Novel manipulation technology of cell sheets for tissue engineering

We have focused on novel cell manipulation techniques as a key technology in tissue engineering. We have developed temperature-responsive polymer-grafted cell culture surfaces that are hydrophobic at 37 degrees C and change to hydrophilic below 32 degrees C. Various cell lines adhere, spread, and proliferate on the grafted surfaces similarly to those on ungrafted commercial tissue culture dishes. By reducing culture temperature, cells are spontaneously liberated only from the grafted surfaces without the need for typical enzymatic digestion. Highly trypsin-susceptible cells such as hepatocytes and retinal pigmented epithelial cells retained higher activities of specific cell functions after recovery by reducing temperature. Confluent cells are recovered from the grafted surfaces as a single contiguous monolayer sheet with intact cell-cell and cell-extracellular matrix junctions. Therefore, viable cell sheets can be transferred from temperature-responsive surfaces to other surfaces of culture dishes or devices. With two-dimensional cell sheet manipulation, we confirmed the apical-basal localization of specific cell membrane proteins. Finally we have three-dimensional cell sheet manipulation to reconstruct tissue architectures from cell sheets, since several cell types are coorganized into defined cell sheet layers in natural tissues. Overlaying two monolayer sheets of hepatocytes and endothelial cells obtained from polymer-grafted culture surfaces provides a viable construct for in vitro fabricated liver lobule-like tissue. Cultured lamellar cell sheets preserve each cell phenotype and basic cell functions. We believe that these two- and three-dimensional cell manipulation techniques will become new revolutionary tools for tissue engineering.