ORIGINAL ARTICLE ‘‘Deep-media culture condition’’ promoted lumen formation of endothelial cells within engineered three-dimensional tissues in vitro Sachiko Sekiya • Tatsuya Shimizu • Masayuki Yamato • Teruo Okano Received: 29 March 2010 / Accepted: 24 November 2010 / Published online: 2 February 2011 Ó The Japanese Society for Artificial Organs 2011 Abstract In the field of tissue engineering, the induction of microvessels into tissues is an important task because of the need to overcome diffusion limitations of oxygen and nutrients within tissues. Powerful methods to create vessels in engineered tissues are needed for creating real living tissues. In this study, we utilized three-dimensional (3D) highly cell dense tissues fabricated by cell sheet technol- ogy. The 3D tissue constructs are close to living-cell dense tissue in vivo. Additionally, creating an endothelial cell (EC) network within tissues promoted neovascularization promptly within the tissue after transplantation in vivo. Compared to the conditions in vivo, however, common in vitro cell culture conditions provide a poor environment for creating lumens within 3D tissue constructs. Therefore, for determining adequate conditions for vascularizing engi- neered tissue in vitro, our 3D tissue constructs were cul- tured under a ‘‘deep-media culture conditions.’’ Compared to the control conditions, the morphology of ECs showed a visibly strained cytoskeleton, and the density of lumen formation within tissues increased under hydrostatic pres- sure conditions. Moreover, the increasing expression of vascular endothelial cadherin in the lumens suggested that the vessels were stabilized in the stimulated tissues com- pared with the control. These findings suggested that deep- media culture conditions improved lumen formation in engineered tissues in vitro. Keywords Cell sheet engineering  Endothelial cell-network  Tissue engineering  Hydrostatic pressure  Simulating in vivo conditions Abbreviations 3D Three-dimensional EC Endothelial cell SMC Smooth muscle cell Introduction Tissue engineering technology produces artificial organs fabricated with cells and provides many benefits for regenerative medicine, including corneal and vesicular regeneration [1, 2]. The aim of tissue engineering is the fabrication of various kinds of functional biocompatible artificial organs. The conventional techniques for tissue engineering always employ scaffolds composed of biode- gradable polymers where living cells can be seeded [3, 4]. While these approaches using scaffolds allow cartilage and other tissues to be reconstructed, they are unfit for fabricating highly cell dense tissues that have complex structures, such as the heart and liver. Therefore, a novel cell-sheet technology has been utilized to reconstruct highly cell dense structures. The cell-sheet technology allows harvesting cells as a single intact sheet from tem- perature-responsive culture dishes by simply reducing the culture temperature without protease [5]. Since adhesive proteins and the extracellular matrix (ECM) are preserved on the basal side of the cell sheets, three-dimensional (3D) tissue-like structures can be reconstructed by simply layering these cell sheets [6, 7]. S. Sekiya (&)  T. Shimizu  M. Yamato  T. Okano (&) Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan e-mail: sgoto@abmes.twmu.ac.jp T. Okano e-mail: tokano@abmes.twmu.ac.jp 123 J Artif Organs (2011) 14:43–51 DOI 10.1007/s10047-011-0553-0