Cell sheets can be prepared by culturing cells at 37 degrees C on temperature-responsive surfaces, modified with poly (N-isopropylacrylamide) at a thickness of 20-30 nm. Cultured cell sheets are successfully harvested and manipulated by simple temperature changes from 37 degrees C to 20 degrees C due to hydrophobic/hydrophilic changes of the dish surfaces. This technology has led to "cell sheet engineering" which has initiated new therapies for tissue engineering and regenerative medicine. Various cell sheets such as corneal epithelium, corneal endothelium, skin, periodontal ligaments, oral mucosal epithelium, urothelium, and cardiomyocyte tissues have already been fabricating using these temperature-responsive cell culture surfaces. Additionally, we have already initiated clinical treatments using both corneal and oral mucosal epithelial cell sheets for the treatment of patients with corneal surface diseases. By creating layered cell sheets, structural and functional connections can be achieved. Therefore, we have successfully prepared synchronously beating heart tissues in vitro. Finally, we are also examining the use of heterogeneously layered cell sheets to create laminar structures that resemble the functional and structural units of complex organs such as the liver and kidney.