Short Communication Three-dimensional cell-dense constructs containing endothelial cell-networks are an effective tool for in vivo and in vitro vascular biology research Sachiko Sekiya, Megumi Muraoka, Tadashi Sasagawa, Tatsuya Shimizu, Masayuki Yamato, Teruo Okano ⁎ Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan abstract article info Article history: Accepted 2 August 2010 Available online 6 August 2010 Keywords: Angiogenesis Temperature responsive culture dish EC-networks Angiogenesis is a complicated natural process, and understanding the mechanism by which it occurs is important for medical, pharmaceutical, and cell biological sciences. Many techniques for investigating angiogenesis have been reported. In this study, we introduced a novel application of a cell culture technique that can be used in in vitro and in vivo vascular biology research. Cultivated endothelial cells (ECs) were harvested from temperature responsive culture dishes by reducing the temperature, without the need for a proteinase treatment. For this technique, the direct contact of ECs with fibroblasts was important for the formation of a capillary-like network in vitro. Moreover, layered cell sheets containing EC-networks produced lumen and vascular structures in the three-dimensional constructs, as well as in the construct transplanted into a living body. Thus, our culture technique was able to create cell sheets and three-dimensional constructs containing EC- networks, because they preserved normal and intrinsic cell-cell direct contact and various cell adhesive factors. Moreover, the thickness of these three-dimensional (3-D) constructs could be controlled by the number of layered cell sheets. These observations indicated that our novel technology contributed to the progress of vascular biology and lead to a new tool that can be used in in vivo and in vitro vascular biology research. © 2010 Elsevier Inc. All rights reserved. Introduction There are many known angiogenesis-related diseases, and many techniques for investigating angiogenesis in vitro have been reported (Montanez et al., 2002; Bishop et al., 1999; Donovan et al., 2001). In vascular biology research, observing the interactions between endothelial cells (ECs) and other cells is crucial, because EC-pericyte, or vascular mural cell, interactions have an important role in angio- genesis and vascular maturation (Gaengel et al., 2009; Yana et al., 2007). Therefore, this study introduced a novel tool for developing 3-D constructs with cell–cell interactions to be used for vascular biology research. The constructs were created using “cell sheet engineering” technology. Using cell sheet technology, confluent cells cultivated on a temperature responsive dish can be harvested as a “cell-sheet,” which can then be layered for fabricating 3-D tissue- like constructs (Shimizu et al., 2002). In this study, 3-D constructs containing EC-networks were created by cell sheet technology and their potential to be used as a tool for vascular biology research in vitro and in vivo was examined. Materials and methods Cell culture and cell manipulation All cells were purchased from Lonza (Basel, Switzerland) and cultured according to the manufacturer's instructions. To prepare a fibroblast (FB) cell sheet, FBs were seeded onto a temperature- responsive culture dish (UpCell®, CellSeed, Tokyo, Japan) at a density of 9×10 4 cells/cm 2 and cultured at 37 °C until they reached confluence. To fabricate an EC-network sheet, FB cells, ECs, and smooth muscle cells were co-cultured at a density of 1.2 × 10 5 cells/ cm 2 (the ratio of cells was FB:EC:smooth muscle cells = 10:1:1) with MCDB131 culture medium (Sigma-Aldrich, St. Louis, MO) containing 10 ng/mL recombinant human vascular endothelial cell growth factor (rhVEGF) (Thermo Fisher Scientific, Waltham, MA, USA) on a temperature-responsive culture dish until they reached confluence. To sandwich ECs between two collagen-gel sheets, or two FB sheets, 0.5 × 10 4 ECs were seeded onto a temperature-responsive culture dish, which was incubated for 3 days with EGM2. A hydrogel-coated plunger was placed on the surface of the confluent cells with the plunger-guiding cover (Sasagawa et al., 2010), and the dish contain- ing the plunger was incubated at 20 °C for harvesting. The ECs were harvested with the hydro-gel and transferred onto a type-I collagen- gel sheet (2.7 mg/mL) (Nitta gelatin, Osaka, Japan) or a FB sheet. Then, Microvascular Research 80 (2010) 549–551 ⁎ Corresponding author. 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. Fax: +81 3 3359 6046. E-mail address: tokano@abmes.twmu.ac.jp (T. Okano). 0026-2862/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.mvr.2010.08.001 Contents lists available at ScienceDirect Microvascular Research journal homepage: www.elsevier.com/locate/ymvre