastructure; D002450:Cell Communication; D005109:Extracellular Matrix / Q000648:ultrastructure*; D006801:Humans; D042601:Lymphatic Vessels / Q000166:cytology / Q000648:ultrastructure*; D008853:Microscopy; D046529:Microscopy, Electron, Transmission; D049329:Nanostructures; D046509:Tissue Culture Techniques / Q000379:methods*; D023822:Tissue Engineering / Q000379:methods
keywords: blood vessel; cell accumulation technique; electron microscopy; lymphatic vessel; tissue engineering; vascular network