Design of prevascularized three-dimensional cell-dense tissues using a cell sheet stacking manipulation technology Tadashi Sasagawa a , Tatsuya Shimizu a , Sachiko Sekiya a , Yuji Haraguchi a , Masayuki Yamato a , Yoshiki Sawa b , Teruo Okano a, * a Institute of Advanced Biomedical Engineering and Science, Tokyo Woman’s Medical University, TWIns, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan b Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan article info Article history: Received 16 October 2009 Accepted 16 November 2009 Available online 3 December 2009 Keywords: Cell sheet Endothelial cell Co-culture Prevascularization Functional anastomosis abstract To survive three-dimensional (3-D) cell-dense thick tissues after transplantation, the improvements of hypoxia, nutrient insufficiency, and accumulation of waste products are required. This study presents a strategy for the initiation of prevascular networks in a 3-D tissue construct by sandwiching endothelial cells between the cell sheets. For obtaining a stable stacked cell sheet construct, a sophisticated 3-D cell sheet manipulation system using temperature-responsive culture dishes and a cell sheet manipulator was developed. When sparsely cultured human umbilical vein endothelial cells (HUVECs) were sand- wiched between two myoblast sheets, the inserted HUVECs sprouted and formed network structures in vitro. Additionally, when myoblast sheets and HUVECs were alternately sandwiched, endothelial cell connections through the layers and capillary-like structures were found in a five-layer construct. Moreover, the endothelial networks in the five-layer myoblast sheet construct were observed to connect to the host vessels after transplantation into the subcutaneous tissues of nude rats, resulted in a neo- vascularization that allow the graft to survive. These results indicated that the prevascularized myoblast sheet constructs could induce functional anastomosis. Consequently, our prevascularizing method using a cell sheet stacking manipulation technology provides a substantial advance for developing various types of three-dimensional tissues and contributes to regenerative medicine. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction Tissue engineering aims to replace or restore the function of damaged or diseased tissues [1]. Engineered 3-D tissue constructs were prepared by seeding cells into pre-made porous scaffolds made from biodegradable polymers as substitute for extracellular matrix (ECM) that is essential for forming tissues. Subsequently, these constructs can be transplanted into patient for restoring or improving the original tissue function. However, insufficient cell migration into the scaffolds and host inflammatory reactions by scaffold degradation give problems. In contrast to the conventional scaffold-based tissue engi- neering, a cell sheet-based tissue engineering has been developed for transplanting cell sheets to treat damaged tissues and recon- structing 3-D multi-layer cell-dense tissues without scaffolds [2–4]. To obtain viable cultured cell sheets, a temperature-responsive culture dish covalently grafted with a temperature-responsive polymer, poly (N-isopropylacrylamide) [5,6] is used. A single cell adheres and proliferates at 37  C in the dish, and the multiplying cells spontaneously detach themselves when temperature is reduced below 32  C without any enzymatic digestion. Therefore, confluent cultured cells are noninvasively harvested as a contig- uous cell sheet with intact cell-to-cell connections by lowering the culture temperature. Additionally, due to the presence of intrinsic ECM that was produced during in vitro culture [7], the harvested cell sheet can easily re-attach to other surfaces such as culture dishes, cell sheets, and host tissues [8–13]. On the other hand, the most successes in tissue engineering have been limited to avascular tissues and thin tissues that can survive through the supply of oxygen and nutrients by simple diffusion without an additional vascular supply, such as cartilage, skin, and large-scale vasculature [14–17]. Vascular capillaries precisely organize themselves throughout nearly all tissues and support normal organ functions via the supply of oxygen and nutrients as well as the removal of various metabolic wastes. In living tissues, cells rely on a capillary network within a perimeter of 100–150 mm [18]. In the case of cultured cell aggregates in vitro, oxygen transport is limited to a diffusion distance of 150–200 mm * Corresponding author. Tel.: þ81 3 5367 9945x6201; fax: þ81 3 3358 6046. E-mail address: tokano@abmes.twmu.ac.jp (T. Okano). Contents lists available at ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials 0142-9612/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2009.11.036 Biomaterials 31 (2010) 1646–1654