The Intelligent Surface Group

Development of Next-Generation Temperature-Responsive Culture Substrates for New Cell-Sheet-Based Regenerative Medicine

 In September 2008, CellSeed Inc. began globally marketing a product named UpCell®, which is based on temperature-responsive culture dishes developed by Tokyo Women's Medical University. This first generation technology allows cultured cells to be harvested as sheet-like cell monolayer"cell sheet", requiring only low-temperature treatment. The cell-sheet-based regenerative medicine has already succeeded in regeneration of the corneal epithelium, esophagus, myocardium and periodontal ligament. We are now developing the next generation of temperature-responsive culture substrates for advanced cell sheet technology.

 To prepare cell sheets comprising various cell types for future regenerative medicine, temperature-responsive surfaces need to be designed on-demand for each cell type. To date, we have succeeded in producing a large variety of temperature-responsive surface technologies customized for specific purposes. These technologies include polymer grafting by electron beam irradiation, polymer brush production by living radical polymerization, and simple polymer coating using only spin coating. Currently we are developing a temperature-responsive culture substrate embedded with a mechanism to biochemically capture a cell by specific interactions with biomolecules introduced to polymers grafted on surface.

 We also have achieved micro-patterning on the surface of temperature-responsive substrates by adeptly utilizing micro-processing technology. Several kinds of cells can be co-cultured on the temperature-responsive culture substrate with micro-patterning, and then be harvested as a cell sheet. It is expected as a new type of culture substrate that can faithfully reproduce such intricately constructed human tissues as liver tissue (Fig.1). We have also succeeded in developing a temperature-responsive culture substrate with micro-patterning that enables to control cell orientation, and produce cell sheets with well-organized cell alignment (Fig.2 and 3). In developing new class of temperature-responsive, we collaborate with Dai Nippon Printing Co., Ltd. was mainly used, and for example, their microfabrication technology was used to design temperature-responsive surface for acceleration of the cell sheet detachment.

 In addition, we are working on a new approach to assess the properties and functions of these microfabricated culture substrates. As part of this approach, we have developing physicochemical assessing techniques for the microfabricated surface by means of an atomic force microscope (AFM) and observation of cellular behavior by using microchannel technology. It is expected that these techniques will result in an effective approach to assess the features of various substrates from a broad range of perspectives.


Co-culture of hepatocytes and vascular endothelial cell on a micropatterned temperature-responsive surface

Red: hepatocyte, Green: Vascular Endothelial Cell


Aligned fibroblasts on the stripe-patterned temperature-responsive surface

Red : Actin, Blue : Nuclei

←Click to enlarge


Detachment of cell sheet with well-organized alignment by low-temperature treatment

Left: Thermally induced cell sheet detachment, Right: Cell sheet showing different shrinking rate depending on cell alignment (harvested from a substrate (20 mm x 20 mm))
←Click to enlarge


Researcher Introduction

Institute of Advanced BioMedical Engineering and Science,
Instructor  Hironobu TAKAHASHI

Hironobu TAKAHASHI Using photolithography technology, we are developing a new class micropatterned culture substrate, which is based on a temperature-responsive polymer brush. Using the micropatterned substrate, we have succeeded in controlling the orientation of fibroblasts, and have harvested anisotropic cell sheets by means of low-temperature cultivation. As it is possible to manipulate the cell orientation in the direction of the pattern merely by cell seeding onto the substrate, this approach holds promise as a means of reproducing in vitro the innate orientation of cells in the body. Moreover, unlike the cells cultured using existing technologies, these orientation-controlled cells can be harvested as cell sheets. Therefore, the combined use of this new technology and cell sheet engineering promises to create complex tissue that requires the three-dimensional control of their anisotropies.



  • Takahashi H, Nakayama M, Itoga K, Yamato M, Okano T. Micropatterned thermoresponsive polymer brush surfaces for fabricating cell sheets with well-controlled orientational structures. Biomacromolecules. 2011;12(5):1414 - 8.
  • Takahashi H, Nakayama M, Yamato M, Okano T. Controlled chain length and graft density of thermoresponsive polymer brushes for optimizing cell sheet harvest. Biomacromolecules. 2010;11(8):1991- 9.
  • Tang Z, Akiyama Y, Yamato M, Okano T. Comb-type grafted poly(N-isopropylacrylamide) gel modified surfaces for rapid detachment of cell sheet. Biomaterials. 2010;31(29):7435 - 43.
  • Elloumi-Hannachi I, Itoga K, Kumashiro Y, Kobayashi J, Yamato M, Okano T. Fabrication of transferable micropatterned-cocultured cell sheets with microcontact printing. Biomaterials. 2009;30(29):5427- 32.
  • Itoga K, Kobayashi J, Tsuda Y, Yamato M, Okano T. Second-generation maskless photolithography device for surface micropatterning and microfluidic channel fabrication. Anal Chem. 2008;80(4):1323 -7.